Category: Scientific

Tomomi Takano, Kumi Satoh, and Tomoyoshi Doki
Original article: Possible Antiviral Activity of 5-Aminolevulinic Acid in Feline Infectious Peritonitis Virus (Feline Coronavirus) Infection; 10.2.2021; 18.4.2021

Abstract

Infectious feline peritonitis (FIP) is a life-threatening infectious disease of cats caused by virulent feline coronavirus (FIP: FIPV). Several effective antivirals have recently emerged for the treatment of FIP, but many are not available for practical use. 5-Aminolevulinic acid (5-ALA) is a low molecular weight amino acid synthesized in plant and animal cells. 5-ALA can be synthesized in large quantities and is widely used in the medical and agricultural industries. We hypothesized that 5-ALA inhibits FIPV infection. Therefore, we evaluated its antiviral activity against FIPV in whole felis catus fetal 4 cells and in primary feline macrophages. FIPV infection was significantly inhibited by 250 μM 5-ALA. Our study suggested that 5-ALA is useful for the treatment and prevention of FIPV infection.

Keywords: FIP, coronavirus, 5-aminolevulinic acid, antiviral drug, cat

Introduction

Feline Infectious Peritonitis (FIP) is a life-threatening infectious disease caused by feline coronavirus (FCoV) in domestic and wild Felidae species. FCoV is highly prevalent in cats throughout the world. FCoV is an enveloped single-stranded positive RNA virus. This virus belongs to the genus Alphacoronavirus of the subfamily Orthocoronavirinae of the family Coronaviridae (1). FCoV is divided into two serotypes according to the amino acid sequence of the spike protein (S), FCoV serotype I and FCoV serotype II (2). Serological and genetic studies have revealed that type I FCoV is dominant worldwide (3-5). FCoV is spread mainly by faecal-oral transmission (6). Most FCoV-infected cats are subclinical. However, several mutations have occurred in the VS protein that have led to the development of a virulent type called feline infectious peritonitis virus (FIPV) (7, 8). Serum in the peritoneal and pleural cavities and pyogranulomatous lesions in several organs are characteristic pathological findings of FIP in cats (9).

FIP is an immune-mediated and difficult-to-treat viral infection. Several effective antivirals for the treatment of FIP have recently been published (10, 11), but many are not available for practical use. Some anti-FCoV drugs, such as itraconazole, are available in animal kinetics, but their therapeutic effects are limited (12). Because FIP is a chronic and systemic disease, clinical remission is difficult to achieve. Therefore, it is desirable that therapeutic drugs for FIP have the following properties: (1) Few side effects for cats, (2) low cost, and (3) low pathogen mutagenesis.

5-Aminolevulinic acid (5-ALA) is a low molecular weight amino acid synthesized in plant and animal cells (13, 14). It is an intermediate in tetrapyrrole biosynthesis. Because 5-ALA is highly soluble in water and has low cytotoxicity, it is widely used in the medical and agricultural industries (15). Several studies have been published on the effects of 5-ALA on infectious diseases. Suzuki et al. stated that when 5-ALA and iron ion were administered orally to mice infected with a malaria parasite (Plasmodium yoelii), the mice survived (16). On the other hand, its effects on viral infection are unclear.

In veterinary medicine, photodynamic therapy (PDT) using 5-ALA has been studied in the treatment of tumors in dogs (17), but to our knowledge the effect of 5-ALA on infectious animal diseases has not been studied. We investigated whether 5-ALA could be administered as an anti-FCoV drug in vitro.

Materials and methods

Cell cultures, animals and viruses

Feline fetal 4-cell Felis catus (fcwf)-4 (kindly provided by Dr. MC Horzinke from Utrecht University) were grown in Eagles’ MEM containing 50% of Leibovitz’s L-15 medium, 5% of fetal calf serum (FCS), 100 U/ml penicillin and 100 μg/ml streptomycin. The maintenance medium had the same composition as the growth medium except for the FCS concentration (2%). Feline primary macrophages were selected for primary macrophages. Feline alveolar macrophages were obtained from four specific pathogen-free (SPF) cats aged 3–5 years by bronchoalveolar lavage with Hank’s balanced salt solution. Feline primary macrophages were maintained in D-MEM supplemented with 10% FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin. SPF cats were maintained in an isolated, temperature-controlled facility. The animal experiment was approved by the President of Kitasato University based on the decision of the Institutional Animal Care and Use Committee of Kitasato University (18-050) and performed in accordance with the Guidelines for Animal Experiments of Kitasato University. Sample sizes were determined based on our previous study and a minimum number of cats were used. The FCoV KU-2 type I strain (FIPV-I KU-2) was isolated in our laboratory. FCoV-II 79-1146 was kindly provided by Dr. MC Horzinek from Utrecht University. These viruses were cultured in fcwf-4 cells at 37°C.

Compounds

5-ALA and sodium ferrous citrate (SFC) were provided by Neopharma Japan (Tokyo, Japan). 5-ALA and SFC were dissolved in culture medium at 200 mM and 50 mM. The SFC solution was used as the 5-ALA solvent. On the day of the experiments, these compounds were diluted to the desired concentrations in the culture medium.

Cytotoxic effects of compounds

Fcwf-4 cells were seeded in 96-well plates. Compounds were loaded into wells in triplicate. After incubation for 96 hours, the culture supernatants were removed, WST-8 solution (Kishida Chemical, Osaka, Japan) was added, and the cells were returned to the incubator for 1 hour. The absorbance of the formazan produced was measured at 450 nm using a 96-well spectrophotometric plate reader, according to the manufacturer's recommendations. Percent cell viability was calculated using the following formula: Cell viability (%) = [(OD of untreated compounds - cells treated with compound) / (OD of cells not treated with compound)] x100. 50% cytotoxicity concentration (CC₅₀ ) was defined as the cytotoxic concentration of each compound that reduced the absorbance of treated cells to 50% compared to the absorption of untreated cells.

Antiviral effects of 5-ALA

Confluent monolayers of fcwf-4 cells were cultured in or without compound media at the indicated concentrations in 24-well multi-plate plates at 37 ° C for 24 or 48 hours. The cells were washed and the virus (MOI 0.01) was adsorbed into the cells at 37 ° C for 1 hour. After washing, cells were cultured in 1.5% carboxymethylcellulose (CMC) -MEM or MEM with or without compounds. For cells cultured in CMC-MEM, cell monolayers were incubated at 37 ° C for 48 hours, fixed and stained with 1% crystal violet solution containing 10% buffered formalin, and then the resulting plaques were counted. The percentage of decrease or increase in plaques was calculated using the following formula: Percent reduction of plaques (%) = [(number of plaques of compounds treated with compound) / (number of plaques of cells not treated with compound)] x100. EC₅₀ was defined as the effective concentration of compounds that reduced the virus titer in the culture supernatant of infected cells to 50% compared to the virus control titer. For cells cultured in MEM, culture supernatants were collected 48 hours after infection and virus titers were measured by TCID assay.₅₀.

Primary feline macrophages were cultured in medium with or without compounds at the indicated concentrations in 24-well multiple plates at 37 ° C for 48 hours. After washing with PBS, FIPV 79-1146 (1 x 10⁴ TCID₅₀) adsorbed into cells at 37 ° C with 5% CO₂ for 1 hour. After washing with PBS, the cells were cultured in medium and the supernatants were collected. Virus titers were measured using the TCID assay₅₀.

Statistical analysis

Data from only two groups were analyzed using Student's t-test (Welch t-test) and data from multiple groups were analyzed by one-way ANOVA followed by Tukey's test. A P value <0.05 was considered significant.

The results

Cytotoxic and antiviral effects of 5-ALA

A cytotoxicity assay was performed to determine the non-toxic concentration of 5-ALA against fcwf-4 cells (Figure 1). More than 75% fcwf-4 cells survived in the presence of 1,000 μM 5-ALA (maximum concentration in this experiment). The vehicle does not show any cytotoxic effects on fcwf-4 cells.

Figure 1.

Cytotoxic effects of 5-ALA on fcwf-4 cells. The viability of fcwf-4 cells was measured using the WST-8 assay. Black ring: 5-ALA. White circle: Vehicle (SFC). The vehicle (control solvent) was the same as the vehicle in the 5-ALA solution at each serial dilution. Results are presented as mean ± SE. The data represent three independent experiments (n = 3).

Effects of 5-ALA on FIPV infection in a feline cell line

The antiviral effects of 5-ALA against FIPV were evaluated in a plaque inhibition assay in fcwf-4 cells. Cells were treated with 5-ALA as follows: 24-hour pre-treatment (pre-24h), 24-hour pre-treatment followed by 49-hour FIPV treatment (pre-24h and co-49h) and 48-hour pre-treatment, after which followed by a 49-hour FIPV treatment (pre-48h and co-49h). At pre-24h, the percent plaque inhibition increased significantly at 500 μM or higher (Figures 2A, B). At pre-24h and co-49h, the percentage of plaque inhibition increased significantly at 125μM or higher (Figures 2C, D). At pre-48h and co-49h, the percentage of plaque inhibition in FIPV type I reached 125 μM 5-ALA 75% (Figures 2E, F). The control vehicle, SFC, showed no plaque inhibitory effects on FCoV under any conditions. According to the titration assay, FIPV type I and type II production was significantly reduced at 250 and 500 μM 5-ALA (Figure 3).

Effects of 5-ALA on FIPV infection in a feline cell line

FIPV-infected macrophages are involved in the progression of FIP symptoms to severe. We investigated whether 5-ALA inhibits FIPV proliferation in macrophages. In this experiment, FIPV 79-1146 type II with a high ability to reproduce in primary cat macrophages was used. Virus production in FIPV-infected macrophages was reduced at 250 μM 5-ALA in three of the four cats (Figure 4).

Discussion

5-ALA is an intermediate in the synthesis of tetrapyrrole in animals, plants and microorganisms (13-15). The potential for 5-ALA efficacy in plants was reported in the 1980s (18), but it was difficult to produce 5-ALA in sufficient quantities for practical use because only a small amount is produced in microorganisms. After Nishikawa et al. introduced the method of mass production of 5-ALA using bacteria (19), the effectiveness of 5-ALA has been confirmed not only in agriculture but also in the medical and biological fields. 5-ALA is an inexpensive drug and is practically used as a supplement to improve animal performance and immune response in veterinary medicine (14, 20).

5-ALA inhibited FIPV growth in fcwf-4 cells. The metal complexes of the 5-ALA metabolite, protoporphyrin IX (PpIX), were found to have antiviral activity (21-23). The PpIX metal complex, heme, inhibits the multiplication of dengue virus (21). On the other hand, the multiplication of Zika virus is not inhibited by heme (23). It is not clear whether heme inhibits FCoV proliferation. Generally, an increase in intracellular heme stimulates the production of hemeoxygenase-1 (HO-1), a heme degrading enzyme. HO-1 has been reported to induce antiviral activity (24, 25). However, in a preliminary experiment, we confirmed that the expression level of HO-1 mRNA in cells treated with 250μM 5-ALA remained unchanged (data not shown). Based on this finding, 5-ALA-induced inhibition of FIPV infection occurs due to a factor other than heme and HO-1.

There are already many studies on therapeutic drugs in FIP. Many drugs effective in FIP have been identified in vitro, and some have been shown to have therapeutic effects when administered to cats with FIP (10-12). However, the effects of all drugs were weak in cases with neurological manifestations. Poor transmission of these drugs to the central nervous system was thought to be the cause; therefore, there is a need for a drug that has antiviral effects against FIPV capable of entering brain tissue. 5-ALA is an amino acid with low molecular weight and possible good penetration ability into brain tissue (26). It is further reported that the diffusion of 5-ALA from the blood into normal brain tissue is very low (27), suggesting that it has fewer adverse effects. FIP can only be unambiguously diagnosed by detecting the FCoV antigen in the lesion (28). However, when treatment is not started until a clear diagnosis has been made, the symptoms have meanwhile progressed to a state where, in many cases, there is no longer a response to treatment. Therefore, if there is a drug that can be given prophylactically before the diagnosis of FIP, it is possible to prevent the progression of symptoms, for which 5-ALA may be an ideal means. However, in our experiment with FIPV target cells, macrophages, the antiviral effects of 5-ALA were not observed in some cats. Therefore, when 5-ALA is used as a therapeutic drug for FIP, anti-FCoV drugs such as GS-441524 (29), GC-376 (30), U18666A (31) and itraconazole (32) or anti-inflammatory drugs such as anti-TNF-alpha antibody (33) should be used concomitantly.

FECV transmission mainly occurs in cats in the field, while horizontal FIPV infection between cats is considered rare (34). FIPV has been found to be generated by a genetic mutation of FECV. Thus, if there are means to prevent FECV infection on a daily basis, the development of FIP can be prevented. No vaccine has been developed to prevent FECV infection. Addie et al. reported that viral gene secretion in faeces ceased in FECV-infected cats treated with a synthetic adenosine analog (35). Therefore, elimination of FECV infecting the gut is expected by administering FECV antivirals to infected cats. However, synthetic adenosine analogs can induce coronavirus gene mutations (36). In addition, although the level of the FECV gene in feces has fallen below the limit of detection in cats treated with a synthetic adenosine analog, it is possible that FECV has latently infected the gut or other tissues. Long-term administration of a synthetic adenosine analog is required to prevent FECV gene mutation and reliably eliminate FECV infection, but this is not realistic due to its high cost. On the other hand, 5-ALA is practically used as a dietary supplement. 5-ALA has low toxicity to animals and plants, which strongly suggests the possibility of long-term administration of 5-ALA to cats. In the future, it is necessary to examine whether 5-ALA is useful as an adjunct to prevent the development of FIP in cats infected with FECV.

In this study, we confirmed the possibility that 5-ALA inhibits FIPV proliferation and TNF-alpha production. Because 5-ALA is an amino acid present in the body, immediate administration is possible. However, it is necessary to administer 5-ALA to cats with FIP and to monitor whether therapeutic effects can be achieved. It is further investigated whether long-term administration of 5-ALA eliminates the virus and inhibits FIP development in FECV-infected cats.

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Luca Ferasin, DVM, Ph.D, Matthieu Fritz, Ph.D, Heidi Ferasin, DVM, Pierre Becquart, Ph.D, Vincent Legros, DVM, Ph.D and Eric M. Leroy
Original article: Myocarditis in naturally infected pets with the British variant of COVID-19; 3/18/2021; Translation 1.4.2021

Abstract

Pets may become infected with SARS-CoV-2, but based on the limited information available so far, it is not known whether the new UK variant B.1.1.7 can more easily infect certain animal species or increase the possibility of human-to-animal transmission. In this study, we report the first cases of infection of domestic cats and dogs by British variant B.1.1.7 SARS-CoV-2 diagnosed at a specialized veterinary hospital in the south-east of England. We further found that many owners and caregivers of these pets developed respiratory symptoms in Covid-19 3-6 weeks before their pets became ill, and also had a positive PCR test for Covid-19. Interestingly, all of these B.1.1.7 infected pets developed atypical clinical manifestations, including severe myocarditis-related cardiac abnormalities and severe deterioration of the patient's overall health, but without any primary respiratory symptoms. Our findings demonstrate for the first time the ability of pet animals to be infected with variant B.1.1.7 SARS-CoV-2 and raise questions about its pathogenicity in these animals. In addition, given the increased infectivity and transmissibility of variant B.1.1.7 for humans, these findings also highlight, more than ever, the risk that pets may potentially play a more important role in the dynamics of SARS-CoV-2 outbreaks than previously thought.

Introduction

The COVID-19 pandemic due to severe acute respiratory syndrome caused by type 2 coronavirus (SARS-CoV-2), characterized by a change in the amino acid spike protein (S) D614G (referred to as variant B. 1), has affected several million cases worldwide. This global situation has favored the occurrence of many genomic mutations, some of which have created variants with selective advantages. ¹. At the end of autumn 2020, three notable variants emerged in several countries, which then spread rapidly around the world, including B.1.1.7 (also known as 20I / N501Y.V1), which was first identified in England. ², B.1.351 (20J / N501Y.V2) first detected in South Africa and recently identified "Brazilian" variants of P.1 (20I / N501Y.V3). These three variants carry a constellation of genetic mutations, including those at the level of the protein S receptor binding domain (RDB), which is necessary for binding to the ACE-2 receptor of the host cell to facilitate virus entry.

It is estimated that variant B.1.1.7, also referred to as the dreaded variant (VOC) 202012/01 or 20I / 501Y.V1, appeared in September 2020 in Kent, a county in the south-east of England, and quickly surpassed other existing ones in England. variants due to increased portability and infectivity ². Multiple evidence suggests that its increased portability is driven by the N501Y mutation and the Δ69 / 70 amino acid deletion in RDB ³. Subsequently, the incidence of B.1.1.7 increased during the national lockdown introduced by the UK Government from 5 November to 2 December 2020, despite severe restrictions, which led to an extraordinary increase in COVID-19 cases, which particularly affected the Greater London area. . As of February 7, 2021, VOC 202012/01 represented approximately 95% new SARS-CoV-2 infections in the United Kingdom and has currently been identified in at least 86 countries.

There have been several cases of SARS-CoV-2 infection in pets (especially cats and dogs) worldwide, and these animals are thought to have been infected by their owners or caregivers. Pet infections have mostly led to unnoticeable to mild digestive and respiratory symptoms such as cough, rhinitis, sneezing and conjunctivitis. ^4-6. Interestingly, despite the uncontrolled increase in COVID-19 cases in the UK since November 2020, no SARS-CoV-2 infections have been reported in pets. More surprisingly, to the best of the authors' knowledge, no natural infection of any B.1.1.7 animal has ever been documented, either in England or anywhere else.

The results

We report a sudden increase in the number of domestic dogs and cats with myocarditis at The Ralph Veterinary Referral Center (RVRC) on the outskirts of London (UK) between December 2020 and February 2021, with an unexpected increase in incidence from 1.4% to 12.8% (8, 5% in cats and 4.3% in dogs). This sudden increase in cases appears to have followed the curve and timeline of the COVID-19 human pandemic in the UK as a result of option B.1.1.7, which began in mid-December 2020 and culminated in late January 2021 before returning to historical levels in mid-February. 2021

None of these myocarditis patients had a history of heart disease and their clinical picture was similar and characterized by acute onset of lethargy, anorexia, tachypnoea / dyspnoea (secondary to congestive heart failure) and in some cases syncopal events. Diagnostic examinations revealed the presence of elevated cardiac troponin-I (median 6.8; range 0.68 to 61.1 ng / ml [normal reference range 0.0-0.2 ng / ml]) accompanied by echocardiographic evidence of myocardial remodeling and / or signs of pleural effusion and / or pulmonary edema, often confirmed on chest radiographs, and / or severe ventricular arrhythmias on electrocardiography (see Additional Figure S1). All affected animals achieved remarkable improvement after cage rest, oxygen therapy, acute diuresis, and in some cases antiarrhythmic treatment with sotalol and fish oil before being discharged for oral treatment after several days of intensive care. Notably, most owners and caregivers of these pets with myocarditis developed respiratory symptoms in Covid-19 within 3-6 weeks before their pets became ill, and many of these owners had a positive PCR test for Covid-19. Due to this coincidence and the interesting simultaneous development of myocarditis in these pets and the outbreak of B1.1.7 COVID-19 in the UK, we decided to investigate SARS-CoV-2 infection in these animals. For this purpose, serum samples as well as oro / nasopharyngeal and rectal swabs were taken from seven animals (six cats and one dog) at the initial presentation at the RVRC between 22 January and 10 February 2021 (Table 1). During the same period, we took blood samples from four other pets (two cats and two dogs) during their recovery, 2-6 weeks after they developed symptoms of myocarditis. None of the 11 myocarditis animals developed any influenza-like symptoms and all improved clinically over several days of intensive care, although one cat (LL) had a relapse of her clinical symptoms characterized by severe lethargy and uncontrolled ventricular tachycardia one week after discharge. its owners to opt for euthanasia. All cats and dogs were neutered and aged from one to 12 years. After collection, all samples were stored at -20 ° C until transport on ice to the MIVEGEC laboratory in Montpellier, France for serological and virological examinations.

Oro / nasopharyngeal and rectal swabs were tested by droplet digital RT-PCR (ddPCR) targeting one region specific for the SARS-CoV-2 N gene and two Spike protein regions specific for the three current predominant SARS-CoV-2 variants, namely 20I / N501Y.V1, 20J / N501Y.V2 and 20I / N501Y.V3. One target region containing the N501Y mutation is common to three variants and the other target region containing the Δ69-70 deletion is specific to variant B.1.1.7. Sera were tested for SARS-CoV-2 specific IgG using three microsphere immunoassays (MIA) that detected IgG binding to N protein, S1-RBD protein or S trimeric protein, as well as a retrovirus-based assay to detect SARS-CoV-2. 2 neutralizing antibodies (see Materials and Methods).

All oro / nasopharyngeal swabs were found to be negative for SARS-CoV-2 ddPCRs. However, positive ddPCR signals were obtained for three areas from rectal swabs from three of the seven animals (two cats and one dog), indicating infection with British variant B.1.1.7. The RNA concentration ranged from 12 to 33 copies / μL of sample, indicating a low viral load (Table 1). In addition, samples from one animal taken during the acute phase of the disease, which showed a negative ddPCR result, as well as from two of the four animals taken during the recovery period, were found to contain SARS-CoV-2 antibodies. Thus, in total, six of our 11 animals tested were shown to be SARS-CoV-2 positive, either ddPCR or serologically. More interestingly, considering only five animals whose owners or caregivers were laboratory confirmed for Covid-19, four showed SARS-CoV-2 positive (Table 1).

Discussion

As far as we know, this is the first report of an infection of cats and dogs by the British variant SARS-CoV-2 B.1.1.7. Given the increased infectivity and transmissibility of human B.1.1.7, the discovery of B.1.1.7 infected cats and dogs emphasizes more than ever the risk that pets may potentially play a significant role in the dynamics of SARS-CoV-2 outbreaks, as previously thought. Therefore, further studies are urgently needed to examine the likelihood of transmission of animal-animal as well as animal-human variant B.1.1.7 and to demonstrate whether the N501Y mutation and the Δ69-70 deletion cause the virus to be more infectious to these animals.

Another remarkable and unexpected finding of our study is the development of unusual clinical manifestations in cats and dogs infected with B.1.1.7, including severe cardiac abnormalities secondary to myocarditis and severe deterioration of the patient's overall health, but without any primary respiratory symptoms. With the exception of the only cat in Spain who experienced cardiorespiratory failure leading to severe respiratory distress ⁷, both natural and experimental SARS-CoV-2 infections have been reported in cats and dogs, either asymptomatic or with mild upper respiratory disease. Although B.1.1.7 infection in humans appears to be associated with higher COVID-19 mortality or clinical severity, the association between myocarditis and B.1.1.7 infection in pets should be recognized and addressed. ⁸ In this context, it is important to emphasize the fact that myocarditis associated with multisystem inflammatory syndrome is also a well-known complication of COVID-19 in humans (both adults and children), probably due to an exaggerated immune response of the host. ^9,10.

Our findings demonstrate the ability of pet animals to become infected with SARS-CoV-2 B.1.1.7 and raise questions about its pathogenicity in these animals. Therefore, there is an urgent need to significantly accelerate and strengthen the investigation and surveillance of animal infections with highly transmissible variants such as British B.1.1.7, South African B1.351 and Brazilian P.1 as part of the global response to the ongoing multi-species pandemic of COVID-19.

Materials and methods

RNA extraction

Rectal and oro / nasopharyngeal swabs were resuspended by vortexing in 300 μL PBS. Total RNA was extracted from 200 μL of rectal swab supernatant and 200 μL of nasopharyngeal swab virus transport medium. Extraction was performed on an IndiMag 48 extraction system (Indical Bioscience) using magnetic bead technology with the IndiMag Pathogen kit according to the manufacturer's instructions. The elution volume was 100 μL.

One-step dRT-PCR

The RT-dPCR procedure was performed according to the manufacturer's instructions using a QIAcuity 8.5 plexus (Qiagen, Germany), a QIAcuity One-Step Viral RT-PCR kit (cat. No. 1123145, Qiagen, Germany) and a 24-well 26K nanoplate ( Cat. No. 250001, Qiagen, Germany). The ddRT-PCR technique showed higher sensitivity and specificity for the diagnosis of COVID-19 compared to RT-qPCR. ¹¹

Briefly, the RT-dPCR reaction mixture was assembled as follows: 4x one-step viral RT-PCR Master Mix 10 μl, 100x multiplex reverse transcription mixture 0.4 μl, 20x primer and probe set 0149, 0130, 0150 (ref IAGE) 2 μl x3 (6 μL), RNase-free water 22.6 μl and RNA template 1 μl, in a final volume of 40 μl. Target 0130 target 2019-nCoV_N2 NC_045512v2 fluorophore HEX, amplicon length 67 bp. Target region 0149: deletion of mutations 69-70, lines B1.1 and B1.258, fluorophore HEX, amplicon length 100 bp. Target region 0150 S: mutation N501Y, line B1.1.7, fluorophore Cy5, amplicon length 133 bp. The sequences are confidential and are stored under number EP20306715.2. The mixture was prepared on a pre-plate and then transferred to a 24-well 26K nanoplate. It was then introduced into the QIAcuity 8, a fully automated system. The procedure included i) a priming and rolling step to generate and isolate the ventricular compartments, ii) an amplification step according to the following cyclic protocol: 50 ° C for 40 minutes for reverse transcription, 95 ° C for 2 minutes for enzyme activation, 95 ° C after for 5 s for denaturation and 60 ° C for 30 s for annealing / extension for 40 cycles and iii) the imaging step was performed by reading in the following channels FAM, HEX and CY5. The entire workflow time for these three steps was approximately 2 hours. Experiments were performed using a negative control (no template control, NTC) and a positive control (the patient sample was confirmed as positive by RT-PCR using our routine diagnostic testing). All reactions had at least 25,400 compartments. Data were analyzed using QIAcuity Suite Software V1.1.3 (Qiagen, Germany) and expressed as copies / μl.

Microsphere immunoassay

Serum samples from cats and dogs were tested by multiplex microscopic immunoassay (MIA). 10 μg of three recombinant SARS-CoV-2 antigens were used to capture specific serum antibodies: nucleoprotein (N), receptor binding domain (RBD) and trimeric spike (tri-S). Separate MagPlex microsphere kits (Luminex Corp) were appropriately coupled to viral antigens using an amine coupling kit (Bio-Rad Laboratories) according to the manufacturer's instructions, while the recombinant human protein-coupled microsphere kit (O6-methylguanine DNA methyltransferase) was used as a control in the assay. . The MIA procedure was performed as described in ¹². Briefly, microsphere mixtures were sequentially incubated protected from light on an orbital shaker with serum samples (1: 400), biotinylated protein A and biotinylated protein G (4 μg / ml each) (Thermo Fisher Scientific) and Streptavidin-R-phycoerythrin (4 μg / ml) (Life technologies). Measurements were performed using a Magpix instrument (Luminex). Relative fluorescence intensities (RFI) were calculated for each sample by dividing the MFI signal measured for the antigen-coated sets of microspheres by the MFI signal obtained for the set of control microspheres to take into account the non-specific binding of the antibodies to the beads. Specific seropositivity limits for each antigen were set to three standard deviations above the mean RFI value of serum samples from 29 dogs and 30 cats collected before 2019. Based on the pre-pandemic population, the MIA specificity for N protein was set at 96.6%. for dogs and cats, 96.6% for RBD for cats and 100% for dogs and 100% for tri-S for cats and 96.6% for dogs.

Measurement of neutralizing activity

An MLV-based pseudo-particle carrying a SARS-CoV2 tip pseudotyped (SARS-CoV-2pp) was used to measure neutralizing activity in cat and dog sera. Each SARS-CoV2 positive sample detected by MIA was treated by a neutralization procedure as described in ¹³. The level of infectivity was expressed as % GFP positive cells and compared to SARS-CoV-2pp infected cells incubated without serum. Pre-pandemic sera from France were used as negative controls and anti-SARS-CoV-2 RBD antibody was used as a positive control.

Declaration of conflict of interest

None of the authors in this study has any conflict of interest (financial or personal).

Financing

The study was funded by the French National Research Agency (ANR-RA-COVID-19; Geographical and Temporal Serological Study of SARS-CoV-2 Pet Infection during the Second COVID-19 Wave in France, CoVet) and the IDEXLYON project from the Université de Lyon as part of the "Investissements d'Avenir Program" (ANR-16-IDEX-0005) and the Institut de Recherche pour le Développement (IRD).

Acknowledgement

We are grateful to the owners of the pets for giving us consent to take samples from their animals. We also thank Dr. Laurent Locquet and Dr. Altin Cala for contributing to the clinical management of these patients. We also thank Franz Durandet (IAGE - Environmental Engineering and Analysis, Grabels, France), Alix de Mont-Marin (Innovative Diagnostics, France) and Afif M. Abdel Nour (Qiagen, France) for their help in molecular biology. We also thank François Loïco Cosset, Solène Denolly, Bertrand Boson of CIRI - Center for International Research in Infectiology, Team EVIR, Univ Lyon, Claude Bernard Lyon 1 University, Inserm, U111, UMR5308, ENS Lyon, for the development of seroneutralization technology. In conclusion, we thank Dr. Thierry Buronfosse for the kind gift of pre-pandemic sera.

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4.3.2021; Translation 16.4.2021
Yiya Yin, Ting Li, Chaohao Wang, Xiaoya Liu, Hehao Ouyang, Wanfeng Ji, Jiahao Liu, Xueyu Liao, Junyi Li, and Changmin Hu

Original article: A retrospective study of clinical and laboratory features and treatment on cats highly suspected of feline infectious peritonitis in Wuhan, China

Abstract

Feline infectious peritonitis (FIP) is a systemic, potentially fatal viral disease. The aim of this study was to investigate the clinical and laboratory characteristics and treatment of cats with highly suspected FIP in Wuhan, China. The clinical records of 127 cats with highly suspicious FIP were reviewed for history, clinical signs, physical findings, and diagnostic test results. Gender, castration status, breed, age and month of onset of the disease were compared with the characteristics of the clinical population. Age and castration status were significantly correlated with suspected FIP. Gender, breed and month of onset of symptoms were not related to FIP. Many more cases of suspected FIP have been reported in young cats or intact males. The effusion was observed in 85.8% cats suspected of having FIP. Elevations in serum amyloid A (SAA) and lymphopenia were common abnormalities in FIP cases. Furthermore, 91.7% cats with highly suspected FIP had an albumin / globulin (A / G) ratio <0.6, while 85.3% had an A / G ratio <0.5. Mortality for cats suspected of FIP was 67% and six cases were confirmed by FIP-specific immunohistochemistry. Of the 30 cats treated with GS-441524 and / or GC376, 29 were clinically cured. The study highlights the diversity of clinical manifestations in the diagnosis of this potentially fatal disease. The A / G ratio and SAA had a higher diagnostic value. GS-441524 and GC376 have been shown to be effective in treating cats suspected of having FIP.

Keywords: Zoology, Diagnosis of infectious diseases

Introduction

Infectious feline peritonitis (FIP) is a worldwide disease of domestic and feral cats. The pathogen of FIP is feline infectious peritonitis virus (FIPV), which was caused by a mutation in feline coronavirus (FCoV) ^1,2. Although extensive FIP research has been conducted, FIP remains one of the most widespread and deadly infectious diseases in cats.

In addition to non-specific clinical symptoms such as malaise, anorexia, fluctuating fever, and weight loss, the clinical picture of FIP is complex and variable. Different clinical signs may be the result of variations in the virus and the nature of the individual host's immune response. FIP can be divided into three types - effusive, non-effusive and mixed - depending on the different clinical features that occur, these types may change over time for each individual cat. Effusive (wet) FIP manifests itself mainly as ascites and / or pleural effusion, while non-effusive (dry) FIP manifests itself mainly by the formation of granulomas in the central nervous system, eyes and abdominal organs and does not produce effusion in the body cavity.³. If the symptoms of effusive and non-effusive form appear simultaneously, it is a mixed FIP⁴. To our knowledge, there are few studies on the clinical and laboratory properties of FIP in China.

Diagnosing FIP before death is a controversial and complicated issue. It is more difficult to diagnose FIP in vivo in non-exuding cats because the clinical signs and laboratory characteristics are relatively unclear. Immunofluorescence staining of FCoV antigen in macrophages is highly specific, but is difficult to detect due to the low number of macrophages in the effusion. Immunohistochemistry (IHC) is currently the gold standard for FIP diagnostics to identify FCoV antigens in affected tissues. However, this invasive method has low operability and requires professional detection equipment and personnel. At the same time, a negative IHC result does not rule out the diagnosis of FIP, which poses another challenge for the diagnosis of this disease.⁵. FIP has an extremely high mortality rate and until recently was considered a fatal disease. In recent years, a 3C protease inhibitor (GC376) and a nucleoside analogue (GS-441524) have been shown to have some therapeutic effect on FIP.^6-9. The purpose of this study is to determine the epidemiological properties and associated risk factors of FIP, to summarize common clinical signs and laboratory characteristics, and to evaluate the therapeutic effects of GS-441524 and GC376.

Materials and methods

Case selection

This study retrospectively evaluated medical records in a database of 12 veterinary hospitals in Wuchan, China, from January 1, 2019 to January 1, 2020. Due to the limitations of diagnostic facilities in veterinary hospitals and the reluctance of cat owners to consent to invasive diagnostic procedures such as biopsy, most cases were not confirmed by the IHC. Therefore, based on previously published literature in this area, the following inclusion criteria have been established for the diagnosis of suspected FIP cases.^5-15. Highly suspected cases of FIP were defined as those that met at least four of the following characteristics: 1) Typical clinical signs of FIP effusion (pleural effusion and / or ascites) or typical clinical signs of non-fusive FIP (ocular and neurological symptoms); 2) Decreased albumin / globulin ratio (A / G <0.6); 3) Rival's test showing positive results in effusion; 4) Positive detection of FCoV RNA in reverse transcription polymerase chain reaction (RT-PCR) effusion; 5) Tissue samples taken at necropsy that show typical histological features of FIP, namely systemic vasculitis and pyogranulomatous lesions.

Of the 20,984 cats registered in 12 veterinary hospitals, 127 cases with a high suspicion of FIP were included according to the above criteria. Through the collection of background information, history, clinical signs, haematological and biochemical analyzes, diagnostic imaging, RT-PCR, Rivalt's test, exploratory laparotomy and histopathology, cases were comprehensively diagnosed and analyzed and detailed information and test results were recorded (see Supplemental Table S1 for FIP case record sheet). All information was collected with the consent and cooperation of veterinarians and cat owners. The protocol was approved by the Animal Management and Ethics Committee of the Center for Laboratory Animals at Huazhong Agricultural University (approval number: HZAUCA-2018-005).

Statistical analysis

Statistical analysis was performed using commercial software (SPSS version 21.0 [IBM]). Descriptive statistics were used for all variables evaluated. Categorical data were analyzed using Pearson's chi-square test (χ²). Fisher's exact two-tailed results were used in 2 × 2 contingency tables with expected cell values <5. The sex, castration, breed, age, and onset of symptoms of suspected FIP were compared to a feline clinical population (20,984 cats) presented from January 2019 to January 2020 from 12 veterinary hospitals to determine if these risk factors were associated with FIP.

The history, clinical signs, and laboratory results of FIP suspicions were retrospectively analyzed to summarize common clinical signs and laboratory abnormalities of the disease. Samples were randomly selected from cats undergoing a laparotomy and sent to LABOKLIN GMBH & CO. KG. KG (Bad Kissingen, Germany) to the IHC to verify that the inclusion criteria for FIP cases are correct and justified. In addition, the therapeutic effect of GS-441524 and GC376 was evaluated.

Ethical approval

All relevant international, national and institutional guidelines for the care and use of animals have been complied with. All procedures performed in animal studies were in accordance with the ethical standards of the institution in which the studies were performed.

The results

Risk factors

No significant correlation was found between FIP and gender (p = 0.083). However, FIP significantly correlated with castration status (p <0.001) and intact males were more susceptible to the disease. Regarding the breed, whether classified as crossbred or purebred (p = 0.069) or specific to each breed (p = 0.246), no correlation was found with the FIP. There was a significant correlation between age and FIP (p <0.001) and young cats were more susceptible. The age of cases of suspected FIP ranged from 1 month to 8 years and 2 months, with a mean age of 13.1 months and a median age of 8 months. In these cases, 40.2% was less than 6 months old, 67% was less than 1 year old, and 90.6% was less than 2 years old; in addition, the prevalence in older cats was significantly lower than the prevalence in young cats (Table 1). The correlation of the month of onset of the disease was studied for the first time and the number of cats suspected of having FIP and the clinic population were counted for each month. The results showed that the highest prevalence was 1% in May. The prevalence in January (0.95%), February (0.87%), September (0.66%) and November (0.77%) was higher than the overall prevalence and the lowest prevalence was 0.35% in July. However, in the statistical analysis, there was no correlation between FIP and month (p = 0.135; Table 1).

Table 1

Pre-diagnosis stress events were documented in 60/127 cats. Because some cats had more stressors, a total of 77 stressors were recorded. Of these events, environmental change was the most common factor leading to stress (40.3%), followed by new domestic pets that coexisted with other diseases, intimidation, and dietary changes (Fig. 1). In addition, information was collected on the density of domestic cats, of which 43 (51.8%) were single-cat households and 40 (48.2%) were single-cat households.

Clinical signs

Of the 127 cats with highly suspicious FIP, 12 (9.4%) developed dry FIP and 109 (85.8%) developed effusive FIP, of which 92 exhibited ascites (84.4%), 11 had pleural effusion (10.1%). and 6 with ascites and pleural effusion (5.5%). In addition, 6 cats (4.7%) had mixed FIP. Some first developed dry FIP, followed by ascites and the development of effusive FIP. Some cats initially showed effusive FIP and developed neurological symptoms several days before death.

The clinical signs of cats with a high suspicion of FIP are listed in Table 2. The most common non-specific signs were weight loss (93.8%), malaise (86.2%) and anorexia (86%). In more than half of the cases, jaundice and 59.4% and 52.9% fever, respectively, were present. Of the 85 cats with highly suspected FIP for whom the temperature was documented on physical examination, 52.9% had a temperature above 39.5 ℃, 36.5% had a temperature above 40 ℃ and 10.6% had a temperature above 40.5 ℃ (see Additional Figure S1.). As a result, the 41.3% cats had a palpable mass on abdominal palpation, which could be either an enlargement of the mesenteric lymph nodes or an increase in the intestinal wall. Diarrhea was less common and occurred in only 17.6%, while dyspnoea occurred in 35.2% and was most common in cats with pleural effusion. In addition, ocular and neurological symptoms generally occurred only in dry or mixed forms of FIP.

Table 2

Hematology and serum biochemistry

As shown in Table 3, hematocrit (HCT) decreased in 40.2% cats, but only in 15.9% cats there was a decrease in red blood cell (RBC) counts. Half of the cats developed lymphocytopenia, 33.9% had an increase in white blood cell (WBC) count, and 34.3% had an increase in neutrophil count (NEU).

Table 3

In serum biochemistry, total protein (TP) levels were mostly normal (75.2%); several (14,7%) cats had elevated levels and very little had decreased levels. More than half of the cats with highly suspected FIP showed hypoalbuminemia (58.7%). Although albumin (ALB) levels in the remaining cats were normal, they were also close to the lower end of the reference range. Hyperglobulinemia was also common in cats with a high suspicion of FIP (57.8%) and the rest of the cats had normal globulin levels (GLOB). The A / G ratio ranged from 0.137 to 0.767, with a mean of 0.399 and a median of 0.385. Among the results, 91.7% was less than 0.6, 85.3% was less than 0.5, and 54.1% was less than 0.4 (see Additional Figure S2). An increase in total bilirubin (TBIL) was observed in almost half of the cats with highly suspected FIP (48.7%) and alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were mostly normal. Creatinine (CRE) decreased in 47.9% cats, while urea decreased in 60.7% cats and rarely increased. Amylase (AMYL) and lipase (LIPA) were mostly negative and positive in only a small number of cats. In addition, feline serum amyloid A (SAA) increased in all cats (Table 3).

Imaging and molecular biology

Of the 127 cats, 90 were examined by ultrasound, which showed varying degrees of ascites, and several cats showed loss of corticomedullar resolution in the kidneys. Radiography was performed on 21 cats that showed pleural effusion and / or ascites (Fig. 2). Of the 109 cats with exudate, 98 exuded the exudate. We then detected FCoV RNA in 98 effusions by RT-PCR, with 83 cats (84.7%) showing FCoV positivity. The FCoV mutation was detected in 34 FCoV-positive effusions with 12 successfully sequenced and obtained partial sequences of the S gene, all of which had M1058L or S1060A mutations. In addition, Rivalta's tests were performed in 85 cases with a positivity rate of 100%; FCoV antibody tests were performed in 12 cases and all were positive. Microscopic examination of swabs from 25 cat effusions showed many pink protein granules and an increase in inflammatory cells.

Histopathology and immunohistochemistry

13 cats underwent an exploratory laparotomy and histopathological diagnosis was performed on 9 cats (for details on 13 cats, see Additional Table S2). After exploratory laparotomy, the samples were subjected to histopathological examination and we found typical pathological changes of FIP, namely systemic vasculitis and pyogranulomatous lesions (Fig. 2). Tissue samples from six cats were randomly sent to LABOKLIN GMBH & CO. KG. KG for IHC. This testing revealed that tissue macrophages showed positive staining for FCoV antigen, confirming the diagnosis of FIP (see Additional Figure S3-S8).

Treatment and results

The results of 88 cats were recorded. Of these, 59 eventually died with a mortality rate of 67%. Thirty-two cats were directly euthanized. Twenty-six cats underwent no treatment or were treated only for symptoms and eventually died naturally. Only one dry FIP was treated with GS-441524 for 4 weeks. After the disease subsided, the owner decided to stop the treatment and the cat eventually relapsed and was killed. In addition, 29 cats (25 wet, 3 dry and 1 mixed) were treated with GS-441524 (2-4 mg / kg, once daily for at least 4 weeks) or GC-376 (6-8 mg / kg) once daily after for at least 4 weeks) with a success rate of 33%. The most visible signs were rapid loss of ascites and / or pleural effusion (within 1 week), recovery of psyche and appetite, and weight gain. The cat with a wet FIP, which was diagnosed on January 1, 2019 and survived for more than a year, survived the longest.

Discussion

In the past, FIP was considered a deadly disease by scientists. In addition, it is difficult to apply the IHC gold standard in FIP diagnostics in China. With the development of research, breakthroughs have gradually been made in the treatment of FIP, such as studies on GS-441524 and GC376 ^6-9.16. However, the side effects of the two medicines are not clear and cannot be used immediately for clinical treatment. Therefore, the establishment of a more accurate diagnostic system is essential for the FIP in China. This paper examined 127 cases of FIP in Wuhan, China, and studied the diagnostic methods of several hospitals. He focused on clinicians' clinical practices in diagnosing the disease and improving the FIP diagnostic system.

This study collected data from 127 cases with highly suspected FIP, where 13 cats underwent exploratory laparotomy, 9 samples from cats with high suspicion of FIP underwent histopathology, and 6 samples underwent IHC, all of which were finally confirmed as FIP. Therefore, the inclusion criteria for the cases in this study were justified. Many studies and studies have been conducted on FIP based on the analysis of highly suspicious cases, which could not be diagnosed by gold standard (IHC) methods, but still provided a high reference value for the diagnosis and treatment of FIP.^17–22. In addition, the implementation of invasive diagnostic methods has been difficult in clinical practice. Even owners of cats who chose euthanasia were not willing to accept the autopsy after death. Thus, if the IHC is not available, a more accurate diagnosis of a case of suspected FIP is a problem that veterinarians are currently paying close attention to.

In this study, the prevalence of cats with highly suspected FIP was 0.61%. Riemer's research in Germany showed that the prevalence of FIP was 1.42% ¹⁴. Previous studies have also shown that the prevalence of FIP in households with one cat or with two cats was 0.02%, while the prevalence in shelters was 5-10%. ^23,24. No correlation was found between gender and FIP. However, some studies have suggested that males are more susceptible to FIP²⁵. The prevalence of intact males was significantly higher than that of other groups and the prevalence of neutered cats was lower, consistent with Rohrbach's findings.²⁶. This may be because sex hormones, especially androgens, have a negative effect on the immune system and increase the risk of viruses spreading and mutating.²⁷. As mentioned above, this study showed that FIP is significantly associated with age, so young cats are more susceptible, probably because the immature immune system and various stressors have often led to a high viral load in young cats. It is possible that the increased rate of uncastrated cats may have been partly due to the young age of the infected cats, which means that the disease occurred before the age of castration. Purebred cats were previously considered more susceptible to FIP. However, a growing number of studies, including this study, showed that the proportion of purebred cats compared to the clinic population did not deviate from the average.¹⁴. This study also examined the regularity of the FIP outbreak month, which was not considered in other FIP-related epidemiological surveys, and ultimately found no significant difference in FIP prevalence by month. This study reported stressors in 60 cats, the most common of which was environmental change. There is no doubt that stress can inhibit immune function and increase the susceptibility of viruses to mutations and multiplication.¹⁰. Housing density was also considered to be one of the main risk factors for FIP, and overcrowding could lead to virus mutations and disease development.¹⁴. In any case, several cats with a high suspicion of FIP in this study came from single-cat households. This may be because these cats were exposed to FCoV when they lived in a multi-cat household as a child and then moved to a single cat household before developing FIP.

Most of the FIP cases presented were effusive (up to 85.8%), most of which were ascites. FIP can affect systemic organs, most commonly abdominal organs, including the intestine, mesenteric lymph nodes, liver, kidney, and spleen. Neurological and ocular symptoms were typical features of dry FIP. A retrospective study of 286 cats with neurological disease showed that FIP was one of the main causes of neurological diseases in cats²⁸. In this study, 52.9% infected cats had a temperature greater than 39.5 ° C and 36.5% had a temperature greater than 40 ° C. Another study showed that the proportion of cats with fever was higher, where 82% cats had a temperature higher than 39.5 ℃ and 39% had a temperature higher than 40 ℃; Fever has also been found to be more common in effusive FIP¹⁴. Cases of pericardial effusion, swelling of the scrotum or skin papules, which are not common but could not be ignored during routine examination, have not been reported. The clinical signs of FIP always change over time (for example, gradual effusion and changes in fundus), so repeated clinical examination was very important to avoid delays in diagnosis and treatment.

Changes in serum hematology and biochemistry could only increase or decrease the suspicion of FIP, but could not be used as a definitive diagnostic method. In this study, there were no apparent signs of haematological changes in the diagnosis of FIP cases, with a decrease in HCT in 40,2% cats and LYM in 50% cats. However, many studies have shown that lymphocytopenia is the most common haematological abnormality of FIP caused by natural or experimental infection.^29,30caused by virus-induced T-cell apoptosis³¹. In this study, we hypothesize that protein testing plays an important role in the diagnosis of FIP cases. ALB decreased in 58.7% cats and GLOB increased in 57.8% cats, so when both occurred simultaneously, TP often remained within the normal range. Therefore, only 14.7% cats showed an increase in TP. Both hyperglobulinemia and hypoalbuminemia may have increased the suspicion of FIP, but the most important serum biochemical abnormality was the A / G ratio. The A / G ratio decreased in almost all cases of FIP. Different studies have yielded different results regarding the effective critical value of the A / G ratio³². According to the A / G ratios in this study (91.7% <0.6, 85.3% <0.5 and 54.1% <0.4), a ratio of 0.5 can be considered a critical value. When the A / G ratio was> 0.8, the probability of FIP was very small; when the ratio was 0.5³³. Therefore, we hypothesize that an increase in SAA may help diagnose FIP.

Compared to serum detection, detection and analysis of body effusion has a better predictive value in cats.³². The positivity rate of the Rivalt test in this study was 100%. The study indicated that the sensitivity of the Rivalt test was 91.3%, the specificity was 65.5%, the positive predictive value was 58.4%, and the negative predictive value was 93.4%. ³⁴. Rival's test is a cheap and fast detection method that does not require expensive instruments and is easy to handle. In view of good sensitivity and negative predictive value, the Rivalt test should be included in the diagnosis of each fed cat.¹⁵. The positive rate of FCoV RNA detected by RT-PCR was 84.7%. However, all coronaviruses, including FCoV, often mutate and recombine, so RT-PCR designed for specific sequences could not amplify all FCoV³⁵ and positive results only represented the presence of FCoV, but not exact FIPV.

In this study, a total of 24 cats were treated with GS-441524, of which 23 were cured. Only one cat relapsed after 4 weeks of treatment and was eventually sacrificed. GS-441524 treatment for more than 8 weeks has been shown to be highly effective. Treatment with GC376 alone or in combination with GS-441524 was also effective. In fact, most of the terminal deaths were due to absence of treatment, purely symptomatic treatment, or euthanasia. In addition, Pruijssers and Denison have suggested that the combination of strong broad-spectrum anti-coronavirus drugs may increase efficacy and reduce the incidence of drug resistance.³⁶.

In conclusion, it can be stated that the FIP did not correlate with gender, breed or month, but significantly correlated with age and castration status. FIP was significantly more common in young cats or intact males. Wet FIP was the most common clinical form. Decreased A / G ratio, increased SAA and lymphopenia were common laboratory abnormalities. GS-441524 and GC376 have been shown to be effective in treating cats with suspected FIP.

Additional information

Complementary pictures. ^{(5.0 million Docx)}

Thanks

The authors would like to thank the veterinarians and owners of the cats, without whose help this study would not have been possible. The study was funded by the National Key Research and Development Plan (No. 2020YFC08845600) and Huazhong Agricultural University. The study was performed in accordance with ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines (https://arriveguidelines.org).

Author's contributions

Study concept and design: CH and Y.Yin. Data collection: YY, HO, WJ, JL, XL and JL Data analysis: YY, TL, CW and XL Manuscript: YY and CH All authors reviewed the manuscript.

Data availability

All data generated or analyzed during this study are included in this published article (and its supplementary information files).

Conflict of interests

The authors are not aware of any conflict of interest.

Footnotes

Publisher Note

Springer Nature remains neutral in terms of jurisdictional claims in published maps and institutional links.

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