Arjun N. Sweet, Nicole M. André, Alison E. Stout, Beth N. Licitra and Gary R. Whittaker
Julia A. Beatty, Academic Editor and Séverine Tasker, Academic Editor

Original article: Clinical and Molecular Relationships between COVID-19 and Feline Infectious Peritonitis (FIP)

Abstract

The emergence of severe acute respiratory syndrome 2 (SARS-CoV-2) has led the medical and scientific community to address questions regarding the pathogenesis and clinical presentation of COVID-19; however, relevant clinical models other than humans are still lacking. In cats, the ubiquitous coronavirus, described as feline coronavirus (FCoV), can manifest as feline infectious peritonitis (FIP), a leading cause of mortality in young cats characterized by severe systemic inflammation. The diverse extrapulmonary symptoms of FIP and the rapidly progressive course of the disease, together with the proximate etiologic agent, represent a degree of overlap with COVID-19. This article reviews the molecular and clinical relationships between FIP and COVID-19. Although there are key differences between the two syndromes, these similarities encourage further investigation of feline coronaviruses as a naturally occurring clinical model for human coronavirus disease.

Keywords: feline infectious peritonitis, SARS-CoV-2, COVID-19, cats

1. Introduction

In the 1960s, feline infectious peritonitis (FIP) was described as a disease in domestic cats and was subsequently found to be of viral etiology, specifically feline coronavirus (FCoV). ^[1,2]. In most cats, infection with FCoV results in mild to inconspicuous clinical signs, but a small proportion of cats develop severe disease and succumb to the systemic form of the disease known as FIP [3]. In the years since the discovery of FCoV, many features of FCoV have remained misunderstood. Similarly, the COVID-19 pandemic, caused by the emergence of SARS-CoV-2, has raised many equally challenging questions regarding pathogenesis, transmissibility, and treatment. The widespread transmission of FCoV/SARS-CoV-2 and the insidious onset of severe symptoms in both FIP and COVID-19 limit the ability to detect the disease early—what may begin as mild or even mild clinical signs or symptoms can quickly lead to systemic disease ^[3,4]. We believe that FIP may represent a valuable, naturally occurring extrapulmonary model of COVID-19.

Both FCoV and SARS-CoV-2 belong to the family Coronaviridae ^[4,5], although to different genera (Figure 1). FCoV, together with similar animal coronaviruses such as canine coronavirus (CCoV) and porcine gastroenteritis virus (TGEV), belong to the genus alpha-coronaviruses. Community respiratory (CAR) human coronaviruses 229E and NL63 are also included in the genus Alphacoronaviruses. ^[6], the latter being associated with the common cold, hail and possibly Kawasaki disease in children ^[7]. In contrast, SARS-CoV-2, together with SARS-CoV (the causative agent of the severe acute respiratory syndrome outbreak in 2002-2003) and the Middle East respiratory syndrome coronavirus (MERS-CoV) belong to the genus betacoronaviruses ^[8], wherein SARS-CoV-2 and SARS-CoV belong to line B (sarbecovirus) and MERS-CoV belong to line C (merbecovirus). Less related beta coronaviruses include human coronavirus CAR OC43 (associated with the common cold), mouse hepatitis virus (MHV), and bovine coronavirus, which is associated with pneumonia and diarrhea in cattle; these viruses are in line A (embekovirus).

FCoV can be classified in two ways, the first of which relates to the form of the disease. Feline enteric coronavirus (FECV) is thought to cause a mild gastrointestinal form of the disease, while feline infectious peritonitis virus (FIPV) is associated with a fatal systemic infection known as FIP. ^[3]. FIPV differs from FECV in its ability to infect and replicate efficiently in monocytes and macrophages ^[9], causing systemic inflammation. FIPV is associated with a spectrum of clinical sequelae. At one end of the spectrum is effusive or “wet” FIP, which progresses rapidly and involves the accumulation of a highly proteinaceous exudate in the abdominal and/or thoracic cavity. At the other end of the spectrum is non-effusive or “dry” FIP, which can affect many organ systems but is usually characterized by neurological and ocular symptoms. Non-effusive FIP generally has a longer course of disease and is less common than its effusive counterpart. FCoV can also be divided into two serotypes – type I or type II – based on major differences in the spike protein of the virus that affect receptor binding and antibody response ^[10]. The receptor for FCoV type II is feline aminopeptidase N (fAPN) ^[11], while the receptor for type I viruses is not identified. Type I FCoV accounts for the vast majority of FIP cases ^[12].

The classification of SARS-CoV-2 virus into different variants based on genetic mutations is still ongoing as the virus continues to evolve. Viral lines that show the potential for increased transmissibility, treatment resistance, vaccine resistance, or increased morbidity and mortality have been identified as VOCs. The spectrum of diseases associated with COVID-19 is broad, ranging from asymptomatic and mild infections to acute respiratory distress syndrome (ARDS), systemic inflammatory response syndrome (SIRS), and multiorgan failure and death. Systemic inflammation in SARS-CoV-2 is not associated with macrophages and monocytes (as in FIP), but is responsible for a wide range of extrapulmonary symptoms. The SARS-CoV-2 receptor, angiotensin converting enzyme-2 (ACE-2), which plays an important role in the renin-angiotensin system and the development of pro-inflammatory status, appears to be involved. ^[13]. Multisystem inflammatory syndrome (MIS) in children and adults, as well as the post-acute course of SARS-CoV-2 infection (PASC), also known as "long COVID", are potential consequences of infection with COVID-19.

2. Transfer

As a group, coronaviruses are known for their ability to cause both respiratory and intestinal diseases and are usually transmitted in one or both ways. While FCoV is considered faecal-oral and SARS-CoV-2 is primarily respiratory, patients with COVID-19 may excrete the infectious virus in the faeces. ^[14], often for a long time, and FCoV can easily become infected by the oronasal route, which is a common method of experimentally vaccinating cats ^[15].

In most cases, FCoV infection is self-limiting, and although the virus can be detected systemically, replication outside the intestinal epithelium is weak. This form of the virus, referred to as FECV, is easily transmitted by the fecal-oral / oronasal route, with common anchors and swallowing of virus particles during purification being common sources of infection. The current understanding of FIP development involves internal mutation: in a small subset of FECV cases, a complex combination of host and viral factors leads to mutation (s) that allow efficient replication in macrophages and monocytes. ^[16]. These lethal variants are classified as FIPV and are associated with systemic inflammation, organ failure, and death. FIPV is generally considered non-transmissible because factors that increase its tropism on troprophages appear to limit its faecal-oral spread. ^[17]. FIP outbreaks were recorded in kennels and shelters. In these situations, congestion stress and high levels of virus in the environment may promote the conversion of FECV to FIPV. There is evidence that some FCoV strains may be more susceptible to this rebirth than others ^[18,19].

SARS-CoV-2 virus infection is primarily targeted at the respiratory epithelium, but as with FCoV, the virus may appear systemically without appropriate symptoms of infection. ^[20,21]. Asymptomatic individuals are a well-documented source of SARS-CoV-2 ^[22,23,24] and delivery includes inhalation of aerosols as well as contact with droplets ^[25]. The incubation times of SARS-CoV-2 and FECV range from 2 to 14 days ^[26]. The incubation time of FIP is highly variable, influenced by the time to internal mutation and the individual's immune response. The onset of FIP can occur several weeks to months after the initial infection ^{[27,28,29,30]}. Multisystem inflammatory syndrome in children (MIS-C), a severe manifestation of SARS-CoV-2, is also delayed by an initial infection with a median onset of 4 weeks. No viral factors have been associated with the development of MIS-C, but an immune-mediated component is thought.

Vertical transmission of FIP via the placenta or milk is considered rare. In the first experimental study in which a suckling cat was infected, one in four kittens succumbed to FIP ^[28]. Maternal antibodies appear to be effective in preventing transmission until approximately six weeks of age, when antibody levels decrease and kittens are susceptible to fecal-oral transmission. ^[31]. However, this maternally acquired immunity can be overcome early in life by high levels of exposure to FCoV – a Swiss study showed that kittens in large herds show infection as early as two weeks of age ^[32,33]. Vertical transmission poses a risk of SARS-CoV-2 infection. Placental transmission is rare but has been documented in fetuses of SARS-CoV-2 infected mothers. ^[34,35,36], as evidenced by virus detection in amniotic fluid, neonatal blood, umbilical cord blood and placental tissue. Transmission cases have been documented in both early and late pregnancies, but infection of the neonate with SARS-CoV-2 may not always occur in the uterus. Infection can also occur during childbirth or in close contact with the mother. The neonatal outcomes of COVID-19 infected mothers remain under study, and it is difficult to distinguish between the effects of SARS-CoV-2 infection and maternal comorbidities. Nevertheless, neonatal infection does not appear to be without sequelae, with one analysis showing that approximately 50 % infected neonates exhibited COVID-19-related clinical symptoms, including fever and respiratory and gastrointestinal symptoms. ^[37].

3. General clinical presentation

Clinical signs associated with both FIP and COVID-19 include fever, diarrhea, depression, weakness, anorexia, and dyspnoea. ^[1]. Typical manifestations of COVID-19 commonly include non-specific symptoms including fever, dry cough, fatigue, dyspnea, and myalgia [38]. Anosmia (loss of smell) and ageusia (loss of appetite) have also been frequently reported in COVID-19 and are more specific symptomatic indicators of the disease. ^[39]. Pneumonia, acute respiratory distress syndrome (ARDS) and sepsis may occur. Men appear to be at higher risk of developing more severe manifestations of COVID-19 ^[40,41], with several small studies confirming the same relationship between males and the development of FIP in cats ^[42,43].

The classic manifestation of FIP is the formation of effusion in the abdomen and / or thoracic cavity; although this manifestation has also been reported in COVID-19 ^[44], is very rare. In addition, FIP is manifested in various bodily systems that are similar to the extrapulmonary manifestations of COVID-19 (Figure 2 and Figure 3). The most similar feature of both diseases is endothelial dysfunction. Vasculitis is a hallmark of FIP pathology ^[45,46] with lesions characterized by perivascular edema and infiltration, vascular wall degeneration and endothelial proliferation ^[47]. In the case of COVID-19, extrapulmonary symptoms are thought to be caused by virus-mediated endothelitis, which leads to vasculitis, especially in veins with minor arterioles ^[48,49]. In the following sections, we will describe these extrapulmonary symptoms and point out key similarities and differences.

4. Biomarkers

Inflammatory biomarkers are important as prognostic markers in COVID-19 and as a means of differentiating FIP from other diseases. In FIP, IL-6 expression appears to be increased in the ascitic fluid of FIP-infected cats, presumably through increased expression in the heart and liver. ^[50,51]. Other acute phase proteins are also elevated in FIP infection. Alpha-1-acid glycoprotein (AGP) has been studied as a diagnostic marker of FIP, but may be elevated in other conditions, thereby limiting its specificity. ^[52,53]. Serum amyloid A (SAA) is another acute phase protein that appears to distinguish between FIPV and FECV infection, with FIPV-infected cats showing higher levels of SAA compared to FECV-infected cats and control cats without SPF. ^[54], but has limited utility in distinguishing FIP from other effusive conditions ^[55].

As with FCoV, subjects with severe COVID-19 have higher SAA levels compared to subjects with milder COVID-19. ^[56]. Higher SAA levels are also reported in patients who died of COVID-19 compared with those who survived ^[57]. C-reactive protein (CRP) is another marker that has been shown to be a promising biomarker in both FCoV and SARS-CoV-2 infections. Liver CRP synthesis is induced by IL-6 expression in response to inflammation ^[58] and is increased in FIP cases ^[59]. Elevated CRP levels in the early stages of COVID-19 are associated with a more severe course of the disease and higher mortality ^[60,61,62], which led to the recommendation to use it as a prognostic indicator in risk assessment in patients hospitalized for COVID-19. In contrast, one meta-survey found that IL-6 levels were elevated, but at least one order of magnitude lower in patients with COVID-19 than in patients with ARDS and non-COVID-19-related sepsis, suggesting a different mechanism of immune dysregulation. ^[63].

The D-dimer, although not specific for COVID-19 or FIP, is another interesting biomarker. D-dimer is released upon fibrin breakdown and is used as a clinical tool to eliminate thromboembolism ^[64]. Thrombotic events have often been documented in COVID-19 in several organ systems ^[65,66] and elevated D-dimer levels are associated with higher morbidity and mortality ^[67,68]. Similarly, thrombotic events can occur in FIP, and high levels of D-dimers, along with other symptoms of disseminated intravascular coagulation (DIC), can be observed in the final stages of FIP in both natural and experimental infections. ^[69,70].

5. Pathophysiology

5.1. Neurological

FIP is one of the major infectious neurological diseases in cats and the symptoms associated with central nervous system (CNS) infection are well documented. ^[71]. CNS symptoms are reported in approximately 40 % cases of dry FIP and may manifest as nystagmus, torticollis, ataxia, paralysis, altered behavior, altered mentoring, and seizures ^[72]. The wide range of symptoms supports the conclusion that the infection is not limited to a specific part of the CNS ^[73]. CNS infection is restricted to the monocyte and macrophage lines and leads to pyogranulomatous and lymphoplasmacytic inflammation, which usually affects leptomening, choroidal plexus and periventricular parenchyma. ^[74].

Documentation of neurological symptoms associated with SARS-CoV-2 CNS infection is limited compared to other coronaviruses ^[75]. The symptoms observed range from headache and confusion to seizures and acute cerebrovascular events. ^[76]. Virus detection in the brain is rare, suggesting that symptoms may not be directly related to CNS infection. Viral particles have been observed in neural capillary endothelial cells and a subset of cranial nerves, although such detection does not correlate with the severity of neurological symptoms. ^[77]. There is often no clear evidence of direct infection. Instead, inflammatory mediators, such as activated microglia, have been reported to contribute to microvascular damage and disease. ^[78,79].

Further comparison of the neuroinflammatory properties of SARS-CoV-2 and FCoV may provide new insights into the neurological manifestations of COVID-19. Further understanding of the neurological symptoms associated with SARS-CoV-2 is necessary to understand the progression of COVID-19 and the extent of CNS infection.

5.2. Ophthalmological

Ocular manifestations of FIP are more common in the dry form of the disease ^[80]. Mydriasis, iritis, retinal detachment, conjunctivitis, hyphema and keratic precipitates have been observed ^[81]. The most common ocular manifestation of FIP is uveitis, which can affect both the anterior and posterior uvea ^[80]. Viral antigen can also be detected in epithelial cells of the nitrating membrane, but viral antigen detection does not distinguish between FECV and FIPV ^[82].

Ocular manifestations of COVID-19 include conjunctivitis, chemosis, epiphora, conjunctival hyperaemia, and increased tear production ^[83]. Uveitis—a common ocular presentation of FIP—has also been observed in SARS-CoV-2 infection ^[84,85]. Tear fluid virus detection has led to concerns about ocular transmission in the first months of the COVID-19 pandemic [83,86]. SARS-CoV-2 RNA was detected in tear secretions and was isolated from ocular secretions, supporting the possibility of ophthalmic transmission ^[87,88]. Interestingly, in the above case study in China, out of 12 patients with ophthalmic symptoms, only 2 patients returned positive conjunctival tests, suggesting limited sensitivity in detecting virus from conjunctival specimens. ^[83].

5.3. Cardiovascular

Pericardial effusion is a less common manifestation of FIP, but is well documented in the literature ^{[26,89,90,91]}. FCoV was detected in the pericardium of cats with recurrent pericardial effusion, which later developed neurological symptoms ^[92]. Direct FCoV infection of the heart was documented in a 2019 case study of FIP-associated myocarditis with severe left ventricular hypertrophy and atrial enlargement. ^[93]. Immunohistochemistry (IHC) revealed the presence of FCoV-infected macrophages and associated pyogranulomatous lesions. ^[26]. Interestingly, severe SARS-CoV-2 infection with evidence of viral replication in the heart and lungs has recently been documented in cats with pre-existing hypertrophic cardiomyopathy (HCM). ^[94].

Unlike FIP, heart damage associated with SARS-CoV-2 infection appears to be much more prevalent. A study of 187 patients found that 27.8 % cases of COVID-19 showed evidence of myocardial damage, as evidenced by elevated cardiac troponin (TnT) levels. ^[95]. High levels of TnT were in turn associated with higher mortality. In a retrospective multicenter study of 68 patients with COVID-19, 27 deaths were attributable to myocardial damage and / or circulatory failure as one of the leading causes of mortality, with elevated C-reactive protein and IL-6 levels associated with higher mortality ^[96]. The increase in such inflammatory biomarkers in the blood suggests that the rapid inflammatory nature of COVID-19 may have a particularly detrimental effect on heart function. Diffuse edema as well as increased wall thickness and hypokinesis have been reported with COVID-19 infection. ^[97]. Cardiac tamponade was also observed in patients with COVID-19, with SARS-CoV-2 levels detectable in pericardial fluid. ^[98]. In contrast to FIP, in which direct invasion of FCoV-infected macrophages into the myocardium was observed in myocarditis, myocardial infection with SARS-CoV-2 virus is not clearly associated with mononuclear cell infiltration or myocarditis. ^[99]. This leads to considerations of multiple systemic factors in adverse cardiac outcomes – particularly dysregulation of inflammatory cytokines. The impact of SARS-CoV-2 infection on the cardiovascular system is an important element in our increasing understanding of the morbidity and mortality associated with COVID-19.

5.4. Gastroenterological

FCoV is excreted in feces and transmitted by the oronasal route. Initial FCoV infection targets the intestinal tract – infection may be subclinical or cats may develop diarrhea and, less commonly, vomiting. The primary infection lasts for several months and the virus can be shed for months to years ^[100,101]. Colonic epithelial cells appear to serve as a reservoir for persistent infection and excretion. ^[21]. The symptoms tend to be mild and spontaneous and only a small proportion of the animals go into the FIP stage. Fibrinous serositis and pyogranulomatous lesions with vasculitis are classic FIP lesions and can be found in the small and large intestines of affected cats. ^[102]. FIP can cause solitary mass lesions in the intestinal wall, although this is considered a rare presentation (26/156 cats in one study) ^[103]. These are usually found in the colon or ileocecal junction and have a pyogranulomatous character.

Gastroenterological symptoms are often reported with COVID-19 infection. ACE2, the cellular receptor for SARS-CoV-2, is widely expressed in glandular cells of the gastric, duodenal and rectal epithelium. Viral RNA and nucleocapsid were detected in these tissues ^[104], which supports their suitability for SARS-CoV-2 replication. Gastrointestinal (GI) symptoms range from general anorexia to diarrhea, nausea, vomiting and abdominal pain ^[105,106]. Excluding a less specific anorexia symptom, multiple meta-analyzes estimate the prevalence of GI symptoms in patients with COVID-19 at approximately 10 % to 20 %, with diarrhea being the most commonly reported symptom. ^{[106,107,108]}. Interestingly, GI symptoms in COVID-19 were observed without accompanying respiratory symptoms ^[105].

Faecal virus excretion is a major concern for COVID-19, as SARS-CoV-2 RNA may continue to be present in faeces even after reaching undetectable levels in upper airway samples. ^[109]. Although the detection of viral RNA in faeces alone does not necessarily indicate the presence of infectious virions, viable viral particles have been detected in faeces. ^[110]. The viral antigen persists in the cells of the gastrointestinal tract as well as in the convalescent phase, up to 6 months after healing ^[20]. In one case study, persistent colonic infection was associated with persistent gastrointestinal symptoms in a case of "long COVID" ^[111], which draws a parallel to the role of the colon epithelium as a reservoir for FCoV.

5.5. Dermatological

Dermatological changes have been reported with both SARS-CoV-2 and FIPV infections. Although papular skin lesions are rare, they are the primary dermatological manifestation of FIP, with several available case reports documenting papules. ^{[81,112,113,114]}. On histological examination, pyogranulomatous dermatitis, phlebitis, periflebitis, vasculitis and necrosis were reported in several FIP cases. ^{[81,112,113,114,115]}.

The first report of dermatological manifestations associated with COVID-19 was recorded at Lecco Hospital in Lombardy, Italy ^[116]. In this study, 18/88 patients (20.4 %) developed a skin disorder, with 8/18 patients observed at the onset of the disease and 10/18 after hospitalization ^[116]. Clinical signs included erythematous rash (14/18 patients), diffuse urticaria (3/18 patients) and smallpox-like vesicles (1/18 patients) ^[116]. Lesions were observed mainly on the trunk (torsion) and pruritus was mild or absent ^[116]. The continuation of the pandemic brought better characteristics of the first observed dermatological symptoms, as well as the identification of rarer presentations. The most common dermatological manifestation of COVID-19 appears to be rash, often characterized by maculopapular lesions. ^[117,118]. Another predominant dermatological symptom also appears to be urticaria ^[118,119]. Importantly, neither rash nor urticaria is specific for COVID-19, which limits their positive predictive value. Varicella-like rash has been observed with SARS-CoV-2 infection and may be more specific due to its low prevalence in viral diseases. In particular, with missing lesions in the oral cavity and pruritus observed in COVID-19-associated rash, together with a previous history of varicella infection, the specificity of this presentation is reinforced. ^[118].

5.6. Teriogenological

Orchiditis and periorchitis with fibrinopurulent or granulomatous infiltrates as well as hypoplastic testes have been observed in several cases of FIP. ^[1,26,120]. Inflammatory mediators from the tuna surrounding the testicles caused enlargement of the testicles in cats with FIP ^[26,120]. In effusive FIP, enlargement of the spinal cord due to edema and peritonitis of tuna was observed ^[16]. Despite the apparent pathology of the male reproductive system in cats, FCoV has not been detected in sperm, which reduces the likelihood of sexual transmission. ^[121]. The pathology of the female reproductive system in FIP is less documented in the literature, but macroscopic lesions present in the ovaries of FIPV-infected cats have been observed. The surrounding uterine and ovarian vessels of these cats were surrounded by lymphocytes, macrophages, plasma cells and neutrophils. ^[122].

As with FIP, the pathology of COVID-19 is manifested in the male reproductive system. One study examining the testes of 12 COVID-19 patients found edema as well as lymphocyte and histiocytic infiltration, consistent with viral orchitis. ^[123]. These samples were also characterized by damage to the seminiferous tubules with a significant effect on Sertoli cells, as well as a reduced number of Leydig cells. In a separate study, germ cell damage was more pronounced despite similar Sertoli cell counts between SARS-CoV-2-infected individuals and uninfected controls, which represents a more direct relationship between infection and fertility. ^[124]. The extent to which SARS-CoV-2 may persist in the male reproductive tract is still being investigated. Although SARS-CoV-2 has been detected in human semen, it is questionable whether this is a true testicular infection or a consequence of a disrupted blood-epididymal / deferent barrier. ^[125,126].

Our knowledge of COVID-19 in the female reproductive system is still limited by the amount of literature and sample size of existing studies. Nevertheless, an understanding of the extent of SARS-CoV-2 in the female reproductive tract is essential to recognize any adverse effects on fertility. ACE2 is expressed in the ovaries, oocytes, and uterus, but limited coexpression of proteases such as TMPRSS2 and cathepsins L and B with ACE2 raises questions about the likelihood of ovarian / uterine infection. ^[127,128]. While in one study of 35 women diagnosed with COVID-19, SARS-CoV-2 was not detected in vaginal fluid or in exfoliated cervical cells, in a case study from Italy, SARS-CoV-2 was detected in vaginal fluid by RT-PCR ( Ct 37.2 on day 7 and Ct 32.9 on day 20 from the onset of symptoms), suggesting that infection of the female reproductive system is possible ^[129,130].

5.7. Immunological response

FIP is classically characterized as an immune-mediated disease based on early observations of complement and immunoglobulin circulation, even in the form of immune complexes. ^[131]. Components of type III and IV immune responses have been described ^[132]. Vasculitis and vasculitis-like lesions are thought to play a role in COVID-19 systemic complications that cannot be explained by direct organ infection, such as microthrombosis in the brain, kidney, spleen and liver. ^[133]. One type III hypersensitivity report has been identified in the COVID-19 literature ^[134]; however, immune complexes do not appear to play an important role in COVID-19 pathology. The mechanism of viral clearance and the inflammatory effects of the immune response are important areas of study for both FIP and COVID-19. Previous work investigating SARS-CoV has demonstrated the need for CD4 + T cells for virus clearance ^[135,136]. T-cell depletion was a recognized consequence of FCoV and was observed to be associated with more severe cases of COVID-19 ^{[137,138,139]}. In addition, FIP reduces both regulatory T cells and NK cells in the blood, mesenteric lymph nodes and spleen. ^[140]. High levels of IL-6 have previously been demonstrated in FIP ascites [50], and similarly, elevated levels of IL-6 appear to be related to disease severity and outcome in patients with COVID-19. ^[141]. The cytokine storm, characterized by overexpression of inflammatory cytokines, has been implicated in the pathogenesis of both infections. In FIP, this pathology is associated with monocyte and macrophage activation, while in COVID-19, the association with macrophages and monocytes is less clear. ^[142]. When considering the balance between cell-mediated immunity and humoral immunity, early reports suggested a link to strong humoral immunity leading to FIP ^[143]. However, humoral immunity may play a more beneficial role in patients with COVID-19 ^[144], especially given the potential clinical benefit of convalescent plasma / serum ^[145].

During the development of the SARS-CoV-2 vaccine, the antibody-dependent infection enhancement process (ADE), in which virus-antibody complexes enhance infection, was particularly important. FIPV has been shown to exhibit ADE in the presence of anti-FIPV antibodies ^[146]. This increase in infection appears to be serotype specific, with passive immunization of cats against FIPV type I or type II leading to ADE only after challenge with the same serotype for which the immunization was performed. ^[147]. As a result, ADE is a major challenge for the development of FIP vaccines. In diseases caused by human coronaviruses, ADE has yet to be fully understood. SARS-CoV has been found to have higher concentrations of anti-spike antibodies having a higher neutralizing effect, while more dilute concentrations are thought to contribute to ADE in vitro. ^[148]. In SARS-CoV-2, ADE was observed in monocyte lines but was not related to the regulation of pro-inflammatory cytokines ^[149]. Spike protein sequence modeling has identified possible ADE mechanisms that involve interaction with Fc receptors on monocytes and adipocytes ^[150]. If ADE played a role in SARS-CoV-2, the most likely mechanism would be overactivation of the immune cascade through Fc-mediated innate immune cell activation. ^[151,152]. There is currently insufficient evidence to suggest an ADE with the pathogenesis of SARS-CoV-2 and further research is needed to assess the true extent of the risk.

6. Molecular similarities between FCoV and SARS-CoV-2 spike proteins

Spike protein is a major factor in tissue and cell tropism and binds the cell receptor ^[153]. It is now well known that SARS-CoV-2 binds angiotensin converting enzyme-2 (ACE-2) as a primary receptor, a common feature of SARS-CoV. There are other binding partners for SARS-CoV-2, including heparan sulfate as a non-specific binding and neuropilin-1 (NRP-1), which may cause viral tropism for the olfactory and central nervous systems. ^[154,155]. In contrast, most alpha-virus viruses, including FCoV type II, use aminopeptidase (APN) virus entry. ^[9,153,156]. The receptor for FCoV type I has yet to be elucidated. The spike protein also mediates membrane fusion, which is activated by a complex process controlled by host cell proteases. ^[153]. While type I FCoV has two protease cleavage activation sites, designated S1 / S2 and S2 ′, type II FCoV has only one cleavage activation site (S2 ′). ^[10]. In comparison, SARS-CoV-2 is similar to FCoV-1 (and currently unique to SARS-related viruses) in that it has two identified cleavage sites (S1 / S2 and S2 ′), the first of which, the furin cleavage site or FCS is considered a significant factor in pandemic spread ^{[157,158,159]}. In both cases, the presence of S1 / S2 cleavage sites distinguishes FCoV-1 and SARS-CoV-2 from their close relatives. The importance of the cleavage activation site appears to be directly related to the proteases required for viral infection, and thus to another component of tissue tropism. In FCoV type I, the transition from FECV to macrophage-tropical FIPV was first demonstrated by amino acid substitutions at the S1 / S2 cleavage site in FIP-confirmed pathological specimens that are thought to reduce proteolytic priming of furin by similar proteases prior to S2-mediated fusion process activation. ^[72,160,161]. In SARS-CoV-2, TMPRSS-2 or other trypsin-like related proteases are a major activator of fusion and entry at S2 ′ ^[162] (Table 1), wherein the furin-like proteases prim the tip and S1 / S2 ^[163] and in particular have been shown to be rapidly regulated after adaptation to Vero E6 cells in culture and possibly also in extrapulmonary human tissues ^[164]. Thus, there appear to be remarkable similarities in host cell adaptation between the two viruses.

7. Prevention and treatment: From social withdrawal to vaccines

Until now, the role of public health / public health measures has been a major driver in mitigating both the spread of FCoV and SARS-CoV-2. ^{[3,31,165,166]}. In this regard, many measures have been introduced for the affected population to reduce social distance, including orders to stay at home, the closure of unnecessary establishments and restrictions on public assemblies. ^[167]. Although not referred to as social distances, similar methods are often introduced or recommended in cat populations. ^[3]. Dreschler et al. summarize the methods that have been recommended in cat populations, especially in a multi-cat environment, including reducing the number of cats per room, frequent cage cleaning, and grouping cats according to excretion and / or serological status. ^[168]. Dreschler argues that quarantining cats exposed to FCoV / FIPV to limit the spread of FCoV in the population is neither effective nor beneficial given the likelihood of widespread FCoV infection in a multi-cat environment as well as the months required for development (and uncertainty in development) FIP. In contrast, quarantine people exposed to SARS-CoV-2 has the potential to reduce the spread of the disease and mortality ^[169]. Regardless of the extent of grouping or separation, the social difficulties caused by separation must be carefully considered for both cats and humans. In the case of cats, especially in connection with the premature weaning of their mothers, special attention must be paid in the weaning process to ensuring adequate socialization of the kittens. Similarly, in the case of COVID-19, the process of quarantine and / or isolation can be psychologically burdensome for individuals. A thorough cost-benefit analysis must often be carried out to compare the benefits of quarantine and isolation for public health with the negative psychological burden on the persons concerned in order to avoid unnecessary / ineffective quarantine. Where appropriate, justification as well as support to improve well-being should be provided ^[170].

Although the FIP vaccine is commercially available (Primucell), the benefit of FIP vaccination is still low. Primucell is an intranasal vaccine that uses an attenuated FIPV serotype 2 isolate. Although the FIP vaccine is commercially available (Primucell), the benefit of FIP vaccination is still low. Primucell is an intranasal vaccine that uses an attenuated FIPV serotype 2 isolate (FIPV-DF2), given in two doses 3 to 4 weeks apart to cats at least 16 weeks old. ^[171]. In a placebo-controlled experimental study in 138 cats, vaccinated cats did not show a significantly lower incidence of FIP compared to controls during the 12-month study period. After adjusting for FCoV titers, cats with lower antibody titers (100 or less) had a significantly lower incidence of FIP at the time of the first vaccination compared to cats with higher titers (400 or more). ^[172]. However, due to the high prevalence of FCoV, especially in multi-cat environments, attempts to alleviate FIP by vaccinating cats that are FCoV-naive at least 16 weeks of age may not be feasible due to the high potential for FCoV infection during the 16 weeks prior to vaccination. As a result, the American Association of Animal Hospitals and the American Association of General Practitioners for Cats do not recommend FIP vaccination. ^[173].

ADE remains a major concern of FIP vaccines. Several studies have attempted to reduce the incidence of FIP in experimentally infected cats with recombinant and other experimental vaccines, but ADEs have been reported repeatedly. In one placebo-controlled study in which purebred British Shorthairs and Specific Pathogen Free (SPF) Domestic Shorthairs were vaccinated with one of two recombinant FIPV type 2 (FIPV-DF2) vaccines, both candidate vaccines showed significantly reduced to no protection against challenge FIPV in non-SPF cats - with the majority of non-SPF animals showing ADE ^[174]. In a separate study, immunization of kittens with a vaccine virus recombined with the FIPV spike glycoprotein gene significantly shortened survival time after FIPV challenge compared to kittens immunized with wild-type vaccine virus. Importantly, low levels of neutralizing antibodies were observed in the group immunized against FIPV ^[175]. Concerns about ADE after FIPV immunization remain a challenging challenge in FIP prevention.

Unlike the FIP vaccination, the COVID-19 vaccines have played a more significant role in reducing the spread of the infection. Several types of vaccines have been produced that have demonstrated safety and efficacy in preventing symptomatic infection, severe disease, and death from COVID-19—including, but not limited to, mRNA vaccines (Pfizer/BioNTech and Moderna), viral vector vaccines (Janssen, AstraZeneca), and inactivated virus vaccines (Bharat Biotech, Sinovac) ^{[176,177,178,179,180,181]}. The first two vaccine platforms use the SARS-CoV-2 spike glycoprotein as the immunogen, while inactivated virus vaccines have the potential to elicit an immune response to viral components other than the spike glycoprotein. Despite the favorable safety profile of COVID-19 vaccines, adverse reactions occurred after vaccination, some of which were mediated by antibodies in analogy to ADE concerns with FIP vaccines. Thrombosis has been a documented problem, especially with AstraZeneca and Janssen vaccines. Although the exact mechanisms are being investigated, the inflammatory response is currently thought to lead to increased levels of platelet-activating antibodies, which bind to platelet factor 4 and lead to a hypercoagulable state. ^[182,183]. In contrast to the higher incidence of ADE in experimental FIP vaccines, the incidence of thrombotic events after administration of COVID-19 is low. ^[184].

In addition to the primary endpoints of vaccine studies, which focused on the prevention of symptomatic infection, serious illness, and death from COVID-19, many phase 3 vaccine studies did not address observations to assess the degree of prevention of asymptomatic infection. Beneficial efficacy against asymptomatic infection is important from a public health perspective, especially given that asymptomatic individuals can transmit COVID-19 and that routine monitoring testing is resource intensive and difficult to coordinate on a large scale. ^[22]. An important contribution towards this field is the real-world studies investigating the efficacy of the vaccine, which show a reduced risk of SARS-CoV-2 infection, as well as a reduced viral load in vaccine "breakthrough" infections. ^{[185,186,187,188]}. Such evidence supports the use of SARS-CoV-2 vaccines as a protective measure not only against severe COVID-19, but also as a crucial contribution in the management of the disease.

8. Clinical care and therapeutic options

In 1963, when the first clinical cases of FIP were described (before understanding the viral etiology), it was found that antibiotic treatment was often tried, but it clearly did not bring any benefit. ^[189]. Since this first report and without an effective vaccine, a number of therapies have been tried in cats with FIP. Ribavirin, a nucleoside analogue, has previously provided promising results against FCoV in an in vitro study ^[190], however, when administered to cats as an experimental treatment, led to poorer results in some cases ^[191]. Similarly, at the onset of the COVID-19 pandemic, ribavirin was used in several doses and in combination with other drugs. ^[192] and a study protocol was designed to examine the benefit in human patients ^[193]. However, another direct-acting antiviral drug (DAA) (remdesivir), a nucleoside analogue that acts as a chain terminator and raises minor toxicity concerns compared to ribavirin, is rapidly gaining prominence in the treatment of hospitalized patients with COVID-19. . Despite initial enthusiasm, remdesivir has not been shown to be effective in patients with such diseases in robust clinical trials; however, several reports have demonstrated the clinical benefit of the related nucleoside analog GS-441524 in the treatment of cats with FIP, including effusive, non-fusive, and neurological forms of the disease. ^{[194,195,196,197]}. At the time of writing, studies on the effectiveness of remdesivir in the treatment of FIP are being conducted in Australia and the United Kingdom. Interestingly, remdesivir is a prodrug form of GS-441524 [195]. Two orally available DAAs have recently entered clinical trials for COVID-19 and are currently awaiting FDA approval; molnupiravir (MK-4482 / EIDD-2801), a modified form of ribavirin, and Paxlovid (a protease inhibitor PF-07321332 in combination with ritonavir, which improves the half-life of PF-07321332) targeting the major protease (Mpro). It is noteworthy that the active substance Paxlovid is related to GC-376 and has previously been shown to be effective in a FIP clinical study. ^[196]. It will be very interesting to follow the development, FDA approval and use of these DAAs in relation to the relevant diseases caused by SARS-CoV-2 and FCoV.

Due to the inflammatory nature of both FIP and COVID-19, treatment often focuses on controlling the immune response. Although cats with FIP are often given glucocorticoids in an effort to alleviate the inflammatory symptoms of the disease, the clinical benefit is negligible. ^[198]. The use of corticosteroids in patients with COVID-19 does not appear to be insignificant, with some studies showing negative profiles ^[199]. However, their administration may be beneficial in severe cases of COVID-19 through the observed reduction in mortality ^[200,201]. Cyclosporine, an immunosuppressive drug that is often used to prevent organ rejection in transplant patients and to treat some autoimmune diseases, has been studied in both FIP and SARS-CoV-2. An in vitro study with cyclosporin A (CsA) using FCoV type II virus showed a reduction in virus replication ^[202], treatment of a 14-year-old cat CsA after unsuccessful IFN treatment resulted in clinical improvement, reduction in viral load and survival of more than 260 days ^[203]. Although there are currently no controlled studies on the use of CsA in the treatment of patients with COVID-19, in addition to safety concerns, potential mechanisms of action have been suggested. ^{[204,205,206]}. In addition, the cyclosporin A analogue, alisporivir, has shown in vitro effects on virus replication ^[207], similar to evidence that cyclophilin A blockade inhibits the replication of other coronaviruses ^[208].

Many antibiotics have been prescribed for both FIP and COVID-19, but not for their antimicrobial properties, but rather for their anti-inflammatory effects. ^[198]. For example, doxycycline may have helped prolong survival in cats with FIP ^[209]. Whether doxycycline would be of benefit to patients with COVID-19 is not currently known, but has been suggested as a possible part of the treatment of the disease. ^[210].

Interferons have also been studied in the treatment of FIP without a clear link to clinical improvement ^[211]. In human patients with COVID-19, combination therapy with interferon-β-1b with lopinavir, ritonavir and ribavirin compared with lopinavir and ritonavir alone was associated with a reduced duration of virus excretion and improved clinical outcomes in mild to moderate cases. ^[212].

Monoclonal antibodies targeting components of the immune response have the potential to reduce inflammatory cytokine levels. A small study in cats experimentally infected with FIPV-1146 demonstrated the benefit of anti-TNF-α in managing the disease ^[213]. Tocilizumab, an anti-IL-6 monoclonal antibody, was administered to patients with COVID-19 [214]. Due to the different clinical results reported, further research is needed with Tocilizumab ^[215,216].

The transfer of knowledge between species will undoubtedly affect cats as well as humans and even other species. Although many compounds are effective when studied in vitro, their use in vivo can lead to different results, including toxicity. In addition, the fact that a compound may show promising results in one species does not mean that the same effect will be observed in other species, especially when comparing similar but different viruses and virus-induced diseases.

9. MIS-C and PASC

In April 2020, the UK's National Health Service issued an alert about an increased incidence of multisystem inflammatory syndrome in children - many of whom tested positive for COVID-19 ^[217]. As the pandemic progressed, studies from other countries examining this inflammatory condition provided more detail towards a clinical understanding of what is now referred to as MIS-C, a rare presentation of COVID-19 in pediatric patients. MIS-C includes several organ systems. Cardiovascular dysregulation in MIS-C is often observed in the form of ventricular dysfunction, pericardial effusion and coronary artery aneurysms ^[218,219]. Gastrointestinal symptoms mimic appendicitis and include abdominal pain, vomiting, and diarrhea. Terminal ileitis is a common finding on imaging tests ^[220]. Many patients also experience neurocognitive symptoms, including headache and confusion. More serious neurological complications, including encephalopathy and stroke, are less common ^[218,221].

One area of significant clinical overlap between FIP and COVID-19 is the rare inflammatory manifestation of SARS-CoV-2 infection – multisystem inflammatory syndrome in children (MIS-C). MIS-C is seen in the pediatric population, just as FIP commonly affects young cats ^[43]. Like FIP, MIS-C has a systemic presentation involving multiple organ systems—including gastrointestinal, cardiovascular, and hematologic abnormalities ^[222]. As with the wet form of FIP, pleural effusions and ascites occur in MIS-C. ^[223]. Both syndromes also show overlap in vascular pathology. FIP shows granulomatous vasculitis, which overlaps with Kawasaki vascular syndrome observed in MIS-C ^[224]. MIS-C is thought to be a post-infectious disease associated with a previous SARS-CoV-2 infection ^[223,225]. FIP also has a delayed onset after the first exposure to FCoV and occurs only in a small subset of cases. Although cats with FIP can still shed FCoV in their feces, the mutations associated with the biotype switch from FECV to FIPV are thought not to be transmissible—supporting some degree of similarity in the limited infectious range of both FIP and MIS-C.

Recently, post-acute sequelae of COVID-19 (PASC) has been defined, which includes memory loss, gastrointestinal distress, fatigue, anosmia, dyspnea, etc. and is more often referred to as "long-term COVID". Along with MIS-C, PASC is a very active subject of research, which has been summarized by others ^[226], and together represent an excellent starting point for the use of feline medicine as a model of coronavirus-induced pathogenesis, possibly in an unexpected way ^[224].

10. SARS-CoV-2 infection in cats

Cats have now become widespread hosts for SARS-CoV-2 infections, in part due to the relative similarity of human and feline ACE2 receptors. Following cases reported in Hong Kong and Belgium in March 2020, the most notable early natural infection occurred at the Bronx Zoo in New York City, USA. In April, four tigers and three lions showed mild respiratory symptoms from their breeders, and SARS-CoV-2 was detected by PCR and sequencing. ^[227]. Consequently, infection of both domesticated and non-domesticated cats has become relatively common in cases where owners and caregivers are positive for SARS-CoV-2. From a clinical point of view, SARS-CoV-2 infection in cats is considered to be predominantly asymptomatic, with some animals showing mild respiratory symptoms. ^{[228,229,230]}. In general, severe respiratory symptoms do not appear to occur in cats, although severe respiratory distress may in some cases be related to the underlying hypertrophic cardiomyopathy (HCM) of cats. ^[94]. An increased incidence of myocarditis in dogs and cats has also been reported in the United Kingdom, associated with a sharp increase in variant B.1.1.7 (Alpha). ^[231]. There is a clear need for further studies in this area, as well as possible links between coronavirus infections in cats and multisystem inflammatory syndrome in children (MIS-C), which, as mentioned above, is a rare manifestation of COVID-19.

Laboratory animal studies have also been key to understanding SARS-CoV-2 infection in cats, which are very susceptible to infection by oronasal challenge. Experimentally infected cats showed mild respiratory symptoms or asymptomatic infection, virus shedding, virus-to-cat virus transmission, and a strong neutralizing antibody response. Recent studies have shown that long-term immunity exists after re-infection of cats, but cats may develop long-term consequences, including persistence of inflammation and other lung lesions. ^[232]. Overall, as with SARS-CoV in 2003, cats in particular can be an important source of information on the pathogenesis and immune responses elicited by SARS-CoV-2.

Thanks

Thanks to Annette Choi for helping with Figure 1 and all Whittaker Lab members for helpful discussions during the preparation of this manuscript.

Author shares

All authors have contributed to this article. All authors have read and agreed to the published version of the manuscript.

Financing

The work in the author's laboratory is partly funded by research grants from the National Institutes of Health, the EveryCat Foundation and the Cornell Feline Health Center. AES was supported by the NIH Comparative Medicine Training Program T32OD011000. Studies on FIP are also supported by the Michael Zemsky Fund for Cat Diseases.

Conflict of interests

The authors do not indicate any conflict of interest.

Footnotes

Publisher Note: The MDPI remains neutral in terms of jurisdictional claims in published maps and institutional affiliation.

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Original article: A review on the diagnosis of feline infectious peritonitis
3.3.2022

Jehanzeb Yousufa | Riyaz Ahmed Bhatb | Shahid Hussain Darb | Alisa Shafib | Snober Irshadc | Mohammad Iqbal Yatoob | Jalal Udin Parrahb | Amatul Muheeb | Abdul Qayoom Mirb

Abstract: Feline infectious peritonitis, or simply FIP, is a coronavirus viral disease in cats, usually less than three years old. It is manifested by an extreme inflammatory reaction in the tissues in the abdomen, kidneys and brain. This review article discusses the various diagnostic tests and their benefits in diagnosing cases of suspected FIP for definitive diagnosis. This review can help compare different diagnostic parameters and also raise awareness of their advantages and disadvantages.

Keywords: acute phase proteins, coronavirus, feline infectious peritonitis, Rivalt's test.

1. Introduction

Feline infectious peritonitis (FIP) is a well-known and widespread coronavirus (CoV) -induced systemic disease in cats, characterized by fibrinous-granulomatous serositis with protein-rich exudates in the body cavities, granulomatosis-announcing phlebitis in the gallbladder and periphels Scott 1981; Kipar et al. 2005). Feline CoV (FCoV) spreads via the faecal-oral route and is primarily infected with enterocytes (Pedersen 1995), but subsequently spreads systemically via monocyte viremia (Meli et al 2004; Kipar et al 2005). The increased ability of the virus to replicate has been shown to be a key feature in the development of FIP, and FIP is also thought to be caused by mutations in common feline enteric coronavirus (FECV), which occurs in cats worldwide and is not a serious infection (Pedersen et al 2009; Healey et al 2022). Approximately 10 % infected cats have mutations that result in feline infectious peritonitis. In large multi-cat situations, FECV is excreted in the faeces of most seemingly healthy cats and the transmission occurs through direct contact with faeces or contaminated litter and other fomits (Pedersen et al 2004). Kittens become infected at approximately 9 weeks of age (Pedersen et al. 2008). The time between the onset of clinical signs and death also varies, but younger cats and cats with effusive disease have a shorter course of disease than older cats and cats with non-effusive disease (Pedersen 2014). Even with severe FIP, some cats can live for months. in multi-feline situations, feline enteric coronavirus (FECV) is extremely common and highly contagious. Almost all cats that come into contact with FECV from secreting cats become ill, but on the other hand, the infection is usually asymptomatic or causes only mild temporary diarrhea (Pedersen et al. 2008; Vogel et al. 2010; Ermakov et al. 2021). On the other hand, feline infectious peritonitis virus (FIPV) is not transmitted via the faecal-oral route, but originates from avirulent FECV in a small percentage of infected cats and causes feline infectious peritonitis (FIP) (Pedersen et al 1981; Vennema et al. 1998). Anorexia, lethargy, weight loss, pyrexia, ocular and neurological symptoms such as gait abnormalities or inappropriate mentoring are non-specific (Giori et al 2011; Kipar et al 2014). The infection takes two forms: "wet" and "dry". The dry form causes inflammatory changes around the vessels, seizures, ataxia and excessive thirst, while the wet form leads to enlargement of the abdomen due to excessive accumulation of fluid in the abdominal cavity. Specificity is always the most important diagnostic value to consider in order to avoid misdiagnosis of FIP in unaffected cats.

2. Diagnostic tests for feline infectious peritonitis

The diagnosis takes into account the age, origin, clinical signs and physical examination of the cat. Abdominal distension with ascites, dyspnoea with pleural effusion, jaundice, hyperbilirubinuria, marked masses of the kidneys and / or mesenteric lymph nodes, uveitis and various neurological symptoms are common in cats with effusive (wet) or non-fusive (dry) forms of FIP. and / or spinal cord. Ocular changes often occur in cats with FIP, with the most common ocular disorders being retinal changes. Retinal cuff cuffs may occur, which appear as blurred gray lines on either side of the vessels. Occasionally, granulomatous changes in the retina occur. FIPV infection has been found to be associated with T-cell depletion by apoptosis, although the virus cannot infect CD4 + and CD8 + T-cells (Haagmans et al. 1996; De Groot et al. 2005). Due to the high mortality rate, many veterinarians and pet owners are cautious about a diagnosis based on "reasonable assurance". The challenge is to decide whether the test increases the likelihood that clinical symptoms are caused by FIP (indirect tests) or offers a definitive diagnosis (direct tests). It is important to note that the sensitivity and specificity of any indirect test will vary depending on the likelihood that the cat is infected due to other factors. This means that a positive predictive value of the test, such as complete blood count (CBC) or albumin: globulin (A: G) ratio, to predict FIP will be much higher in cats with FIP-like signaling than in cats with non-FIP signaling. It should be noted that the results of other indirect tests are only estimates and the results of additional indirect tests have the potential to confuse and support the diagnostic process.

3. Diagnostic tests

The problem with FIP diagnostics is that non-invasive tests are not reliable enough. In general, effusion tests have a significantly higher predictive value than blood tests (Stranieri et al. 2018; Hartmann et al. 2003). As a result, the identification of FIP ante mortem in cats without significant effusion is particularly difficult. The most useful ante-mortem indicators are positive anti-Corona (IgG) antibody titers in cerebrospinal fluid (CSF), high total serum protein, and MRI changes such as periventricular contrast enhancement, ventricular dilatation, and hydrocephalus. However, monoclonal antibodies from affected tissues and coronavirus-specific polymerase chain reaction (PCR) are valuable in post mortem evaluations (Foley et al. 1998). As a clear diagnosis cannot be made on the basis of symptoms, medical history and clinical and laboratory indicators alone, these factors should always be considered as a whole, sometimes in combination with other factors such as molecular or even more invasive diagnostic procedures.

3.1. Analysis of effusion samples

If FIP with effusion is suspected, the effusion sample can be incredibly helpful in making a diagnosis and then in hematological findings, so obtaining effusion samples should always be a top priority. In the case of ascites, the sample can be obtained by ultrasound-guided thin-needle aspiration or the "flying cat" technique. To identify small amounts of fluid in the chest and abdomen, ultrasonography provides useful assistance in locating effusion bags in the abdomen, while evidence of pericardial effusions can be obtained through suppressed cardiac echoes and electrocardiographic changes. Ultrasonography should be used repeatedly to identify any small volume effluents, and ultrasonography may also be used to guide the sampling of small bags of fluid. In cats with pericardial effusions, auscultation of the heart detects muffled sounds and the ECG reveals typical changes.
FIP effusions are often clear, viscous / sticky, straw yellow and rich in protein (cytology often describes a dense eosinophilic protein background) with a total protein concentration> 35 g / l (> 50 % globulins). Chlootic effusions are rarely described. FIP effusions are often pyogranulomatous in nature with macrophages, non-degenerate neutrophils and a relatively small number of lymphocytes. As a result, effusions are often referred to as modified transudates based on cell number (<5 × 109 cells / L), but exudates based on protein concentration (greater than 35 g / L). Typical FIP effusions have a low A: G ratio (see above) and an increased AGP content, which is similar to the serum content. A recent study (Hazuchova et al. 2017) found that AGP concentrations in effusions (> 1.55 mg / ml) are more useful (sensitivity and specificity 93 %) in distinguishing FIP cases from cases without FIP than serum AGP levels or other APPs.
The rival test is a simple test that can be used to distinguish transudate from exudate in a effusion sample (Barker and Tasker 2020). Positive results simply suggest that the effluent is exudate and not specific to FIP; positive transudate results have been documented in situations other than FIP (eg, bacterial / septic peritonitis and lymphoma) (Fischer et al. 2012).

3.2. Serum biochemistry

Although the changes in blood biochemistry observed in FIP cases are variable and often non-specific, there are several key anomalies that need to be addressed in order to confirm the diagnosis of FIP.

3.2.1. Acute phase proteins

Many inflammatory and non-inflammatory diseases produce acute phase proteins (APPs) in the liver in response to cytokines released by macrophages and monocytes (particularly interleukins 1 and 6 and tumor necrosis factor α).
AGP is an abbreviation for α1-acid glycoprotein and its examination may help in the diagnosis of FIP. Although the increase in AGP levels (> 0.48 mg / ml) is not specific for FIP, patients with FIP often have significantly high AGP levels (> 1.5 mg / ml). As a result, the magnitude of the increase may be valuable in helping to diagnose FIP, with higher levels more effectively increasing the suspicion index (Giori et al 2011; Hazuchova et al 2017).

3.2.2. Hyperglobulinemia

In 89% cases, hyperglobulinemia is present; often in association with hypoalbuminemia or low-normal serum albumin levels (observed in 64.5 % cases) (Riemer et al. 2016). Hyperproteinemia may not always occur due to the existence of hypoalbuminemia. The albumin: globulin (A: G) ratio is low in hyperglobulinemia and hypoalbuminemia (low-normal albumin concentration) and this parameter can be used to assess the likelihood of FIP in a particular case.

3.2.3. Hyperbilirubinemia

Hyperbilirubinemia occurs in 21-63 % cases of FIP and is more common in effusive FIP, where alanine aminotransferase (ALT), alkaline phosphatase (ALP) and γ-glutamyltransferase enzymes are commonly high (although these may be slightly increased in FIP cases). FIP is rarely associated with hyperbilirubinemia due to immune-mediated hemolytic anemia (IMHA) (Norris et al. 2012) and cats are often not severely anemic. In the absence of high liver enzyme activity or severe anemia, the presence of hyperbilirubinaemia should suggest FIP (note that sepsis and pancreatitis may cause hyperbilirubinaemia without increased liver enzyme activity). Based on a stepwise evaluation of cats with FIP, it was documented that hyperbilirubinemia was more typically recognized in cats just before death or euthanasia than in the first presentation (Harvey et al. 1996). In addition, higher bilirubin levels were observed in cats just before death or euthanasia than in the first presentation.

3.3. Hematology

In FIP, hematological changes are non-specific; however, there are several abnormalities that need to be checked to confirm the diagnosis. Lymphopenia is the most common change (55 - 77%) in cases, with a recent study (Riemer et al. 2016) revealing lymphopenia in only 49.5 % cases of FIP, with neutrophilia (39 - 57 %), left-sided and mild to severe normocytic, normochromic anemia (37-54 %) (Riemer et al. 2016; Norris et al. 2012). Recently, an association between FIP and microcytosis (with or without anemia) has been discovered. FIP can cause severe IMHA with concomitant regenerative anemia; however, this is an unusual phenomenon.

3.4. Serology

ELISAs, indirect immunofluorescence antibody assays, and rapid immunocompromination assays are the most common assays for anti-FCoV antibodies in serum (Addie et al. 2015). In most studies, cells infected with CoV pigs or cats are used as substrate and titers are measured in multiples of serum dilutions. A positive anti-FCoV antibody test means that the cat has been infected with FCoV and has seroconverted (which lasts 2-3 weeks after infection). The tests are therefore of limited clinical importance. Breed-dependent differences in anti-FCoV antibody titers were found, which could indicate differences in the breed's response to FCoV infection (Meli et al. 2013).
Although cats with FIP had higher anti-FCoV antibody titers than cats without FIP, no difference was found between the median anti-FCoV antibody titers in healthy cats and cats with suspected FIP. As a result, the titer in one animal is only marginally useful in identifying cats with FIP (Bell et al. 2006). Many clinically healthy cats (especially those in multi-cat households) have positive and often very high anti-FCoV antibody titers, while 10 % cats with FIP are seronegative, which could be due to virus binding to the antibody and its inaccessibility for serological testing. which also points to problems with interpretation (Meli et al 2013). A negative FCoV antibody test if dry FIP is suspected may be more effective at excluding FIP (Addie et al. 2009). Nevertheless, negative results have been observed in neurological FIP situations (Negrin et al 2007). As a result, doctors disagree on whether to perform a serological test in suspected cases, even though a positive result almost always means FCoV exposure.

3.5. Current trends in diagnostics

The use of anti-coronavirus antibody testing in cerebrospinal fluid (CSF) for diagnosis in cases involving the central nervous system is another breakthrough in which IgG is detected in the CSF. However, in most cases, the antibody was detected only in cats with high serum IgG titers (Boettcher et al. 2007)
An important difference between feline coronavirus and FIP infection is the behavior of the NSP3c gene. The infected tissue isolates from the second case were found to have a disrupted gene 3c, while in the first case the gene was intact. Mutation of the S1 / S2 locus and modulation of the furin recognition site, which is normally present in the S-gene of the enteric coronavirus (Levy and Hutsell 2019), is also a crucial contributing factor.
The diagnostic utility of cerebrospinal fluid immunocytochemistry is also used to diagnose FIP manifested by severe central nervous system involvement. Immunocytochemical staining (ICC) of feline coronavirus antibodies in cerebrospinal fluid macrophages is a highly sensitive test, especially for the diagnosis of ante mortem with a sensitivity of 85 % and a specificity of 83.3 % (Gruendl et al 2017).

4. Conclusions

In cats with suspected FIP, anamnesis, clinical signs and clinicopathological examinations should be correlated. The dry form is more difficult to diagnose than the wet form. In the wet form, laboratory analysis of the fluid can be performed, such as the Rivalt's test. If the test is negative, the probability of FIP is small, but if the test is positive, additional diagnostic tests should follow to confirm FIP. In FIP, the A: G ratio is low because hyperglobulinemia and hypoalbuminemia (low normal albumin concentration) are present, and this parameter can be used to assess the likelihood of FIP in a particular case. Patients with FIP often have significantly high levels of AGP (α1-acid glycoprotein). When distinguishing FIP cases from non-FIP cases, AGP concentrations in effusions (> 1.55 mg / ml) have a sensitivity and specificity of 93 %.

Conflict of interests

The authors declare that they do not have a conflict of interest.

Financing

No financial support was provided from any institute or other source.

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Ying-Ting Wang,¹ Bi-Ling Su,² Li-En Hsieh,¹ and Ling-Ling Chueh¹
Original article: An outbreak of feline infectious peritonitis in a Taiwanese shelter: epidemiologic and molecular evidence for horizontal transmission of a novel type II feline coronavirus
Czech translation partially taken from: Results Confirmation of the FIP outbreak in the cat shelter - Sevaron
13.7.2013

Abstract

Infectious feline peritonitis (FIP) is a fatal disease caused by feline coronavirus (FCoV) infection. FCoV can be divided into serotypes I and II. The virus that causes FIP (FIPV) is said to occur sporadically and does not often spread from one cat to another. An outbreak in one animal shelter in Taiwan was recently confirmed. FCoV from all cats in this shelter was analyzed to determine the epidemiology of this outbreak. Thirteen of the 46 (28,2%) cats with typical FIP symptoms were identified. Of these, FIP was confirmed in seven cats by necropsy or histopathological examination. Despite the fact that in this environment with more cats, more than one FCoV was identified, eight cats with symptoms of FIP were reliably found to be infected with FCoV type II. Sequence analysis revealed that FIPV type II, found from feline faeces, body effusions and granulomatous tissue homogenate from cats that underwent FIP, contained identical recombination in all cases. WITH gene. Two cats that succumbed to FIP were found to have an identical nonsense mutation in 3c gene. The excretion of this type II virus in faeces of the effusive form of FIP can be detected up to six days before the animal dies. In general, our data demonstrate that horizontal transmission of FIPV is possible and that FIP cats may pose a potential risk to other cats living in the same environment.

Introduction

Infectious feline peritonitis (FIP) is a fatal disease of cats caused by feline coronavirus (FCoV) infection. FCoV is an enveloped RNA virus that belongs to the species Alphacoronavirus, family Coronaviridae and in order Nidovirales. The size of the FCoV genome is approximately 28.9 kb, including the nonstructural replication gene; four structural genes that encode spike (S), envelope, membrane, and nucleocapsid proteins; and five helper / nonstructural genes 3abca 7ab[1].

Feline coronaviruses cause mild, invisible, and transient bowel infections and are ubiquitous among cat populations worldwide [2]. They occur in two serotypes, I and II [man]3]. Type I FCoV predominates here, while type II virus represents only 2-30% infections [4–8]. Following the accumulation of genetic evidence, it is apparent that FCoV type II was formed by two homologous recombinations between FCoV type I and canine coronavirus CoV (CCoV) [9,10]. Both serotypes can mutate in the host, lead to macrophage tropism and a systemic disease called infectious feline peritonitis [cat]2,11,12]. Due to poor virus shedding in FIP studies in cats, mutant FIP viruses (FIP-inducing FCoV, FIPV) appear to be contained only in diseased tissues and are not naturally transmitted in cat-to-cat contact [2,11,13,14].

In this article, we report an epizootic FIP in a shelter in Taiwan that was caused by a new Type II FCoV. Epidemiological and molecular examination of isolates from various healthy and sick cats from this shelter strongly suggests that the virus was introduced by moving kittens from another shelter with subsequent horizontal spread to adult cats with which the new kittens shared the shelter.

Materials and methods

Animals and sampling

A total of 46 cats from a private shelter were included in this study, which ran from September 2011 to August 2012. This shelter houses adult cats and from time to time a few kittens. All the cats were either strayed or rescued, and some of them were obtained from the homes of various private rescue stations where the rescued cats were temporarily housed. Before the onset of the disease, all cats lived together in an indoor environment without cages, sharing food, drink and toilets. Some cats were siblings, others were not related to them (Table 1).

Faeces or rectal samples were taken from all asymptomatic cats at least once to monitor for FCoV. Body swabs, blood samples, swab specimens, including rectal, nasal, oral and conjunctival specimens, were taken as standard from cats that already showed signs of the disease or were suspected of having FIP. In addition to supportive care, cats with suspected FIP were treated with prednisolone (Prelon®, YF Chemical Corp., New Taipei City, Taiwan), benazepril (Cibacen®, Novartis, Barbera del Valles, Spain) and recombinant human interferon alpha (Roferon®-A). , Roche, Basel, Switzerland). Cats that succumbed to the disease were necropsied for pathological confirmation. During necropsy, body exudates were first removed with a needle and syringe, followed by swabs, blood, urine and granulomatous lesions on the internal organs. All samples were frozen at -20 ° C until use. All samples were tested for FCoV nested reverse transcription polymerase chain reaction (RT-nPCR) [man]15]. Samples with positive results were subsequently subjected to further analysis.

Sample preparation and reverse transcription

Swab samples were suspended in 1 ml of water treated with 0.1% diethyl pyrocarbonate (DEPC). Stool samples were suspended with 9x treated water 0.1% DEPC by vortexing. The suspension was centrifuged and the supernatant was transferred to a new tube. About 0.5 g of tissue was frozen and then crushed with a mortar and pestle in the presence of 2 ml of Trizol [16]. Total RNA was extracted from 300 μl of swab suspension, whole blood, faeces suspension, tissue homogenate and body effusion using Trizol. Twenty-one microliters of isolated RNA was reverse transcribed with specific primer N1 (5′-gctacaattgtatcctcaac-3 ′) or P211 [15] with Moloney mouse leukemia reverse transcription (Invitrogen, CA, USA). The reaction was incubated at 37 ° C for 60 min, at 72 ° C for 15 min and finally at 94 ° C for 5 min.

FCoV type determination by nested PCR

Nested PCR was performed for FCoV typing according to the procedures of Addie et al. [5] with a slight modification. After reverse transcription, 5 μl of complementary DNA was added to 25 μl of PCR mix (Invitrogen, CA, USA) according to the manufacturer's instructions for the following primer sets: S1 and Iffs to determine FCoV type I and S1 and Icfs to determine FCoV type II. Nested PCR was performed on 2 μl of the first PCR product using nested primers. The expected size of the second PCR achieved for type I and type II FCoV was 360 and 218 bp. RT-nPCR products were electrophoresed and then the target DNA fragments were purified (Geneaid Biotech, Ltd, Taipei) and sequenced (Mission Biotech, Taipei, Taiwan) - from both orientations.

Gene amplification, sequencing and analysis 3a and 3c  from FCoV type II

For amplification 3a of the FCoV type II gene from FIP cats, a set of specific primers was designed that is able to amplify from WITH type II gene to gen 3a. Complementary DNA, amplified with a primer set, targeted the 3 'end WITH FCoV type II gene (Icfs) and 5 ′ end 3a FCoVe gene (3aR2: 5′-caccaaaacctatacacacaag-3 ′). The temperature cycle was as follows: 5 minutes preheating at 94 ° C; 35 cycles of denaturation at 94 ° C for 20 s, annealing at 50 ° C for 20 s and extension at 72 ° C for 30 s; and final extension at 72 ° C for 5 minutes. This was followed by a second series of amplification using primers nIcfs and 3aR2; the expected product size was about 600 bp. Amplicons were electrophoresed, purified, and sequenced from both orientations to confirm nucleotide sequences.

For amplification 3c of the FCoV type II gene from FIP cats, a set of specific primers was designed that is able to amplify from WITH type II gene to gen 3c. Complementary DNA was amplified with forward primer (Icfs) and reverse primer (E68R: 5′-aatatcaatataattatctgctgga-3 ′ and N21R: 5′-gttcatctccccagttgacg-3 ′, respectively). The temperature cycle was as follows: 5 minutes preheating at 94 ° C; 40 cycles of denaturation at 94 ° C for 30 s, annealing at 46 ° C for 30 s and extension at 72 ° C for 90 s; and final extension at 72 ° C for 7 minutes. Following a second series of amplification using primers nIcfs and E68R, the products were electrophoresed, purified and sequenced from both orientations to confirm nucleotide sequences.

Phylogenetic analysis and recombinant analysis of FCoV type II

Several sequence alignments were performed using ClustalW 2.0 with manual editing in EditSeq (DNASTAR, Madison, USA). Phylogenetic analyzes were performed using MegAlign, version 7.2.1 (DNASTAR, Madison, USA). Bootscan and similar graphs were compiled using SimPlot 3.5.1 software (SCRoftware, Baltimore, USA).

The results

Confirmation of the FIP outbreak in the cat shelter

The shelter has been operating for three and a half years. Prior to August 2011, there were no records of FIP. The kittens (cats 1, 3, 4, 8 and 10) were moved to this shelter between June and July 2011. After arrival, these kittens played together and lived together with adult cats that lived here before. Prior to the outbreak, the kittens were individually taken to a veterinarian for vaccination and adoption visits. Fever was first detected in four kittens (cats 1, 3, 4, 5) within a few days (from 15 to 18 August) (Table 1). Clinical symptoms, e.g. fever, anorexia, neurological symptoms, shortness of breath and enlargement of the abdomen were observed over the next two months and the kittens gradually died between 1 September and 22 October (Table 1). Shelters from the shelter asked for our help on September 27. All cats housed in the shelter for a long time were immediately examined for FCoV using the RT-nPCR method. All FCoV-positive cats were isolated and kept separately. Nevertheless, starting in September, adult cats with FIP (cats 7-13) showed clinical signs similar to kittens, and all of these cats later died.

Six kittens (cats 1-6) with body effusions or neurological symptoms that succumbed in the first two months were not confirmed for necropsy (Table 1). Cat 1 was once brought to our teaching hospital and ascites (free fluid in the abdominal cavity) was taken from her. In cats 7-13, typical symptoms were found, namely ascites or pleural effusions in the body cavity (effusive FIP) and granulomatous lesions in some organs, especially in the kidneys, nuclei, lungs, omentum (forecourt) and eyes (non-effusive FIP). In cats 9, 11 and 12, necropsy showed a mixed form of the disease (Table 2) 1).

A total of 13 of the 46 cats (28.3%) died between September 2011 and April 2012 at FIP. At this time, 33 cats (71.7%) appeared to be clinically healthy and 26 of these asymptomatic cats (78.7%) were positive at least once for FCoV - detected from faeces using the RT-nPCR method. The other seven of these asymptomatic cats were negative for FCoV (Table 2) 2).

FIPV type II was found in all cats that succumbed to FIP

In order to further investigate the relationship between these seven histopathologically confirmed FIP cats, the amplified DNA was typed, sequenced and analyzed. FIPV type II was detected in all eight animals that succumbed to FIP, from swabs, faeces, urine, body effusions, cerebrospinal fluid, and tissue homogenates (Table 3). Type II viruses that cause FIP have been found not only in diseased tissue but also in faeces samples (cats 7, 11, 12 and 13), nasal / oral / conjunctival swab samples (cats 7, 8, 9, 11 and 12). ) and in urine collected by cystocentesis (cat 11) (Table 3). Although no necropsy was performed, ascites from cat 1 - the first cat to die in the shelter at FIP - were available for analysis. This cat was confirmed to be infected with type II virus. In healthy animals, only type I or FCoV was detected from faeces samples without type determination (Table 2) 2). Cats 8, 9 and 13 were infected with both types of FCoV (Table 2) 3). Although it has been found that in this environment with many cats there is more than one type of FCoV, ie. type I, II or non-typed viruses, FCoV type II infection was found in all eight FIP cats, whereas this was not the case in healthy animals (Tables 2 and33).

FIPV type II of the same origin was found in cats that succumbed to FIP

To further investigate the relationship of these disease-causing type II viruses, which were isolated from cats that succumbed to FIP, sets of specific primers capable of specifically amplifying from the 3 'end were used to analyze viral sequences. WITH the type II gene has a subsequent gene. The identity of the 620 bp amplicons derived from the seven FIPV type II was approximately 98.7% to 99.8%. Phylogenetic analysis found that the type II FCoVs derived from the outbreak described above were all grouped into a separate cluster, which differs from the other four type II FCoVs currently available at GenBank, i. FIPV 79-1146 (GenBank: {“Type”: ”entrez-nucleotide”, “attrs”: {“text”: ”DQ010921 ″,” term_id ”:” 63098796 ″}} DQ010921), FCoV 79-1683 (GenBank: {“Type”: ”entrez-nucleotide”, “attrs”: {“text”: ”JN634064 ″,” term_id ”:” 384038902 ″}} JN634064), FCoV DF-2 (GenBank: {“Type”: ”entrez-nucleotide”, “attrs”: {“text”: ”DQ286389 ″,” term_id ”:” 87242672 ″}} DQ286389) and FCoV NTU156 (GenBank: {“Type”: ”entrez-nucleotide”, “attrs”: {“text”: ”GQ152141 ″,” term_id ”:” 240015188 ″}} GQ152141) (data not shown).

Recombination at the 3 ′ end WITH of the putative recombination site at nucleotide 4250 was determined in all FCoV type II animals obtained from body effusions and tissue homogenates in cats 1, 7, 9, 10, 11, 12 and 13 (Additional set 1) (Table 3). Sequences above this site show greater similarity to CCoV, whereas sequences beyond this site were more similar to type I FCoV (Fig. 1). 1). Indeed, these findings suggest that FCoV type II, found in all FIP cats, has a common origin.

Identical nonsensical mutation on 3c The gene was found in two cats that succumbed to FIP

In order to further analyze the relationship of these FIPVs, they were 3c genes, a proposed virulence-associated FIP, are amplified from the disease-causing FCoV type II. Mutated 3c genes with identical premature stop codon at nucleotides 628-630 (amino acids 210, G210 *) were found in two FIP cats, cat 9 (ascites, spleen and brain) and 12 (ascites and rectal swabs from the day the cat died and four days previously) (Fig. 2A). It is worth noting that FIPV, obtained from cat 12, showed the same nonsense mutation as the virus in its ascites. Intact 3c the genes were discovered in cats 1, 7 and 10, which had previously succumbed to FIP. Two other clear / different nonsense mutations were found in cats 11 (E47 *) and 13 (Q218 *) (Fig. 1). 2AB, Table 3).

FIPV type II excretion can be detected in the terminal phase in FIP cats

The occurrence of FCoV was continuously analyzed to elucidate a possible route of FIPV secretion and transmission. Disease-associated FCoV type II was found to be excreted by the nasal / oral / conjunctival route and faeces (Table 4). Faecal and nasal / oral / conjunctival type II shedding can be detected from day 6 (cat 11) and from day 4 (cat 12) before death. Viremia can be detected during the terminal stage in cats suffering from FIP up to 18 days before death, and concomitant faecal excretion was detected in one cat (cat 12) (Table 4).

Discussion

The possibility of horizontal transmission is generally questioned in FIP because (i) the occurrence of FIP is sporadic and it is common for only one of them to develop FIP in an environment with a large number of cats [2]; (ii) internal mutation theory, which describes that FIPV is a mutant generated from enteric FCoV in one cat [12,17]; (iii) there is insufficient evidence that the mutant FIPV is eliminated from FIP cats; and (iv) mutations 3c gene is unique for every FIP cat [man]11,13,18]. The current belief is that cats that have succumbed to FIP do not excrete and pass FIPV to other cats [11,13,14,18–20]. Our data indicate that this outbreak of FIP was caused by viruses of the same origin. First, all cats that died of FIP had a type II infection, and recombination of these seven type II viruses was located at the same site. Recombination of type II viruses currently available in the genetic bank, i.e. FIPV 79-1146, FCoV 79-1683 and FCoV NTU156, were all unique, specific and occurred independently [9,10]. Second, FIPV, found in three kittens that died within the first two months after the onset of fever, had an intact 3c gene, whereas viruses from cats that survived longer (died four to eight months later) all contained a nonsensical mutation, i. G210 * (cats 9 and 12), E47 * (cat 11) and Q218 * (cat 13). Because the three nonsense mutations found in FIPV in these animals were all located at different sites, the viruses that originally infected these cats should be intact. 3c gene - similar to the virus found in kittens that died earlier. Following infection, local mutations occurred during virus replication in individual cats, resulting in FIPV with 3c a gene that carries meaningless mutations in different places. The finding that viruses, which were identified not only in tissues but also in faecal samples in two cats (cats 9 and 12), had an identical mutation in 3c gene, further confirmed that there was a horizontal transfer (Table 2) 3). Taken together, all of these findings demonstrated that highly virulent FIPV spread horizontally from one animal to another.

This is the first report of an FIPV type II outbreak with evidence of horizontal disease-causing FCoV transmission. The FIP broke out after five kittens (cats 1, 3, 4, 8 and 10) entered this shelter between June and July 2011. Because causative type II viruses with a specific genetic marker in the S gene have been confirmed as feline and canine coronavirus recombination, and some of the kittens that died earlier were found to have lived together or next to dogs between rescue and transport to the shelter. of these kittens may have been the source of this type II virus. Dogs and especially young dogs often shed large amounts of canine coronavirus in their faeces in shelters, and recombination between feline-canine and canine-feline coronavirus is already well documented [man]21–23]. In addition, type II causative viruses have been detected in a number of excreta and secretions in cats that have died of FIP (Table 3), demonstrating that it is possible to spread between cats.

Although immediately after the first examination of all animals from this FCoV shelter, FCoV-secreting cats were housed in separate cages and transmission subsequently ceased, mortality at the onset of the disease was high (28%, 13/46). The results of three studies that looked at the outbreak of FIP have been reported earlier. The results of a four-year study conducted at a nearby cat kennel showed an average mortality of 17.3% [24]; the mortality rate from a ten-year study conducted at a nearby kennel was 29.4% (5/17) [25]. Another epidemic study conducted in seven kennels / shelters revealed >10% mortality [20]. The high incidence of FIP in these closed breeding stations could be influenced by genetically predisposed breeding animals. In our study, only a few FIP cats in this shelter were siblings and the other cats were not genetically related. Our study shows that even without the influence of genetic predisposing factors, FIP mortality can be high in a confined environment with a large number of cats if the spread of FCoV, which causes the disease, remains undetected.

In this environment with a large number of cats, three FIP cats were infected not only with FCoV type II, but also co-infected with FCoV type I (Table 3). Type I FCoV was found only in faecal samples, while type II FCoV was found in various samples, including body effusions, granulomatous tissue homogenates, and cerebrospinal fluid. This finding indicates that FCoV type II was a major cause of FIP in these doubly infected animals. This finding is consistent with our previous finding that FCoV type II infection is significantly associated with FIP [4].

The presence of FCoV in whole blood in the terminal phase has been identified previously [26,27]; however, to our knowledge, the presence of FIPV in faeces prior to the final stage of the disease was not published anywhere until our study. The excretion of this type II virus in faeces and by the nasal / oral / conjunctival route can be detected in the effusive form of FIP up to six days before the death of the animal. Another experimental study of the infection showed that inoculated viruses could not be detected until about two weeks after inoculation, before clinical signs of the disease developed [14]. In summary, FIPV transmission could occur at the beginning, before the manifestations of the disease and in the terminal phase. When the disease broke out in our case, all the cats were initially placed together in an open room. After seven cats gradually succumbed to the disease, all FCoV-positive cats were housed separately in cages and kept separately. Isolation probably inhibited disease transmission. This outbreak of disease, which killed 13 cats, allowed us to make it clear that FIPV can be transmitted horizontally and to show that the isolation of sick cats should be taken into account in an environment where more cats are present.

Competitive interests

The authors claim that they have no competitive interests.

Contributions and contributions of authors

YTW performed sampling and preparation, FCoV detection, type determination, amplification 3c gene and other analyzes and compiled a manuscript. The BLS supervised the sampling and treatment of all FIP animals and contributed to the compilation of the manuscript. LEH participated in the amplification 3c gene, genetic analysis and manuscript preparation. The LLC devised the study, participated in the design of the study, coordinated and participated in the preparation of the manuscript. All authors read and approved the final version of the manuscript.

Additional material

Additional file 1:

Thanks

The authors would like to thank the caregivers in the mentioned cat shelter, without whose help this study would not have been possible.

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