The emergence and ensuing dominance of COVID-19 on the world stage has emphasized the urgency of efficient animal models for the development of therapeutics for and assessment of immune responses to SARS-CoV-2 infection. Shortcomings of current animal models for SARS-CoV-2 include limited lower respiratory disease, divergence from clinical COVID-19 disease, and requirements for host genetic modifications to permit infection. In this study, n = 12 specific-pathogen-free domestic cats were infected intratracheally with SARS-CoV-2 to evaluate clinical disease, histopathologic lesions, and viral infection kinetics at 4 and 8 days post-inoculation; n = 6 sham-inoculated cats served as controls. Intratracheal inoculation of SARS-CoV-2 produced a significant degree of clinical disease (lethargy, fever, dyspnea, and dry cough) consistent with that observed in the early exudative phase of COVID-19. Pulmonary lesions such as diffuse alveolar damage, hyaline membrane formation, fibrin deposition, and proteinaceous exudates were also observed with SARS-CoV-2 infection, replicating lesions identified in people hospitalized with ARDS from COVID-19. A significant correlation was observed between the degree of clinical disease identified in infected cats and pulmonary lesions. Viral loads and ACE2 expression were also quantified in nasal turbinates, distal trachea, lungs, and other organs. Results of this study validate a feline model for SARS-CoV-2 infection that results in clinical disease and histopathologic lesions consistent with acute COVID-19 in humans, thus encouraging its use for future translational studies.

Continued emergence of SARS-CoV-2 variants highlights the critical need for adaptable and translational animal models for acute COVID-19. Limitations to current animal models for SARS CoV-2 (e.g., transgenic mice, non-human primates, ferrets) include subclinical to mild lower respiratory disease, divergence from clinical COVID-19 disease course, and/or the need for host genetic modifications to permit infection. We therefore established a feline model to study COVID-19 disease progression and utilized this model to evaluate infection kinetics and immunopathology of the rapidly circulating Delta variant (B.1.617.2) of SARS-CoV-2. In this study, specific-pathogen-free domestic cats (n = 24) were inoculated intranasally and/or intratracheally with SARS CoV-2 (B.1.617.2). Infected cats developed severe clinical respiratory disease and pulmonary lesions at 4- and 12-days post-infection (dpi), even at 1/10 the dose of previously studied wild-type SARS-CoV-2. Infectious virus was isolated from nasal secretions of delta-variant infected cats in high amounts at multiple timepoints, and viral antigen was co-localized in ACE2-expressing cells of the lungs (pneumocytes, vascular endothelium, peribronchial glandular epithelium) and strongly associated with severe pulmonary inflammation and vasculitis that were more pronounced than in wild-type SARS-CoV-2 infection. RNA sequencing of infected feline lung tissues identified upregulation of multiple gene pathways associated with cytokine receptor interactions, chemokine signaling, and viral protein–cytokine interactions during acute infection with SARS-CoV-2. Weighted correlation network analysis (WGCNA) of differentially expressed genes identified several distinct clusters of dysregulated hub genes that are significantly correlated with both clinical signs and lesions during acute infection. Collectively, the results of these studies help to delineate the role of domestic cats in disease transmission and response to variant emergence, establish a flexible translational model to develop strategies to prevent the spread of SARS-CoV-2, and identify potential targets for downstream therapeutic development.

Feline Immunodeficiency Virus (FIV) causes progressive immune dysfunction in cats similar to human immunodeficiency virus (HIV) in humans. Although combination antiretroviral therapy (cART) is effective against HIV, there is no definitive therapy to improve clinical outcomes in cats with FIV. This study therefore evaluated pharmacokinetics and clinical outcomes of cART (2.5 mg/kg Dolutegravir; 20 mg/kg Tenofovir; 40 mg/kg Emtricitabine) in FIV-infected domestic cats. Specific pathogen free cats were experimentally infected with FIV and administered either cART or placebo treatments (n = 6 each) for 18 weeks, while n = 6 naïve uninfected cats served as controls. Blood, saliva, and fine needle aspirates from mandibular lymph nodes were collected to quantify viral and proviral loads via digital droplet PCR and to assess lymphocyte immunophenotypes by flow cytometry. cART improved blood dyscrasias in FIV-infected cats, which normalized by week 16, while placebo cats remained neutropenic, although no significant difference in viremia was observed in the blood or saliva. cART-treated cats exhibited a Th2 immunophenotype with increasing proportions of CD4⁺CCR4⁺ cells compared to placebo cats, and cART restored Th17 cells compared to placebo-treated cats. Of the cART drugs, dolutegravir was the most stable and long-lasting. These findings provide a critical insight into novel cART formulations in FIV-infected cats and highlight their role as a potential animal model to evaluate the impact of cART on lentiviral infection and immune dysregulation.

There is a complex interplay between viral infection and host innate immune response regarding disease severity and outcomes. Neutrophil hyperactivation, including excessive release of neutrophil extracellular traps (NETs), is linked to exacerbated disease in acute COVID-19, notably in hospitalized patients. Delineating protective versus detrimental neutrophil responses is essential to developing targeted COVID-19 therapies and relies on high-quality translational animal models. In this study, we utilize a previously established feline model for COVID-19 to investigate neutrophil dysfunction in which experimentally infected cats develop clinical disease that mimics acute COVID-19. Specific pathogen-free cats were inoculated with SARS-CoV-2 (B.1.617.2; Delta variant) (n = 24) or vehicle (n = 6). Plasma, bronchoalveolar lavage fluid, and lung tissues were collected at various time points over 12 days post-inoculation. Systematic and temporal evaluation of the kinetics of neutrophil activation was conducted by measuring markers of activation including myeloperoxidase (MPO), neutrophil elastase (NE), and citrullinated histone H3 (citH3) in SARS-CoV-2-infected cats at 4 and 12 days post-inoculation (dpi) and compared to vehicle-inoculated controls. Cytokine profiling supported elevated innate inflammatory responses with specific upregulation of neutrophil activation and NET formation-related markers, namely IL-8, IL-18, CXCL1, and SDF-1, in infected cats. An increase in MPO-DNA complexes and cell-free dsDNA in infected cats compared to vehicle-inoculated was noted and supported by histopathologic severity in respiratory tissues. Immunofluorescence analyses further supported correlation of NET markers with tissue damage, especially 4 dpi. Differential gene expression analyses indicated an upregulation of genes associated with innate immune and neutrophil activation pathways. Transcripts involved in activation and NETosis pathways were upregulated by 4 dpi and downregulated by 12 dpi, suggesting peak activation of neutrophils and NET-associated markers in the early acute stages of infection. Correlation analyses conducted between NET-specific markers and clinical scores as well as histopathologic scores support association between neutrophil activation and disease severity during SARS-CoV-2 infection in this model. Overall, this study emphasizes the effect of neutrophil activation and NET release in SARS-CoV-2 infection in a feline model, prompting further investigation into therapeutic strategies aimed at mitigating excessive innate inflammatory responses in COVID-19.

Cytauxzoonosis is a tick-borne infectious disease affecting domestic cats with high mortality and limited treatment modalities. Because early diagnosis and therapeutic intervention are crucial to survival of infected cats, the objective of this study was to develop an ELISA capable of detecting cytauxzoonosis and differentiating acute vs. chronic infection in clinical feline blood samples. A microsphere immunoassay (MIA) was developed to evaluate the production of Cytauxzoon felis-specific IgM and IgG antibodies in serial plasma samples from cats with experimental C. felis infection by targeting a C. felis-specific transmembrane protein (c88). Recombinant c88 protein was utilized to develop indirect ELISAs to detect IgM and IgG antibodies in clinical plasma samples from: PCR-positive cats with acute C. felis infection (n = 36), C. felis-negative cats with pyrexia (n = 10), healthy C. felis-negative cats (n = 22), and chronic C. felis carriers (n = 4). Anti-c88 IgM antibodies were detectable at day 12 post-tick infestation in cats with experimental C. felis infection (within 24 hours of developing clinical signs), while anti-c88 IgG was detectable at day 15 post-tick infestation – indicating IgM could be used to detect early infection. Using a cut-off value of 19.85 percent positive, the C. felis IgM ELISA detected acute cytauxzoonosis in 94.44% (34/36) of cats presented with clinical signs of acute cytauxzoonosis with 100% specificity (indicating a “Strong Positive” result). When a lower cutoff of 8.60 percent positive was used, cytauxzoonosis was detected in the 2 remaining PCR-positive cats with 87.88% specificity (indicating of a “Weak Positive” result). One C. felis-negative, febrile cat had high IgG, and chronic carriers had variable IgM and IgG results. Combined interpretation of IgM and IgG ELISAs did not reliably differentiate acute vs. chronic infection. While further validation on assay performance is needed, the C. felis IgM ELISA is a promising test to detect acute cytauxzoonosis and can be utilized to develop a point-of-care test for clinical use.

Cytauxzoonosis is caused by Cytauxzoon felis (C. felis), a tick-borne parasite that causes severe disease in domestic cats in the United States. Currently, there is no vaccine to prevent this fatal disease, as traditional vaccine development strategies have been limited by the inability to culture this parasite in vitro. Here, we used a replication-defective human adenoviral vector (AdHu5) to deliver C. felis-specific immunogenic antigens and induce a cell-mediated and humoral immune response in cats. Cats (n = 6 per group) received either the vaccine or placebo in two doses, 4 weeks apart, followed by experimental challenge with C. felis at 5 weeks post-second dose. While the vaccine induced significant cell-mediated and humoral immune responses in immunized cats, it did not ultimately prevent infection with C. felis. However, immunization significantly delayed the onset of clinical signs and reduced febrility during C. felis infection. This AdHu5 vaccine platform shows promising results as a vaccination strategy against cytauxzoonosis.