Spillover of highly pathogenic avian influenza H5N1 virus to dairy cattle.

The highly pathogenic avian influenza (HPAI) H5N1 virus clade 2.3.4.4b has caused the death of millions of domestic birds and thousands of wild birds in the USA since January 2022 (refs. 1-4). Throughout this outbreak, spillovers to mammals have been frequently documented5-12. Here we report spillover of the HPAI H5N1 virus to dairy cattle across several states in the USA. The affected cows displayed clinical signs encompassing decreased feed intake, altered faecal consistency, respiratory distress and decreased milk production with abnormal milk. Infectious virus and viral RNA were consistently detected in milk from affected cows. Viral distribution in tissues via immunohistochemistry and in situ hybridization revealed a distinct tropism of the virus for the epithelial cells lining the alveoli of the mammary gland in cows. Whole viral genome sequences recovered from dairy cows, birds, domestic cats and a raccoon from affected farms indicated multidirectional interspecies transmissions. Epidemiological and genomic data revealed efficient cow-to-cow transmission after apparently healthy cows from an affected farm were transported to a premise in a different state. These results demonstrate the transmission of the HPAI H5N1 clade 2.3.4.4b virus at a non-traditional interface, underscoring the ability of the virus to cross species barriers.

Lipid fingerprint-based histology accurately classifies nevus, primary melanoma, and metastatic melanoma samples.

Probably, the most important factor for the survival of a melanoma patient is early detection and precise diagnosis. Although in most cases these tasks are readily carried out by pathologists and dermatologists, there are still difficult cases in which no consensus among experts is achieved. To deal with such cases, new methodologies are required. Following this motivation, we explore here the use of lipid imaging mass spectrometry as a complementary tool for the aid in the diagnosis. Thus, 53 samples (15 nevus, 24 primary melanomas, and 14 metastasis) were explored with the aid of a mass spectrometer, using negative polarity. The rich lipid fingerprint obtained from the samples allowed us to set up an artificial intelligence-based classification model that achieved 100% of specificity and precision both in training and validation data sets. A deeper analysis of the image data shows that the technique reports important information on the tumor microenvironment that may give invaluable insights in the prognosis of the lesion, with the correct interpretation.

Validation of a rapid collagenase activity detection technique based on fluorescent quenched gelatin with synovial fluid samples.

BACKGROUND: Measuring collagenase activity is crucial in the field of joint health and disease management. Collagenases, enzymes responsible for collagen degradation, play a vital role in maintaining the balance between collagen synthesis and breakdown in joints. Dysregulation of collagenase activity leads to joint tissue degradation and diseases such as rheumatoid arthritis and osteoarthritis. The development of methods to measure collagenase activity is essential for diagnosis, disease severity assessment, treatment monitoring, and identification of therapeutic targets. RESULTS: This study aimed to validate a rapid collagenase activity detection technique using synovial fluid samples. Antibody microarray analysis was initially performed to quantify the levels of matrix metalloproteinase-9 (MMP-9), a major collagenase in joints. Subsequently, the developed gelatin-based test utilizing fluorescence measurement was used to determine collagenase activity. There was a significant correlation between the presence of MMP-9 and collagenase activity. In addition, Lower Limit of Detection and Upper Limit of Detection can be preliminary estimated as 8 ng/mL and 48 ng/mL respectively. CONCLUSIONS: The developed technique offers a potential point-of-care assessment of collagenase activity, providing real-time information for clinicians and researchers. By accurately quantifying collagenase activity, healthcare professionals can optimize patient care, improve treatment outcomes, and contribute to the understanding and management of joint-related disorders. Further research and validation are necessary to establish the full potential of this rapid collagenase activity detection method in clinical practice.

Impact of membrane lipid polyunsaturation on dopamine D2 receptor ligand binding and signaling.

Increasing evidence supports a relationship between lipid metabolism and mental health. In particular, the biostatus of polyunsaturated fatty acids (PUFAs) correlates with some symptoms of psychiatric disorders, as well as the efficacy of pharmacological treatments. Recent findings highlight a direct association between brain PUFA levels and dopamine transmission, a major neuromodulatory system implicated in the etiology of psychiatric symptoms. However, the mechanisms underlying this relationship are still unknown. Here we demonstrate that membrane enrichment in the n-3 PUFA docosahexaenoic acid (DHA), potentiates ligand binding to the dopamine D2 receptor (D2R), suggesting that DHA acts as an allosteric modulator of this receptor. Molecular dynamics simulations confirm that DHA has a high preference for interaction with the D2R and show that membrane unsaturation selectively enhances the conformational dynamics of the receptor around its second intracellular loop. We find that membrane unsaturation spares G protein activity but potentiates the recruitment of ß-arrestin in cells. Furthermore, in vivo n-3 PUFA deficiency blunts the behavioral effects of two D2R ligands, quinpirole and aripiprazole. These results highlight the importance of membrane unsaturation for D2R activity and provide a putative mechanism for the ability of PUFAs to enhance antipsychotic efficacy.

Fermentation of whey-derived matrices by Kluyveromyces marxianus: alcoholic beverage development from whey and fruit juice mixes.

This research paper addresses the hypotheses that Kluyveromyces marxianus can be cultured with good alcohol production on different whey-derived matrices, and that the fermented product can be used in order to develop alcoholic beverages with acceptable sensory characteristics by mixtures with yeast-fermented fruit-based matrices. Growth and fermentative characteristics of Kluyveromyces marxianus LFIQK1 in different whey-derived matrices were explored by culturing (24 h, 30°C) on reconstituted whey, demineralized whey, heat-treated whey and milk permeate media. High lactose consumption, ethanol production and yield were observed. Reconstituted whey matrix was selected for mixing with orange or strawberry juices fermented using Saccharomyces cerevisiae to obtain alcoholic beverages (W-OR and W-ST, respectively). Consumer evaluation of beverages was performed using acceptability and Check-All-That-Apply (CATA) questions. Good acceptance was observed, significantly higher for W-ST than for W-OR. CATA questions gave information about organoleptic characteristics of beverages. Penalty analysis showed W-R and W-ST were positively associated with smooth/refreshing and fruity/natural, respectively. Liking was represented, accordingly with penalty analysis, by natural/refreshing. A novel alternative for utilization of whey and whey-related matrices by alcoholic beverages production with natural ingredients is presented.

Inflammatory Bowel Disease: A Comprehensive Analysis of Molecular Bases, Predictive Biomarkers, Diagnostic Methods, and Therapeutic Options.

In inflammatory bowel diseases (IBDs), such as Crohn's disease (CD) and ulcerative colitis (UC), the immune system relentlessly attacks intestinal cells, causing recurrent tissue damage over the lifetime of patients. The etiology of IBD is complex and multifactorial, involving environmental, microbiota, genetic, and immunological factors that alter the molecular basis of the organism. Among these, the microbiota and immune cells play pivotal roles; the microbiota generates antigens recognized by immune cells and antibodies, while autoantibodies target and attack the intestinal membrane, exacerbating inflammation and tissue damage. Given the altered molecular framework, the analysis of multiple molecular biomarkers in patients proves exceedingly valuable for diagnosing and prognosing IBD, including markers like C reactive protein and fecal calprotectin. Upon detection and classification of patients, specific treatments are administered, ranging from conventional drugs to new biological therapies, such as antibodies to neutralize inflammatory molecules like tumor necrosis factor (TNF) and integrin. This review delves into the molecular basis and targets, biomarkers, treatment options, monitoring techniques, and, ultimately, current challenges in IBD management.

Serological Antibodies against Kidney, Liver, and Spleen Membrane Antigens as Potential Biomarkers in Patients with Immune Disorders.

Immune disorders arise from complex genetic and environmental factors, which lead to dysregulation at the cellular and inflammatory levels and cause tissue damage. Recent research highlights the crucial role of reactive antibodies in autoimmune diseases and graft rejection, but their complex determination poses challenges for clinical use. Therefore, our study aimed to ascertain whether the presence of reactive antibodies against membrane antigens in tissues from both animal models and humans could serve as biomarkers in patients with autoimmune disorders. To address this issue, we examined the binding profile of serological antibodies against a diverse panel of cell membranes from the spleen, liver, and kidney tissues of monkeys, rats, and humans. After developing the cell membrane microarrays, human sera were immunologically assayed. The study was first conducted on sera from two groups, healthy subjects and patients with inflammatory and autoimmune disorders, and then optimized for kidney transplant patient sera. A significant increase in antibody reactivity against specific monkey kidney and spleen membranes was observed in the serum of patients with lupus nephritis, while kidney transplant patients showed a significant enhancement against human tissues and human embryonic kidney 293 cells. These results show the potential importance for clinical and basic research purposes of studying the presence of specific IgG against membrane antigens in patients' serum as potential biomarkers of immune disorders. However, it is important to note that these results need to be verified in further studies with a larger sample size to confirm their relevance.

From Lipid Signatures to Cellular Responses: Unraveling the Complexity of Melanoma and Furthering Its Diagnosis and Treatment.

Recent advancements in mass spectrometry have significantly enhanced our understanding of complex lipid profiles, opening new avenues for oncological diagnostics. This review highlights the importance of lipidomics in the comprehension of certain metabolic pathways and its potential for the detection and characterization of various cancers, in particular melanoma. Through detailed case studies, we demonstrate how lipidomic analysis has led to significant breakthroughs in the identification and understanding of cancer types and its potential for detecting unique biomarkers that are instrumental in its diagnosis. Additionally, this review addresses the technical challenges and future perspectives of these methodologies, including their potential expansion and refinement for clinical applications. The discussion underscores the critical role of lipidomic profiling in advancing cancer diagnostics, proposing a new paradigm in how we approach this devastating disease, with particular emphasis on its application in comparative oncology.

Neuroinflammation in the Evolution of Motor Function in Stroke and Trauma Patients: Treatment and Potential Biomarkers.

Neuroinflammation has a significant impact on different pathologies, such as stroke or spinal cord injury, intervening in their pathophysiology: expansion, progression, and resolution. Neuroinflammation involves oxidative stress, damage, and cell death, playing an important role in neuroplasticity and motor dysfunction by affecting the neuronal connection responsible for motor control. The diagnosis of this pathology is performed using neuroimaging techniques and molecular diagnostics based on identifying and measuring signaling molecules or specific markers. In parallel, new therapeutic targets are being investigated via the use of bionanomaterials and electrostimulation to modulate the neuroinflammatory response. These novel diagnostic and therapeutic strategies have the potential to facilitate the development of anticipatory patterns and deliver the most beneficial treatment to improve patients' quality of life and directly impact their motor skills. However, important challenges remain to be solved. Hence, the goal of this study was to review the implication of neuroinflammation in the evolution of motor function in stroke and trauma patients, with a particular focus on novel methods and potential biomarkers to aid clinicians in diagnosis, treatment, and therapy. A specific analysis of the strengths, weaknesses, threats, and opportunities was conducted, highlighting the key challenges to be faced in the coming years.

Immunisation with Neospora caninum subunits rsNcSAG4 and rsNcGRA1 (NcSAG4 and NcGRA1 epitopes construct) in BALB/c mice: the profile of the immune response and controlling the vertical transmission.

Neospora caninum is an apicomplexan protozoan that causes neosporosis, which has a high economic impact on cattle herds with no available vaccine. During infection, the secretion of dense granules and the expression of surface antigens play an important role in hosting immunomodulation. However, some epitopes of those antigens are immunogenic, and using these fractions could improve the subunit antigens in vaccine design. This study evaluates the recombinant peptides rsNcGRA1 and rsNcSAG4 derived from NcGRA1 and NcSAG4 native antigens as vaccine candidates produced by a fermentative process in the yeast culture system of Komagataella phaffii strain Km71, confirmed by colony PCR, SDS-PAGE, and western blotting. The assay was conducted in BALB/c mice using the peptides at low (25 µg) and standard (50 µg) dosages in monovalent and combined administrations at three time points with saponin as an adjuvant assessing the immunogenicity by antibodies response and cytokine production. We challenge the females after pregnancy confirmation using 2 × 105 NC-1 tachyzoites previously propagated in Vero cells. We assessed the chronic infection in dams and vertical transmission in the offspring by PCR and histopathology. Mice, especially those immunised with combined peptides and monovalent rsNcGRA1 at a standard dose, controlling the chronic infection in dams with the absence of clinical manifestations, showed an immune response with induction of IgG1, a proper balance between Th1/Th2 cytokines and reduced vertical transmission in the pups. In contrast, dams inoculated with a placebo vaccine showed clinical signs, low-scored brain lesions, augmented chronic infection with 80% positivity, 31% mortality in pups, and 81% vertical transmission. These findings indicate that rsNcGRA1 peptides in monovalent and combined with rsNCSAG4 at standard dose are potential vaccine candidates and improve the protective immune response against neosporosis in mice.

Microarrays, Enzymatic Assays, and MALDI-MS for Determining Specific Alterations to Mitochondrial Electron Transport Chain Activity, ROS Formation, and Lipid Composition in a Monkey Model of Parkinson's Disease.

Multiple evidences suggest that mitochondrial dysfunction is implicated in the pathogenesis of Parkinson's disease via the selective cell death of dopaminergic neurons, such as that which occurs after prolonged exposure to the mitochondrial electron transport chain (ETC) complex I inhibitor, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrine (MPTP). However, the effects of chronic MPTP on the ETC complexes and on enzymes of lipid metabolism have not yet been thoroughly determined. To face these questions, the enzymatic activities of ETC complexes and the lipidomic profile of MPTP-treated non-human primate samples were determined using cell membrane microarrays from different brain areas and tissues. MPTP treatment induced an increase in complex II activity in the olfactory bulb, putamen, caudate, and substantia nigra, where a decrease in complex IV activity was observed. The lipidomic profile was also altered in these areas, with a reduction in the phosphatidylserine (38:1) content being especially relevant. Thus, MPTP treatment not only modulates ETC enzymes, but also seems to alter other mitochondrial enzymes that regulate the lipid metabolism. Moreover, these results show that a combination of cell membrane microarrays, enzymatic assays, and MALDI-MS provides a powerful tool for identifying and validating new therapeutic targets that might accelerate the drug discovery process.

Screening System of Cannabis sativa Extracts Based on Their Mitochondrial Safety Profile Using Cytochrome c Oxidase Activity as a Biomarker

The development of Cannabis sativa strains with high cannabidiol (CBD) and low tetrahydrocannabinol (THC) content is a growing field of research, both for medical and recreational use. However, the mechanisms behind clinical actions of cannabinoids are still under investigation, although there is growing evidence that mitochondria play an important role in many of them. Numerous studies have described that cannabinoids modulate mitochondrial activity both through activation of mitochondrial cannabinoid receptors and through direct action on other proteins such as mitochondrial complexes involved in cellular respiration. Thus, the aim of this study was to determine the actions of a panel of extracts, isolated from high-CBD varieties of Cannabis sativa, on the activity of the mitochondrial electron transport chain complex IV, cytochrome c oxidase (CCO), in order to select those with a safer profile. After demonstrating that Cannabis sativa strains could be identified by cannabinoids content, concentration-response curves were performed with a collection of extracts from strains with high-CBD and low-THC content using bovine CCO. The CCO rate was clearly modified by specific extracts of Cannabis sativa plants compared to others. Half maximal inhibitory concentrations (IC50) of extracts and the inhibitory effects evoked at 1 × 10-4 g/mL displayed a significant correlation with the THC. Therefore, the screening of extracts based on CCO activity provides a powerful and rapid methodology to identify those plants with higher mitochondrial toxicity or even mito-protective actions.

Review of Technological Challenges in Personalised Medicine and Early Diagnosis of Neurodegenerative Disorders.

Neurodegenerative disorders are characterised by progressive neuron loss in specific brain areas. The most common are Alzheimer's disease and Parkinson's disease; in both cases, diagnosis is based on clinical tests with limited capability to discriminate between similar neurodegenerative disorders and detect the early stages of the disease. It is common that by the time a patient is diagnosed with the disease, the level of neurodegeneration is already severe. Thus, it is critical to find new diagnostic methods that allow earlier and more accurate disease detection. This study reviews the methods available for the clinical diagnosis of neurodegenerative diseases and potentially interesting new technologies. Neuroimaging techniques are the most widely used in clinical practice, and new techniques such as magnetic resonance imaging (MRI) and positron emission tomography (PET) have significantly improved the diagnosis quality. Identifying biomarkers in peripheral samples such as blood or cerebrospinal fluid is a major focus of the current research on neurodegenerative diseases. The discovery of good markers could allow preventive screening to identify early or asymptomatic stages of the neurodegenerative process. These methods, in combination with artificial intelligence, could contribute to the generation of predictive models that will help clinicians in the early diagnosis, stratification, and prognostic assessment of patients, leading to improvements in patient treatment and quality of life.

Characterization of the Antitumor Potential of Extracts of Cannabis sativa Strains with High CBD Content in Human Neuroblastoma.

Cannabis has been used for decades as a palliative therapy in the treatment of cancer. This is because of its beneficial effects on the pain and nausea that patients can experience as a result of chemo/radiotherapy. Tetrahydrocannabinol and cannabidiol are the main compounds present in Cannabis sativa, and both exert their actions through a receptor-mediated mechanism and through a non-receptor-mediated mechanism, which modulates the formation of reactive oxygen species. These oxidative stress conditions might trigger lipidic changes, which would compromise cell membrane stability and viability. In this sense, numerous pieces of evidence describe a potential antitumor effect of cannabinoid compounds in different types of cancer, although controversial results limit their implementation. In order to further investigate the possible mechanism involved in the antitumoral effects of cannabinoids, three extracts isolated from Cannabis sativa strains with high cannabidiol content were analyzed. Cell mortality, cytochrome c oxidase activity and the lipid composition of SH-SY5Y cells were determined in the absence and presence of specific cannabinoid ligands, with and without antioxidant pre-treatment. The cell mortality induced by the extracts in this study appeared to be related to the inhibition of the cytochrome c oxidase activity and to the THC concentration. This effect on cell viability was similar to that observed with the cannabinoid agonist WIN55,212-2. The effect was partially blocked by the selective CB1 antagonist AM281, and the antioxidant α-tocopherol. Moreover, certain membrane lipids were affected by the extracts, which demonstrated the importance of oxidative stress in the potential antitumoral effects of cannabinoids.

Acute Stress-Induced Changes in the Lipid Composition of Cow's Milk in Healthy and Pathological Animals.

Producers of milk and dairy products have been faced with the challenge of responding to European society's demand for guaranteed animal welfare production. In recent years, measures have been taken to improve animal welfare conditions on farms and evaluation systems have been developed to certify them, such as the Welfare Quality® protocol. Among the markers used for this purpose, acute phase proteins stand out, with haptoglobin being one of the most relevant. However, the diagnostic power of these tools is limited and more sensitive and specific technologies are required to monitor animal health status. Different factors such as diet, stress, and diseases modify the metabolism of the animals, altering the composition of the milk in terms of oligosaccharides, proteins, and lipids. Thus, in order to study oxidative-stress-associated lipids, a collection of well-characterized milk samples, both by veterinary diagnosis and by content of the acute stress biomarker haptoglobin, was analyzed by mass spectrometry and artificial intelligence. Two lipid species (sphingomyelin and phosphatidylcholine) were identified as potential biomarkers of health status in dairy cows. Both lipids allow for the discrimination of milk from sick animals and also milk from those with stress. Moreover, lipidomics revealed specific lipid profiles depending on the origin of the samples and the degree of freedom of the animals on the farm. These data provide evidence for specific lipid changes in stressed animals and open up the possibility that haptoglobin could also affect lipid metabolism in cow's milk.

Defects in efferent duct multiciliogenesis underlie male infertility in GEMC1-, MCIDAS- or CCNO-deficient mice.

GEMC1 and MCIDAS are geminin family proteins that transcriptionally activate E2F4/5-target genes during multiciliogenesis, including Foxj1 and Ccno Male mice that lacked Gemc1, Mcidas or Ccno were found to be infertile, but the origin of this defect has remained unclear. Here, we show that all three genes are necessary for the generation of functional multiciliated cells in the efferent ducts that are required for spermatozoa to enter the epididymis. In mice that are mutant for Gemc1, Mcidas or Ccno, we observed a similar spectrum of phenotypes, including thinning of the seminiferous tubule epithelia, dilation of the rete testes, sperm agglutinations in the efferent ducts and lack of spermatozoa in the epididymis (azoospermia). These data suggest that defective efferent duct development is the dominant cause of male infertility in these mouse models, and this likely extends to individuals with the ciliopathy reduced generation of multiple motile cilia with mutations in MCIDAS and CCNO.

A cannabinoid link between mitochondria and memory.

Cellular activity in the brain depends on the high energetic support provided by mitochondria, the cell organelles which use energy sources to generate ATP. Acute cannabinoid intoxication induces amnesia in humans and animals, and the activation of type-1 cannabinoid receptors present at brain mitochondria membranes (mtCB1) can directly alter mitochondrial energetic activity. Although the pathological impact of chronic mitochondrial dysfunctions in the brain is well established, the involvement of acute modulation of mitochondrial activity in high brain functions, including learning and memory, is unknown. Here, we show that acute cannabinoid-induced memory impairment in mice requires activation of hippocampal mtCB1 receptors. Genetic exclusion of CB1 receptors from hippocampal mitochondria prevents cannabinoid-induced reduction of mitochondrial mobility, synaptic transmission and memory formation. mtCB1 receptors signal through intra-mitochondrial Gαi protein activation and consequent inhibition of soluble-adenylyl cyclase (sAC). The resulting inhibition of protein kinase A (PKA)-dependent phosphorylation of specific subunits of the mitochondrial electron transport system eventually leads to decreased cellular respiration. Hippocampal inhibition of sAC activity or manipulation of intra-mitochondrial PKA signalling or phosphorylation of the Complex I subunit NDUFS2 inhibit bioenergetic and amnesic effects of cannabinoids. Thus, the G protein-coupled mtCB1 receptors regulate memory processes via modulation of mitochondrial energy metabolism. By directly linking mitochondrial activity to memory formation, these data reveal that bioenergetic processes are primary acute regulators of cognitive functions.

Malignancy Rates by Bethesda Subcategory in the Pediatric Population.

INTRODUCTION: While the Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) is the most widely used method for categorizing thyroid nodules, its applicability to children is often debated. We describe our institution's experience utilizing the TBSRTC and examine the rates of malignancy in our population. METHODS: We conducted a retrospective chart review of eligible patients undergoing primary thyroidectomy at a high-volume tertiary care pediatric hospital. All patients had pre-operative fine needle aspiration. RESULTS: Of the 112 patients in our cohort, 85 (76%) were female. The median age was 15.1 years. The patients were divided into groups based on the Bethesda categorization of the fine needle aspirations of their nodules. The percentages of patients whose resection specimens showed evidence of malignancy on the surgical pathology reports were recorded as follows: category I (n = 5): 20%, category II (n = 11): 0%, category III (n = 30): 17%, category IV (n = 13): 31%, category V (n = 17): 94% and category VI (n = 36): 100%. CONCLUSION: Our findings indicate that the malignancy rates at our institution are comparable to those reported by other high-volume studies. When compared with the 2017 TBSRTC data, we found that our results were similar in many categories, with the exception of categories I and V.

Yokenella regensburgei, a novel pathogen in farmed American alligators.

Increased acute mortality of farmed American alligators (Alligator mississippiensis) was observed in various pens from 2 different farms in Louisiana over 2 years (2019-2021). A total of 14 alligators from multiple events of increased mortality were subjected to postmortem investigations. Except for one alligator with acute neurologic signs, no premonitory signs were observed. All animals had pneumonia (14/14), coelomitis (14/14), and intravascular short Gram-negative bacilli (14/14). Myocarditis (13/14) was common. Yokenella regensburgei was isolated from all alligators tested (13/13). These data suggest the respiratory tract may be a primary target system and could be involved in transmission, either through exhaled bacteria or through swallowing of contaminated respiratory fluids with passage through the feces. Available sensitivity data for Y. regensburgei in this study indicates in vitro sensitivity to aminoglycosides, fluoroquinolones, chloramphenicol, and trimethoprim/sulphamethoxazole antibiotics. Yokenella regensburgei should be included in the differential diagnosis of septicemia and acute death in alligators.

Investigation of SARS-CoV-2 infection and associated lesions in exotic and companion animals.

Documented natural infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in exotic and companion animals following human exposures are uncommon. Those documented in animals are typically mild and self-limiting, and infected animals have only infrequently died or been euthanized. Through a coordinated One Health initiative, necropsies were conducted on 5 animals from different premises that were exposed to humans with laboratory-confirmed SARS-CoV-2 infection. The combination of epidemiologic evidence of exposure and confirmatory real-time reverse transcriptase-polymerase chain reaction testing confirmed infection in 3 cats and a tiger. A dog was a suspect case based on epidemiologic evidence of exposure but tested negative for SARS-CoV-2. Four animals had respiratory clinical signs that developed 2 to 12 days after exposure. The dog had bronchointerstitial pneumonia and the tiger had bronchopneumonia; both had syncytial-like cells with no detection of SARS-CoV-2. Individual findings in the 3 cats included metastatic mammary carcinoma, congenital renal disease, and myocardial disease. Based on the necropsy findings and a standardized algorithm, SARS-CoV-2 infection was not considered the cause of death in any of the cases. Continued surveillance and necropsy examination of animals with fatal outcomes will further our understanding of natural SARS-CoV-2 infection in animals and the potential role of the virus in development of lesions.