Subject and sample collection
Two members of a family of four with 8-year-old male castrated Sphynx cat owners were tested SARS-CoV-2 RT-PCR positive by 13 April 2021 and allocated quarantine site by the government. However, the other two family members were tested SARS-CoV-2 RT-PCR negative but according to the government COVID-19 pandemic regulation they were classified as contacted family members and allocated different quarantine site. The elder family member who has co-morbidity (obesity and diabetes) with respiratory problems was taken to intensive care unit at the government state pandemic hospital. The youngest family member had back pain, cough and fewer as COVID-19 symptoms. The cat stayed together in the same room with one of them. The SARS-CoV-2 RT-PCR results were positive to previously negative detected family members by 17 April 2021 with back pain, cough and loss of smell. Since then, the cat was separated from the owner and relocated to a home, which was cared by a family friend for 3 days. The owners contacted Near East University Animal Hospital to report the situation. This study was approved by the Near East University Animal Experiments Local Ethics Committee (Approval number: 2020/120) and written informed consent was obtained from the owners for clinical and molecular analysis.
For this purpose, oropharyngeal, third eyelid inner side conjunctival and rectal swabs were taken from the cat at certain intervals, and that treated into viral transfer medium was collected from the cat every 3 days for 12 days for SARS-CoV-2 RT-PCR test for traditional microbiological tests.
Blood samples were collected through the cephalic vein into vacuum tubes with a K3EDTA (0.5 mL, Ref. 450530, MiniCollect Tube, Greiner bio-one, Kremsmünster, Austria) as an anticoagulant for complete blood count (CBC), a serum separator tube (5 mL, Ref 10175, BD Vacutainer, Plymouth, UK) for serum biochemistry and serology, and a lithium heparin tube (4 mL, Ref 368884, BD Vacutainer) for blood gas analysis. Serum separator tube blood sample was then centrifuged at 1500 g for 10 min after complete blood clot formation, and serum was separated.
Molecular microbiological and biochemical analyses
Nucleic acid extraction kit for viral DNA and RNA extraction and its automatic system (Ascend Biotechnology, Henan, China) was used to isolate nucleic acid from swab samples. Diagnovital® HS SARS-CoV-2 real-time PCR kit (RTA Laboratories Inc., Gebze, Kocaeli, Turkey) was used to detect the SARS-CoV-2 directly from swab samples and extracted nucleic acids. The kit contains primer and probe set mixes particularly designed for SARS-Cov-N1 and N2 genes (FAM). Twenty microlitres of RT-PCR reaction mix was prepared according to manufacturer's guidelines. Hibrigen® SARS-CoV-2 and N501Y mutation detection kit (Hibrigen Biotechnology AR-GE San Tic Ltd Sti., Gebze, Kocaeli, Turkey) was used for the detection of the presence of N501Y mutation. The kit contains primer and probe sets specifically designed for SARS-CoV-2 RdRp gene (FAM) and nucleotide sequence harbouring the N501Y mutation (HEX). Twenty microlitre reaction mix was prepared with 10 μL of 2× One Step RT-PCR Mix, 4 μL of Primer Probe Mix and 6 μL RNA sample. A positive control and a negative control, which were provided by the kit, were included for each run. RT-qPCR was carried out using Insta Q96™ Plus Real-time PCR Detection System (HiMedia Laboratories Pvt. Ltd., Mumbai, India). Analyses were carried out according to the manufacturer's instructions, and samples that have Ct (cycle threshold) values in both FAM and HEX channels were considered positive for the SARS-CoV-2 and the SARS-CoV-2 N501Y mutation, respectively. The confirmation of the B.1.1.7 variant was conducted by the detection of spike protein 69/70 deletion and N501Y variants by SNP SARS-CoV-2 real-time PCR kit (SNP Biotechnology, Ankara, Turkey) according to manufacturer instructions.
CBC, serum biochemistry and serology were done at Diagnostic Laboratory, Animal Hospital, Near East University, using fresh blood samples. CBC was performed using an automatic analyser calibrated for veterinary use (BC-2800Vet, Mindray, Shenzen, China). The assessed parameters were white blood cells (in ×109/L), including lymphocytes (in ×109/L), monocytes (in ×109/L) and granulocytes (Gran, in ×109/L); erythrocytes (red blood cells, in ×1012/μL), haemoglobin (in g/dL), haematocrit (in %), mean corpuscular volume (in fL); mean corpuscular haemoglobin (in pg); mean corpuscular haemoglobin concentration (in g/dL), platelets (in ×109/μL) and mean platelet volume (in fL). Serum biochemistry was performed using commercially available assay kits (Mindray Chemistry Reagents, Shenzen, China) and an automated clinical chemistry analyser (BS120, Mindray, Shenzen, China). Total protein (biuret method, in g/dL), albumin (bromocresol green method, in g/dL), total bilirubin (VOX method, in mg/dL), blood urea nitrogen (Urea/2.14; urease method, in mg/dL), total cholesterol (CHOD-POD method, in mg/dL), creatinine (Jaffe method, in mg/dL) and triglycerides (GPO-POD method, in mg/dL) were measured as serum metabolites, and also serum phosphorous concentration was measured (phosphomolybdate method, in mg/dL). Serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, creatine kinase, γ-glutamyl transferase, lactate dehydrogenase, α-amylase and lipase activities were quantified using the International Federation of Clinical Chemistry methods in U/L. Besides, pH, pCO2 (in mmHg), bicarbonate (HCO3−, in mmol/L), base excess (in mmol/L), sodium (Na+, in mmol/L), potassium (K+, in mmol/L), ionized calcium (Ca++, in mmol/L), chloride (Cl−, in mmol/L), anion gap (in mmol/L), glucose (in mg/dL) and lactate (in mmol/L) levels were measured using veterinary calibrated Epoc blood gas analyses system (Ref 10736516, Epocal Inc., Ottawa, ON, Canada).
Indirect immunofluorescence antibody test (IFAT) was performed to detect feline coronavirus specific IgG antibodies (FCoV IgG). Standardised assay kits were used for this purpose, supplied by MEGACOR Diagnostik GmbH, Austria (Ref 821K10FK1). Indirect immunofluorescence antibody test titres greater than or equal to 1:100 were considered seropositive according to assay instructions. A rapid immunoassay (SNAP Combo FeLV Ag/FIV ab, Ref 99-06033, IDEXX, Westbrook, ME, USA) was performed to detect the presence of feline leukaemia virus p27 antigen (FeLV Ag) and antibodies specific to feline immunodeficiency virus (FIV Ab). The tests were assayed following manufacturers' instructions.
Clinical examination
After the necessary precautions were taken, the cat was brought to the Near East Animal Hospital for a detailed clinical examination. In the general examination, routine clinical examination methods, inspection, palpation and auscultation examination methods were applied. Blood pressure, pulse, heart rhythm measurements were made, and then routine eye examination of the bulbus oculi and eye attachment organs was started. For this purpose, penlight was used to evaluate direct and indirect pupillary light reflexes; direct ophthalmoscope (Riester Otoscope/Ophtalmoscope, Germany) was used for cornea, anterior chamber, lens and fundus examination. Schirmer tear test (STT) for tear function evaluation, fluorescent staining test for corneal erosions and rebound tonometry (Icare Tonovet, Finland) for intraocular pressure measurement were conducted. Ocular reflex examinations were performed, including the manage response.
Subsequently, three-sided direct radiographs of the patient, thoracic and abdominal, were taken (ECORAY HF-525 PLUS VET, South Korea). Standard left parasternal apical imaging, right parasternal apical long axis and short axis imaging, colour Doppler, tissue Doppler imaging and modified Simpson methods were used for echocardiographic evaluation. Echocardiographic examination was performed with a GE LOGIQ e R7 CONSOLE ultrasound machine with built-in DTI capacity equipped with a 4–7 MHz flat phased array probe.
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