key: cord-0905714-g0zccwz2
authors: André, Nicole M; Miller, Andrew D; Whittaker, Gary R
title: Feline infectious peritonitis virus-associated rhinitis in a cat
date: 2020-06-23
journal: JFMS Open Rep
DOI: 10.1177/2055116920930582
sha: 95b63d95f84434892d072546621dc2fc7f2667a7
doc_id: 905714
cord_uid: g0zccwz2

CASE SUMMARY: This report describes a cat with initial respiratory signs prior to developing fulminant feline infectious peritonitis (FIP) after adoption from an animal shelter. Histologic examination of the tissues revealed typical lesions associated with FIP in the lung, liver, large intestine and small intestine. Histologic examination of the nasal cavity revealed pyogranulomatous rhinitis. Immunohistochemistry with monoclonal antibody FIPV3-70 targeting FIP antigen in macrophages confirmed FIP and molecular analysis identified a spike protein mutation (R793S) consistent with the presence of an FIP virus. Pathological changes, immunolabeling and molecular analysis provide evidence that respiratory infection by feline coronavirus is part of the spectrum of FIP-associated disease. RELEVANCE AND NOVEL INFORMATION: This report highlights nasal pathology associated with FIP through a combination of histopathology, immunohistochemistry and molecular characterization of the virus. Our work supports a little-appreciated role of the respiratory tract in FIP.

Coronaviruses have been implicated in respiratory and gastrointestinal disease in many animal species. 1 In cats, feline coronavirus (FCoV) consists of two bio types, commonly referred to as feline enteric corona virus (FECV) and feline infectious peritonitis virus (FIPV). 2, 3 FECV is generally considered to be associated with a mild, selflimiting gastrointestinal infection but can cause mild respiratory signs. 4 Initial infection with FCoV is thought to occur by oronasal exposure to the virus in fecal material or fecalcontaminated fomites. 4 In some FCoVinfected cats, mutation of the viral genome leads to an alteration in the host cell tropism of FCoV from enterocytes to macrophages, leading to the systemic infection known as feline infectious peritonitis (FIP). 2, 3, 5, 6 FIP is one of the most important infectious diseases affecting domestic and wild cat populations. There are multiple presentations of FIP: effusive (wet); noneffusive (dry); and a mixed form, which can include a combination of effusion and pyogranuloma tous lesions. 4, 7 Clinical signs can be variable in all presentations and comprise anorexia, pyrexia, lethargy, diarrhea and weight loss. 4, 7, 8 Early studies suggested that upper respiratory infections characterized by con junctivitis or rhinitis preceded the development of FIP, 7, 9, 10 although FCoV is not routinely considered a common respiratory pathogen in cats. 4 This case report describes a cat with respiratory signs prior to developing systemic FIP. The pathological changes with marked FIPV immunolabeling supports previous work suggesting that respiratory infection is part of the spectrum of FIPassociated disease.

An 8weekold spayed female domestic shorthair cat was adopted from a shelter into a singlecat home. The cat received three feline viral rhinotracheitis, calicivirus and panleukopenia vaccinations, and was not vacci nated against rabies. The cat was previously dewormed and was feline leukemia virus negative. The diet con sisted of a commercial dry and canned food.

At 10 weeks of age, the cat presented to the general practitioner (GP) for a wellness visit. On physical exami nation, a mild mucopurulent discharge was noted in both eyes, and the cat was sneezing. The remainder of the examination was normal. A fecal centrifugation was performed on formed feces and no ova or parasites were detected. Tobramycin eye drops were dispensed, and one drop was administered to both eyes every 12 h for 10 days. In addition, lysine (Enisyl F) was dispensed and one pump was administered by mouth every 12 h for 10 days. Fenbendazole suspension was administered orally at 0.6 ml once daily for 5 days.

At 14 weeks of age, the cat was returned to the GP for evaluation of sneezing, diarrhea, a poor appetite and a distended abdomen. On physical examination, epaxial muscle loss was noted and the abdominal dis tension was confirmed. A fecal centrifugation was per formed on soft feces, and no ova or parasites were detected. Metronidazole suspension was dispensed and 20 mg was administered orally twice daily for 10 days and 25 mg (0.4 ml) of amoxicillin clavulanic acid 62.5 mg/ml (Clavamox drops; Zoetis) was adminis tered orally for 10 days.

The cat was returned to the GP 2 days later with hyporexia, polydipsia, lethargy and a reluctance to ambulate. A moderately distended abdomen was noted on physical examination. Cytological analysis of fluid obtained via abdominocentesis revealed a high protein nonseptic exudate with mixed inflammation (Table 1) . A complete blood count (CBC) and chemistry panel (Tables 2-6) were performed. The CBC with electronic differential revealed a slight anemia and neutrophilia ( Table 2 ). The manual differential revealed abnormal red blood cell (RBC) and white blood cell (WBC) morphology (Tables 3-5) . RBC evaluation revealed occasional poikilocytosis, anisocytosis, microcytosis and polychromasia, with Heinz bodies (Table 4 ). WBCs showed the following occasional changes in neutro phils: basophilic, vacuolated and foamy cytoplasm, swollen nucleus, indented nuclear margins and Döhle bodies ( Table 5 ). The chemistry panel showed mild ele vations in total protein and glucose, and moderate ele vations of globulin and triglycerides. A decrease in alanine aminotransferase, alkaline phosphatase, cre atine kinase and albumin were noted (Table 6) . A tenta tive diagnosis of FIP was made based on clinical signs, fluid analysis and laboratory abnormalities. Predni solone oral solution 3 mg/ml was dispensed and 1.5 mg (0.5 ml) was administered twice daily.

At 15 weeks of age, the cat was returned to the GP for reevaluation. An FCoV ELISA was performed and found to be positive at 1.228 (>1.20 = positive, <0.90 = negative). Lactated Ringer's fluids were administered 50 ml subcutaneously (SC). Vitamin B12 injections were dispensed and 0.1 ml was administered SC weekly. A week later, the cat was presented for an abdominocente sis prior to starting feline omega interferon. A total of 215 ml of strawcolored viscous fluid was removed. The cat's appetite was decreased. Mirtazapine 7.5 mg/ml suspension was dispensed and 1.88 mg was adminis tered (0.25 ml) every 48-72 h to increase appetite. Histologic examination revealed a diffuse pyogranu lomatous rhinitis that partially obliterated the ethmo turbinates ( Figure 1 ). Additional FIPVassociated lesions were found in lung, liver, large intestine, mesenteric lymph node and small intestine. No lesions were pre sent in the brain or kidneys.

Respiratory samples were collected using two sterile flocked swabs from the conjunctiva, nasal cavity and the oropharynx. The samples were pooled and were sent to the Animal Health Diagnostic Center, Cornell University College of Veterinary Medicine, for the feline respiratory panel and the pooled sample was found to be low positive for Mycoplasma felis ( Table 7) . All other respiratory agents screened in panel were negative within the pooled sample (Table 7) .

PCR and Sanger sequencing were performed on a subset of tissues as described. 11 Briefly, 25 μl reverse transcription PCRs were performed with qScript XLT 1Step RT PCR kit (Quantbio). PCR conditions were 20 mins at 50°C, 3 mins at 95°C and 40 cycles of 10 s at 95°C, 20 s at 55°C, 40 s at 72°C and then 10 mins at 72°C. 11 Molecular analysis of the viral spike protein showed an amino acid change from an arginine to a serine (RS) at an essential P1 cleavage activation position at residue 793 ( Figure 2 ). 12 This mutation was observed in all sam ples; that is, lung, liver, mesenteric lymph node, small intestine and large intestine.

This case report illustrates a cat with pathology in the nasal cavity with immunohistochemistry staining of macrophages associated with FIP. The cat was ini tially presented to the veterinarian for a wellness visit, where an upper respiratory tract infection was primar ily observed and FIP was not considered a differential at the time. The respiratory signs resolved; however, clini cal signs associated with FIP subsequently surfaced and ultimately caused the deterioration and euthanasia of the cat. FIP has previously been observed in the nasal and oral cavities. 9 In a noneffusive case of FIP, small granu lomas were observed on the frenulum of the tongue. 9 In an FIP case coinfected with toxoplasmosis, histologic examination of the nasal submucosa showed a severe diffuse lymphoplasmacytic rhinitis with perivascular aggregations of neutrophils and macrophages, which immunochemistry revealed as FCoV antigen within macrophages. 13 In an experimental study, when the virus was aerosolized, lesions were frequently found in the nasal turbinates, lungs, pleura and tracheobronchial lymph nodes. 14 The functionally relevant amino acid change (R793S) was found through the molecular analysis of the viral spike protein in all tissues in this cat ( Figure 2 ). This muta tion (R793S), an alteration from a charged to an uncharged amino acid, is predicted to eliminate the ability for furin to proteolytically process the S1/S2 cleavage activation site, which can ultimately affect viral entry. 12 FIP has been also associated with other mutations in the spike gene (1058) 15 and the 3c gene. 16, 17 Additional sequencing of these regions is summarized in Table 8 , and showed an M1058L conversion and truncated 3c, as expected.

The observations and findings in this case suggest that the respiratory system is a potential route of trans mission for FCoV, given the significant rhinitis present in this young cat's nasal cavity. Other coronaviruses, such as mouse hepatitis virus, infectious bronchitis virus in chickens and porcine respiratory coronavirus, transmit via the respiratory route prior to disseminating or target ing a specific organ system. The respiratory disease early in the course of disease in this cat may also be an early indicator of FIP. It is unlikely that an underlying immu nodeficiency is the cause of the respiratory disease as it resolved and did not continue as other conditions devel oped. FCoV present in the nasal cavity may also suggest a role of significant hematogenous spread of the virus as the virus produces a vasculitis and the nasal cavity is extremely vascular.

Examination of the nasal cavities of FIP cats at nec ropsy could be performed to further support the region's role in FIP. Currently, with the historical information, experimental studies and this case study, we confirm that the nasal cavity is involved in the pathogenesis of FIP; however, to further elucidate the precise mecha nism, additional experiments are necessary. In addition, Figure 2 Molecular analysis of the spike gene. A 156 base pair region of the feline coronavirus (FCoV) spike gene is shown and represented in single amino acid code, with variant residues and amino acid positions noted. The activation site between the S1 and S2 domains (S1/S2) is indicated and boxed amino acid positions are based on that for FCoV RM spike (Genbank accession # ACT10854.1) as a prototype sequence. Sequences were analyzed using Geneious Prime 2020.05 

This case report describes a kitten with FIP where the nasal cavity was extensively involved and respiratory signs were observed early on prior to clinical signs asso ciated with FIP. A respiratory panel was performed on pooled respiratory swabs to exclude any common path ogens associated with feline respiratory disease com plex (FRDC). While the swabs were low positive for M felis, no other FRDC pathogens were detected such as feline herpesvirus. While FIP is a systemic disease once it mutates, FCoV is not routinely thought of as a respiratory pathogen. This case suggests that the res piratory tract may be a mode of transmission for feline coronavirus.

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We thank Dr Alison Stout for clini cal consultation and critical reading of the manuscript, the Section of Anatomic Pathology at the Animal Health Diagnos tic Center for help with processing and training, Wendy Win gate for help with sample collection, and all the members of the Whittaker laboratory for helpful comments and support.

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Center.Ethical approval This work involved the use of non experimental animals only (including owned or unowned animals and data from prospective or retrospective studies). Established internationally recognised high standards ('best practice') of individual veterinary clinical patient care were followed. Ethical approval from a committee was therefore not necessarily required.Informed consent Informed consent (either verbal or written) was obtained from the owner or legal custodian of all animal(s) described in this work (either experimental or nonexperimental animals) for the procedure(s) undertaken. No animals or humans are identifiable within this publica tion, and therefore additional informed consent for publica tion was not required.