Comparative sequence analysis of the accessory and nucleocapsid genes of feline coronavirus strains isolated from cats diagnosed with effusive feline infectious peritonitis.

Feline infectious peritonitis (FIP) is a lethal infectious disease of domestic cats caused by feline coronavirus (FCoV) infection. Feline infectious peritonitis virus (FIPV) is a mutant type of FCoV that is characterized by causing fibrinous serositis with effusions in the pleural and abdominal cavities (wet form) and/or granulomatous-necrotizing inflammatory lesions in several organs (dry form). There have been numerous studies on FIP worldwide, whereas information about this disease in Thailand is still limited. Most studies involving molecular surveillance and evaluation of FCoV field strains have examined the genetic diversity of the spike and accessory ORF3c coding regions. Of these, the S gene is more divergent and is responsible for the two FCoV serotypes, while ORF3c harbors mutations that result either in early termination or destruction of the protein. In this study, we investigated the genetic diversity and genetic relationships among the current Thai and global FCoV strains in the accessory and nucleocapsid genes using a virus-specific PCR method. Comparative sequence analysis suggested that the Thai FCoV isolates were most closely related to strains reported in the Netherlands, the USA, and China. In the ORF3ab sequences, some Thai strains were more than 99% identical to the DF-2 prototype strain. Truncation of the 3a gene product was found in Thai FCoV strains of group 2. Amino acid deletions were observed in the N, ORF3c, and ORF7b proteins of Thai FCoV sequences. The accessory gene sequence divergence may be responsible for driving the periodic emergence and continued persistence of FCoVs in Thai domestic cat populations. Our findings provide updated information about the molecular characteristics of the accessory and nucleocapsid genes of FCoV strains in circulation that were not previously documented in this country.

Molecular survey of selected viral pathogens in wild leopard cats (Prionailurus bengalensis) in Taiwan with an emphasis on the spatial and temporal dynamics of carnivore protoparvovirus 1.

The leopard cat (Prionailurus bengalensis) was listed as an endangered species under the Wildlife Conservation Act in Taiwan in 2009. However, no study has evaluated the possible direct or indirect effects of pathogens on the Taiwanese leopard cat population. Here, we targeted viral pathogens, including carnivore protoparvovirus 1 (genus Protoparvovirus), feline leukemia virus (FeLV), feline immunodeficiency virus (FIV), coronaviruses (CoVs), and canine distemper virus (CDV), through molecular screening. The spatial and temporal dynamics of the target pathogens were evaluated. Through sequencing and phylogenetic analysis, we clarified the phylogenetic relationship of viral pathogens isolated from leopard cats and domestic carnivores. Samples from 23 live-trapped leopard cats and 29 that were found dead were collected from 2015 to 2019 in Miaoli County in northwestern Taiwan. Protoparvoviruses and CoVs were detected in leopard cats, and their prevalence (95% confidence interval) was 63.5% (50.4%-76.6%) and 8.8% (0%-18.4%), respectively. Most of the protoparvovirus sequences amplified from Taiwanese leopard cats and domestic carnivores were identical. All of the CoV sequences amplified from leopard cats were identified as feline CoV. No spatial or temporal aggregation of protoparvovirus infection in leopard cats was found in the sampling area, indicating a wide distribution of protoparvoviruses in the leopard cat habitat. We consider sympatric domestic carnivores to be the probable primary reservoir for the identified pathogens. We strongly recommend management of protoparvoviruses and feline CoV in the leopard cat habitat, particularly vaccination programs and population control measures for free-roaming dogs and cats.

Development of a recombinase polymerase amplification fluorescence assay to detect feline coronavirus.

Feline coronavirus (FCoV) is classified into two pathotypes: the avirulent feline enteric coronavirus (FECV), and the virulent feline infectious peritonitis virus (FIPV). Rapid pathogen detection, which is efficient and convenient, is the best approach for early confirmatory diagnosis. In this study, we first developed and evaluated a rapid recombinase polymerase amplification (RPA) detection method for FCoV that can detect FCoV within 15 min at 39 °C. The detection limit of that assay was 233 copies/µL DNA molecules per reaction. The specificity was high: it did not cross-react with canine distemper virus (CDV), canine coronavirus (CCoV), canine adenovirus (CAV), feline calicivirus (FCV), feline herpesvirus (FHV), or feline parvovirus (FPV). This assay was evaluated using 42 clinical samples (30 diarrhea samples and 12 ascites samples). The coincidence rate between FCoV-RPA and RT-qPCR for detection in clinical samples was 95.2%. In summary, FCoV-RPA analysis provides an efficient, rapid, and sensitive detection method for FCoV.

Rapid Resolution of Non-Effusive Feline Infectious Peritonitis Uveitis with an Oral Adenosine Nucleoside Analogue and Feline Interferon Omega.

This is the first report of a successful treatment of a non-effusive feline infectious peritonitis (FIP) uveitis case using an oral adenosine nucleoside analogue drug and feline interferon omega, and alpha-1 acid glycoprotein (AGP) as an indicator of recovery. A 2-year-old male neutered Norwegian Forest Cat presented with uveitis, keratic precipitates, mesenteric lymphadenopathy and weight loss. The cat was hypergammaglobulinaemic and had a non-regenerative anaemia. Feline coronavirus (FCoV) RNA was detected in a mesenteric lymph node fine-needle aspirate by a reverse-transcriptase polymerase chain reaction-non-effusive FIP was diagnosed. Prednisolone acetate eye drops were administered three times daily for 2 weeks. Oral adenosine nucleoside analogue (Mutian) treatment started. Within 50 days of Mutian treatment, the cat had gained over one kilogram in weight, his globulin level reduced from 77 to 51 g/L and his haematocrit increased from 22 to 35%; his uveitis resolved and his sight improved. Serum AGP level reduced from 3100 to 400 µg/mL (within normal limits). Symmetric dimethylarginine (SDMA) was above normal at 28 µg/dL, reducing to 14 µg/dL on the cessation of treatment; whether the SDMA increase was due to FIP lesions in the kidney or Mutian is unknown. Mutian treatment stopped and low-dose oral recombinant feline interferon omega begun-the cat's recovery continued.

Molecular and serological investigation of cat viral infectious diseases in China from 2016 to 2019.

In order to analyse the prevalence of cat viral diseases in China, including feline parvovirus (FPV), feline calicivirus (FCV), feline herpesvirus 1 (FHV-1), feline leukaemia virus (FeLV), feline immunodeficiency virus (FIV) and feline infectious peritonitis virus (FIPV), a total of 1,326 samples of cats from 16 cities were investigated from 2016 to 2019. Collectively, 1,060 (79.9%) cats were tested positive for at least one virus in nucleotide detection, and the positive rates of cat exposure to FeLV, FPV, FHV-1, FCV, FIV and FIPV were 59.6%, 19.2%, 16.3%, 14.2%, 1.5% and 0.5%, respectively. The prevalence of FHV-1 and FPV was dominant in winter and spring. Cats from north China showed a higher positive rate of viral infection than that of cats from south China. The virus infection is not highly correlated with age, except that FPV is prone to occur within the age of 12 months. In the serological survey, the seroprevalences of 267 vaccinated cats to FPV, FCV and FHV-1 were 83.9%, 58.3% and 44.0%, respectively. Meanwhile, the seroprevalences of 39 unvaccinated cats to FPV, FCV and FHV-1 were 76.9% (30/39), 82.4% (28/34) and 58.6% (17/29), respectively. This study demonstrated that a high prevalence of the six viral diseases in China and the insufficient serological potency of FCV and FHV-1 remind the urgency for more effective vaccines.

Detection of feline coronavirus RNA, spike gene mutations, and feline coronavirus antigen in macrophages in aqueous humor of cats in the diagnosis of feline infectious peritonitis.

Uveitis is common in cats, and is often a feature of feline infectious peritonitis (FIP). We evaluated 3 tools for detection of feline coronavirus (FCoV) in aqueous humor: 1) a 7b gene reverse-transcription real-time PCR (7b-RT-rtPCR) assay to detect FCoV RNA, 2) a spike gene mutation RT-rtPCR (S-RT-rtPCR) assay to detect 2 point mutations in the spike gene of FCoV in cats positive by 7b-RT-rtPCR, and 3) immunocytochemistry (ICC) for detection of FCoV antigen in aqueous humor macrophages. We studied 58 cats, including 31 cats with FIP and 27 control cats. FIP was excluded by postmortem examination and negative immunohistochemistry (IHC). Aqueous humor samples obtained postmortem were assessed using 7b-RT-rtPCR in all cats, and positive samples were evaluated with S-RT-rtPCR. ICC evaluation of aqueous humor samples from 36 of the 58 cats was done using an avidin-biotin complex method and monoclonal anti-FCoV IgG 2A. Sensitivity, specificity, and negative and positive predictive values were calculated including 95% CIs. 7b-RT-rtPCR had a specificity of 100.0% (95% CI: 87.2-100.0) and sensitivity of 35.5% (95% CI: 19.2-54.6). Specificity of S-RT-rtPCR could not be determined because there were no FCoV 7b-RT-rtPCR-positive samples in the control group. Sensitivity of S-RT-rtPCR was 12.9% (95% CI 3.6-29.8). Sensitivity and specificity of ICC were 62.5% (95% CI: 40.6-81.2) and 80.0% (95% CI: 44.4-97.5), respectively. The combination of 7b-RT-rtPCR and IHC could be useful in diagnosing FIP; S-RT-rtPCR did not add value; and ICC of aqueous humor samples cannot be recommended for the diagnosis of FIP.

Epicarditis in a cat caused by feline infectious peritonitis virus: case report / Epicardite em um gato causada pelo vírus da peritonite infecciosa felina: relato de caso

Feline Infectious Peritonitis (FIP) is a progressive and fatal disease in domestic and wild cats, caused by Feline Infectious Peritonitis Virus (FIPV). The disease is characterized by an immunomediated reaction against the virus in various organs. This work described a case report of fibrinous epicarditis caused by FIPV. A male cat, three years old, died and was received to be necropsied. Grossly, soft, multifocal to coalescing, whitish fibrinous exudate, measuring up the 2 centimeters of thickness, was observed in the epicardium, mostly at the apex of the heart. Microscopically, severe, multifocal to coalescing inflammatory infiltrate was observed in the epicardium, composed mainly by macrophages, plasmocytes and lymphocytes, associated with fibrin deposition. Immunohistochemistry was performed for FIPV and was positive in the areas of inflammation in the epicardium. To the author´s knowledge, this is the second report of epicarditis due to FIPV in a cat. Therefore, epicarditis should be considered a differential diagnosis of cardiac diseases in Feline Medicine.(AU)

A Peritonite Infecciosa Felina (PIF) é uma doença progressiva e fatal de felinos domésticos e selvagens, causada pelo vírus da Peritonite Infecciosa Felina (FIPV). A doença é caracterizada por uma reação imunomediada contra o vírus em vários órgãos. Este trabalho descreveu um relato de caso de epicardite fibrinosa causada pelo FPIV. Um gato macho, com três anos de idade, veio a óbito e foi recebido para necropsia. Macroscopicamente, foi observado exsudato fibrinoso esbranquiçado, friável, multifocal, medindo até 2 centímetros de espessura, no epicárdio, principalmente no ápice cardíaco. Microscopicamente, foi observado no epicárdio um infiltrado inflamatório misto, multifocal, composto por macrófagos, plasmócitos e linfócitos, associado a deposição de fibrina. A imuno-histoquímica foi positiva para o FIPV nas áreas de inflamação no epicárdio. Os autores descrevem o segundo relato de caso na literatura científica de epicardite causada pelo FIPV em um gato. Portanto, a epicardite deve ser considerada como diagnóstico diferencial em doenças cardíacas em Medicina Felina.(AU)

Multiplex fluorescent immunocytochemistry for the diagnosis of feline infectious peritonitis: Determining optimal storage conditions.

BACKGROUND: Feline Infectious Peritonitis (FIP) is a fatal disease of cats that can be very difficult to definitively diagnose antemortem. Multiplex fluorescent immunocytochemical (MF-ICC) assays are emerging as useful diagnostic tests in veterinary medicine, particularly for fluid samples. OBJECTIVE: We aimed to develop and optimize an MF-ICC assay to detect feline coronavirus within macrophages, with the primary goal of determining the allowable/recommended sample storage conditions for clinical use of this assay. METHODS: A feline macrophage cell line was infected with the FIP virus. Following harvest into EDTA tubes (simulating typical clinical collection of effusion), cells were stored at 4℃, 22℃, and 37℃. For each temperature condition, slides for MF-ICC were made at 0, 1, 2, 3, and 5 days post-collection. To assess the stability of immunoreactivity following fixation, freshly harvested infected cells were fixed onto slides and maintained at 4℃ for 1, 2, 4, and 12 weeks. All slides were analyzed by MF-ICC for the presence of mononuclear cells with co-expression of vimentin and coronaviral antigen. RESULTS: MF-ICC confirmed that cells tested positive for coronavirus at 4℃ through 3 days post-harvest, 22℃ through 48 hours post-harvest, and 37℃ through 24 hours post-harvest. The MF-ICC assay was successfully performed on fixed slides through the 12-week time point. This assay also demonstrated positive results on a clinical sample of abdominal fluid from a cat later confirmed to have FIP. CONCLUSIONS: The MF-ICC assay described here offers a potentially specific and relatively stable antemortem diagnostic test for feline infectious peritonitis. Evaluation of this assay in clinical samples is ongoing.

Effect of cat litters on feline coronavirus infection of cell culture and cats.

OBJECTIVES: Feline infectious peritonitis (FIP) is caused by infection with feline coronavirus (FCoV). FCoV is incredibly contagious and transmission is via the faecal-oral route. FCoV infection, and therefore FIP, is most common in breeder and rescue catteries, where many cats are kept indoors, using litter trays. Whether it is possible to break the cycle of FCoV infection and reinfection using cat litters has never been investigated. The aim of the study was to examine the effect of cat litters on FCoV infectivity and virus load in multi-cat households, and transmission frequency. METHODS: Fifteen cat litters were mixed and incubated with FCoV, centrifuged and the supernatants tested in vitro for the ability to prevent virus infection of cell culture. To test applicability of in vitro results to real life, virus load was measured in two households in a double crossover study of four Fuller's earth-based cat litters by testing rectal swabs using FCoV reverse transcriptase quantitative PCR. RESULTS: Four litters abrogated FCoV infection of cell culture, nine reduced it to a greater or lesser extent and two had no effect. One brand had different virus inhibitory properties depending on where it was manufactured. Fuller's earth-based litters performed best, presumably by adsorbing virus. In the field study, there appeared to be less virus shedding on one Fuller's earth-based cat litter. CONCLUSIONS AND RELEVANCE: The in vitro study successfully identified cat litters that inactivate FCoV; such litters exist so do not need to be developed. Fuller's earth-based litters best prevented infection of cell culture, but did not completely abrogate FCoV transmission in two multi-cat households. A dust-free clumping Fuller's earth litter appeared to fare best, but virus shedding also varied on the control litters, complicating interpretation. Sawdust-based cat litters are not useful in FCoV-endemic households because they track badly and have a poor effect on virus infection.

Evaluation of polymorphisms in inflammatory mediator and cellular adhesion genes as risk factors for feline infectious peritonitis.

OBJECTIVES: Feline infectious peritonitis (FIP) is a high mortality infectious disease. Single nucleotide polymorphisms (SNPs) in the genes encoding interferon gamma (IFNG), tumour necrosis factor alpha (TNFA) and dendritic cell-specific intercellular adhesion molecule-grabbing non-integrin (DC-SIGN; CD209) have been associated with increased and decreased risk of developing FIP. This study was designed to determine whether these associations were present in a UK population of pedigree cats using samples from cats euthanased with a confirmed diagnosis (FIP, n = 22; non-FIP, n = 10) or clinically healthy cats over 11 years of age (n = 3). METHODS: DNA was extracted from tissue (n = 32) or blood (n = 3) and PCR performed for regions of IFNG, TNFA and CD209. PCR amplicons were sequenced, each SNP genotype was determined, and genotype/allele frequency for each SNP and FIP status were compared. RESULTS: No significant association was found between the genotype and FIP status for any SNP analysed. There was a trend for the heterozygous CT genotype at both IFNG g.401 and IFNG g.408 to be associated with FIP (P = 0.13), but this genotype was also found in a substantial proportion of non-FIP cats. There was also a trend for the heterozygous CT genotype at IFNG g.428 to be associated with FIP (P = 0.06), although most cats with FIP had the CC genotype at this locus. No associations were found between any allele at TNFA g.-421, CD209 g.1900, CD209 g.2276, CD209 g.2392 and CD209 g.2713 and FIP. CONCLUSIONS AND RELEVANCE: The use of the IFNG, TNFA and CD209 SNPs described to predict the risk of FIP cannot currently be recommended.

Differential recognition of peptides within feline coronavirus polyprotein 1 ab by sera from healthy cats and cats with feline infectious peritonitis.

The aim of the study was to identify peptides within the polyprotein (Pp) 1 ab that are differentially recognised by cats with either enteric or systemic disease following infection with feline coronavirus. Overlapping 12-mer peptides (n = 28,426) across the entire Pp1ab were arrayed on peptide chips and reacted with pooled sera from coronavirus seropositive cats and from one seronegative cat. Eleven peptides were further tested in ELISA with individual serum samples, and three were selected for further screening. Two peptides (16433 and 4934) in the nsp3 region encoding the papain 1 and 2 proteases were identified for final testing. Peptide 4934 reacted equally with positive sera from healthy cats and cats with feline infectious peritonitis (FIP), while peptide 16433 was recognized predominantly by FIP-affected cats. The value of antibody tests based on these peptides in differentiating between the enteric and FIP forms of feline coronavirus infection remains to be determined.

Cerebral toxoplasmosis in a cat with feline leukemia and feline infectious peritonitis viral infections.

A diarrheic young cat died after neurological involvement. Biochemistry pointed to feline infectious peritonitis (FIP). The final diagnosis was severe multifocal meningoencephalitis due to Toxoplasma gondii. The presence of the parasite in the brain was confirmed using immunohistochemical staining. Concomitant feline leukemia virus (FeLV) and FIP were possible contributors to the clinical, fatal outcome.

Toxoplasmose cérébrale chez un chat atteint des infections virales de leucémie féline et de péritonite infectieuse féline. Un jeune chat diarrhéique est mort après des symptômes neurologiques. La biochimie a signalé une péritonite infectieuse féline (FIP). Le diagnostic final a été une méningo-encéphalite multifocale grave causée par Toxoplasma gondii. La présence du parasite dans le cerveau a été confirmée à l'aide de la coloration immunohistochimique. La présence concomitante du virus de la leucémie féline (FeLV) et de la FIP sont des facteurs possibles ayant contribué au résultat clinique mortel.(Traduit par Isabelle Vallières).

Cross-sectional quantitative RT-PCR study of feline coronavirus viremia and replication in peripheral blood of healthy shelter cats in Southern California.

Objectives The objectives of this study were to determine the prevalence of feline coronavirus (FCoV) viremia, and its replication in peripheral blood using quantitative RT-PCR (qRT-PCR) methodology in a population of 205 healthy shelter cats in Southern California, as well as to assess any possible connection to longitudinal development of feline infectious peritonitis (FIP). Methods The study was performed on buffy-coat samples from EDTA-anticoagulated whole blood samples of 205 healthy shelter cats. From 50 of these cats, fecal samples were also examined. FCoV genomic and subgenomic RNA in the buffy coats was amplified by a total FCoV RNA qRT-PCR. Evidence for FCoV replication in peripheral blood and feces was obtained by M gene mRNA qRT-PCR. Results Nine of 205 cats (4.4%) were viremic by the total FCoV RNA qRT-PCR, and one of these cats had evidence of peripheral FCoV blood replication by an FCoV mRNA qRT-PCR. The single cat with peripheral blood replication had a unique partial M gene sequence distinct from positive controls and previously published FCoV sequences. Neither seven of the nine viremic cats with follow-up nor the single cat with replicating FCoV with positive qRT-PCR results developed signs compatible with FIP within 6 months of sample collection. Conclusions and relevance FCoV viremia and peripheral blood replication in healthy shelter cats have a low prevalence and do not correlate with later development of FIP in this study population, but larger case-control studies evaluating the prognostic accuracy of the qRT-PCR assays are needed.

Utility of an immunocytochemical assay using aqueous humor in the diagnosis of feline infectious peritonitis.

OBJECTIVE: In cats suffering from feline infectious peritonitis (FIP) without effusion, antemortem diagnosis is challenging. Uveitis is common in these cats. It was the aim of this study to evaluate sensitivity and specificity of an immunocytochemical assay (ICC) in aqueous humor of cats suspected of having FIP. ANIMALS STUDIED: The study included 26 cats with immunohistochemically confirmed FIP and 12 control cats for which FIP was suspected due to similar clinical or laboratory changes, but which suffered from other diseases confirmed via histopathology. PROCEDURES: All aqueous humor samples were collected postmortem by paracentesis. ICC was carried out as avidin-biotin complex method. Sensitivity, specificity, and the overall accuracy including 95% confidence intervals (95% CI) were calculated. RESULTS: Immunocytochemistry was positive in 16 of 25 cats with FIP and 2 of 11 control cats (one cat with lymphoma, one with pulmonary adenocarcinoma). Aqueous humor samples of one cat with FIP and of one control cat were excluded from statistical analysis. Sensitivity was 64.0% (95% CI: 42.5-82.0); specificity 81.8% (95% CI: 48.2-97.7); and overall accuracy 69.4% (95% CI: 51.9-83.7). CONCLUSIONS: As false-positive results occurred and specificity is most important in the diagnosis of FIP, the diagnostic utility of ICC in aqueous humor is limited. Further studies are required to clarify the origin of false-positive ICC results.

Molecular detection of viral agents in free-ranging and captive neotropical felids in Brazil.

We describe molecular testing for felid alphaherpesvirus 1 (FHV-1), carnivore protoparvovirus 1 (CPPV-1), feline calicivirus (FCV), alphacoronavirus 1 (feline coronavirus [FCoV]), feline leukemia virus (FeLV), feline immunodeficiency virus (FIV), and canine distemper virus (CDV) in whole blood samples of 109 free-ranging and 68 captive neotropical felids from Brazil. Samples from 2 jaguars ( Panthera onca) and 1 oncilla ( Leopardus tigrinus) were positive for FHV-1; 2 jaguars, 1 puma ( Puma concolor), and 1 jaguarundi ( Herpairulus yagouaroundi) tested positive for CPPV-1; and 1 puma was positive for FIV. Based on comparison of 103 nucleotides of the UL24-UL25 gene, the FHV-1 sequences were 99-100% similar to the FHV-1 strain of domestic cats. Nucleotide sequences of CPPV-1 were closely related to sequences detected in other wild carnivores, comparing 294 nucleotides of the VP1 gene. The FIV nucleotide sequence detected in the free-ranging puma, based on comparison of 444 nucleotides of the pol gene, grouped with other lentiviruses described in pumas, and had 82.4% identity with a free-ranging puma from Yellowstone Park and 79.5% with a captive puma from Brazil. Our data document the circulation of FHV-1, CPPV-1, and FIV in neotropical felids in Brazil.

Comparison of real-time reverse transcriptase polymerase chain reaction of peripheral blood mononuclear cells, serum and cell-free body cavity effusion for the diagnosis of feline infectious peritonitis.

Objectives Diagnosis of feline infectious peritonitis (FIP) remains challenging, especially in cats without effusions. The objective of this study was to evaluate the sensitivity and specificity of a real-time reverse transcriptase polymerase chain reaction (RT-PCR) detecting feline coronavirus (FCoV) RNA in peripheral blood mononuclear cells (PBMCs) and serum in comparison with the same real-time RT-PCR in cell-free body cavity effusion. Methods This prospective case-control study included 92 cats. Forty-three cats had a definitive diagnosis of FIP, established either by histopathological examination (n = 28) or by positive immunofluorescence staining of FCoV antigen in macrophages of effusions (n = 11), or by both methods (n = 4). Forty-nine control cats had other diseases but similar clinical signs. Real-time RT-PCR was performed on PBMCs of 37 cats (21 cats with FIP, 16 controls), on serum of 51 cats (26 cats with FIP, 25 controls) and on cell-free body cavity effusion of 69 cats (36 cats with FIP, 33 controls). Sensitivity, specificity, positive and negative predictive value, including 95% confidence intervals (CI), were calculated. Results Real-time RT-PCR of PBMCs, serum and cell-free body cavity effusion showed a specificity of 100% (95% CI 79.4-100% in PBMCs, 86.3-100% in serum, 89.4-100% in cell-free body cavity effusion) and a sensitivity of 28.6% (95% CI 11.3-52.2%) in PBMCs, 15.4% (95% CI 4.4-34.9%) in serum and 88.9% (95% CI 73.9-96.9%) in cell-free body cavity effusion to diagnose FIP. Conclusions and relevance Although it is known that RT-PCR can often provide false-positive results in healthy cats, this real-time RT-PCR was shown to be a specific tool for the diagnosis of FIP when applied in a clinical setting. Sensitivity in cell-free body cavity effusion was high but low in PBMCs and serum. PBMC samples showed a higher sensitivity than serum samples, and are therefore a better choice if no effusion is present.

Detection of feline coronavirus spike gene mutations as a tool to diagnose feline infectious peritonitis.

Objectives Feline infectious peritonitis (FIP) is an important cause of death in the cat population worldwide. The ante-mortem diagnosis of FIP in clinical cases is still challenging. In cats without effusion, a definitive diagnosis can only be achieved post mortem or with invasive methods. The aim of this study was to evaluate the use of a combined reverse transcriptase nested polymerase chain reaction (RT-nPCR) and sequencing approach in the diagnosis of FIP, detecting mutations at two different nucleotide positions within the spike (S) gene. Methods The study population consisted of 64 cats with confirmed FIP and 63 cats in which FIP was initially suspected due to similar clinical or laboratory signs, but that were definitively diagnosed with another disease. Serum/plasma and/or effusion samples of these cats were examined for feline coronavirus (FCoV) RNA by RT-nPCR and, if positive, PCR products were sequenced for nucleotide transitions within the S gene. Results Specificity of RT-nPCR was 100% in all materials (95% confidence interval [CI] in serum/plasma 83.9-100.0; 95% CI in effusion 93.0-100.0). The specificity of the sequencing step could not be determined as none of the cats of the control group tested positive for FCoV RNA. Sensitivity of the 'combined RT-nPCR and sequencing approach' was 6.5% (95% CI 0.8-21.4) in serum/plasma and 65.3% (95% CI 50.4-78.3) in effusion. Conclusions and relevance A positive result is highly indicative of the presence of FIP, but as none of the control cats tested positive by RT-nPCR, it was not possible to confirm that the FCoV mutant described can only be found in cats with FIP. Further studies are necessary to evaluate the usefulness of the sequencing step including FCoV-RNA-positive cats with and without FIP. A negative result cannot be used to exclude the disease, especially when only serum/plasma samples are available.

Feline coronavirus quantitative reverse transcriptase polymerase chain reaction on effusion samples in cats with and without feline infectious peritonitis.

Objectives The aim of the study was to determine whether feline coronavirus (FCoV) RNA in effusion samples can be used as a diagnostic marker of feline infectious peritonitis (FIP); and in FCoV RNA-positive samples to examine amino acid codons in the FCoV spike protein at positions 1058 and 1060 where leucine and alanine, respectively, have been associated with systemic or virulent (FIP) FCoV infection. Methods Total RNA was extracted from effusion samples from 20 cats with confirmed FIP and 23 cats with other diseases. Feline coronavirus RNA was detected using a reverse transcriptase quantitative polymerase chain reaction assay (qRT-PCR), and positive samples underwent pyrosequencing of position 1058 with or without Sanger sequencing of position 1060 in the FCoV spike protein. Results Seventeen (85%) of the effusion samples from 20 cats with FIP were positive for FCoV RNA, whereas none of the 23 cats with other diseases were positive. Pyrosequencing of the 17 FCoV-positive samples showed that 11 (65%) of the cats had leucine and two (12%) had methionine at position 1058. Of the latter two samples with methionine, one had alanine at position 1060. Conclusions and relevance A positive FCoV qRT-PCR result on effusions appears specific for FIP and may be a useful diagnostic marker for FIP in cats with effusions. The majority of FCoVs contained amino acid changes previously associated with systemic spread or virulence (FIP) of the virus.

Diagnostic utility of cerebrospinal fluid immunocytochemistry for diagnosis of feline infectious peritonitis manifesting in the central nervous system.

Objectives The aim of the study was to evaluate whether an ante-mortem diagnosis of central nervous system (CNS) feline infectious peritonitis (FIP) is possible via immunocytochemical staining (ICC) of feline coronavirus antigen (FCoV) within macrophages of cerebrospinal fluid (CSF). Methods Prospectively, CSF samples of 41 cats were investigated, including cats with histopathologically confirmed FIP and neurological signs (n = 10), cats with confirmed FIP without CNS involvement (n = 11), cats with neurological signs but another confirmed CNS disease (n = 17), and cats without neurological signs and a disease other than FIP (n = 3). ICC staining of CSF macrophages was performed in all cats. Sensitivity, specificity, positive (PPV) and negative predictive values (NPV) of CSF ICC were calculated. Results Of 10 samples from cats with CNS FIP, eight had detectable CSF macrophages, seven of which were positive for FCoV. Ten of 11 samples from cats with confirmed FIP without neurological signs had macrophages in the CSF, with all 10 being ICC-positive. In cats with other CNS disorders, 11/17 had macrophages, two of which stained positively. In cats with diseases other than FIP and without neurological disorders, 2/3 revealed macrophages, with one cat showing positive ICC staining. Diagnosis of FIP via CSF ICC had a sensitivity of 85.0% and a specificity of 83.3%. PPV and NPV were 85.0% and 83.3%. Conclusions and relevance CSF ICC is a highly sensitive test for ante-mortem diagnosis of FIP manifesting in the CNS. However, CNS ICC specificity is too low to confirm FIP and the method should only be applied in conjunction with other features such as CSF cytology. CNS ICC could be helpful to discover pre-neurological stages of CNS FIP.

Investigation into the utility of an immunocytochemical assay in body cavity effusions for diagnosis of feline infectious peritonitis.

Objectives Feline coronaviruses (FCoVs) exist as two biotypes, feline enteric coronavirus and feline infectious peritonitis virus. Although feline infectious peritonitis (FIP) is a very common disease, the ante-mortem diagnosis of this disease still remains a challenge. Immunofluorescence staining of FCoV in macrophages in effusion has been considered as the reference standard for the diagnosis, but recently this method has been shown to have lower specificity than previously reported. In addition, this method is not widely available and requires the use of fluorescence microscopes. Therefore, it was the aim of this study to evaluate the diagnostic potential of an immunocytochemical (ICC) assay using body cavity effusion. Methods Effusion samples from 27 cats with immunohistochemically confirmed FIP and 29 cats with suspected FIP but a definitive diagnosis of another disease were examined. ICC specimens were evaluated with respect to positive immunostaining. In addition, effusion samples were stained with haematoxylin and eosin and evaluated cytologically. Results A diagnostic sensitivity of 85.2% was recorded for effusion specimens (95% confidence interval [CI] 66.3-95.8), while the diagnostic specificity was only 72.4% (95% CI 52.8-87.3). Conclusions and relevance Once the clinical disease of FIP develops in a cat, it always leads to death, and most of the cats are euthanased within a few days or weeks. As false-positive results might lead to euthanasia of cats suffering from potentially treatable diseases, the diagnostic specificity of a diagnostic tool is the most important factor in a fatal disease like FIP. Thus, the diagnostic utility of this test proved to be insufficient and positive ICC results should be interpreted with caution. Nevertheless, full-body necropsy could not be performed in 13/29 control cats. It is possible that these cats actually suffered from early-stage FIP and that this fact might have influenced the diagnostic specificity of the ICC. Based on the results of the present study, however, ICC of effusion samples currently cannot be recommended to confirm a suspicion of FIP.