Selective targeting of chemically modified miR-34a to prostate cancer using a small molecule ligand and an endosomal escape agent.

Use of tumor-suppressive microRNAs (miRNAs) as anti-cancer agents is hindered by the lack of effective delivery vehicles, entrapment of the miRNA within endocytic compartments, and rapid degradation of miRNA by nucleases. To address these issues, we developed a miRNA delivery strategy that includes (1) a targeting ligand, (2) an endosomal escape agent, nigericin and (3) a chemically modified miRNA. The delivery ligand, DUPA (2-[3-(1,3-dicarboxy propyl) ureido] pentanedioic acid), was selected based on its specificity for prostate-specific membrane antigen (PSMA), a receptor routinely upregulated in prostate cancer-one of the leading causes of cancer death among men. DUPA was conjugated to the tumor suppressive miRNA, miR-34a (DUPA-miR-34a) based on the ability of miR-34a to inhibit prostate cancer cell proliferation. To mediate endosomal escape, nigericin was incorporated into the complex, resulting in DUPA-nigericin-miR-34a. Both DUPA-miR-34a and DUPA-nigericin-miR-34a specifically bound to, and were taken up by, PSMA-expressing cells in vitro and in vivo. And while both DUPA-miR-34a and DUPA-nigericin-miR-34a downregulated miR-34a target genes, only DUPA-nigericin-miR-34a decreased cell proliferation in vitro and delayed tumor growth in vivo. Tumor growth was further reduced using a fully modified version of miR-34a that has significantly increased stability.

Development of NP-Based Universal Vaccine for Influenza A Viruses.

The nucleoprotein (NP) is a vital target for the heterosubtypic immunity of CD8+ cytotoxic T lymphocytes (CTLs) due to its conservation among influenza virus subtypes. To further enhance the T cell immunity of NP, autophagy-inducing peptide C5 (AIP-C5) from the CFP10 protein of Mycobacterium tuberculosis was used. Mice were immunized intranasally (i.n.) with human adenoviral vectors, HAd-C5-NP(H7N9) or HAd-NP(H7N9), expressing NP of an H7N9 influenza virus with or without the AIP-C5, respectively. Both vaccines developed similar levels of NP-specific systemic and mucosal antibody titers; however, there was a significantly higher number of NP-specific CD8 T cells secreting interferon-gamma (IFN-γ) in the HAd-C5-NP(H7N9) group than in the HAd-NP(H7N9) group. The HAd-C5-NP(H7N9) vaccine provided better protection following the challenge with A/Puerto Rico/8/1934(H1N1), A/Hong Kong/1/68(H3N2), A/chukkar/MN/14951-7/1998(H5N2), A/goose/Nebraska/17097/2011(H7N9), or A/Hong Kong/1073/1999(H9N2) influenza viruses compared to the HAd-NP(H7N9) group. The autophagy transcriptomic gene analysis of the HAd-C5-NP(H7N9) group revealed the upregulation of some genes involved in the positive regulation of the autophagy process. The results support further exploring the use of NP and AIP-C5 for developing a universal influenza vaccine for pandemic preparedness.

Hemotropic Mycoplasmas (Hemoplasmas) in Free-Ranging Azara's Agoutis (Dasyprocta azarae) from an Urban Area of Southern Brazil.

Hemotropic mycoplasmas (hemoplasmas) are opportunistic bacteria that attach to the erythrocyte surface, causing infectious anemia in several mammalian species, including rodents. Studies surveying native Azara's agoutis (Dasyprocta azarae) in Brazil are lacking. Accordingly, the present study aimed to assess hemoplasmas infection in free-ranging agoutis from an urban environmental conservation area in Curitiba, southern Brazil. Overall, 11/35 (31.43%) agoutis were positive to hemoplasmas by quantitative PCR (cycle threshold≤34.4). Sequencing of the 16S ribosomal RNA gene indicated Mycoplasma haemomuris infection, closely related to M. haemomuris subsp. ratti, suggesting hemoplasma transmission from urban rats to agoutis. Because the main route of M. haemomuris transmission has been direct rodent-to-rodent infection, the relatively lower positivity that we detected may be the result of low intraspecies contact due to the smaller social units of agoutis, generally consisting of two to four individuals, and low interspecies contact due to only sporadic agouti-rat interactions in urban settings, compared with other rodent species interactions. Further studies should be conducted to determine whether the hemoplasma infection that we found can cause clinical onset and life-threatening anemia in agoutis.

Effects of intravenous administration of ascorbic acid (vitamin C) on oxidative status in healthy adult horses.

BACKGROUND: Ascorbic acid (AA) is an antioxidant that might be beneficial for adjunctive treatment of sepsis in horses. The optimal dose and effects on oxidative status are unknown. HYPOTHESIS: Ascorbic acid administration will increase plasma AA concentrations and decrease determinants of reactive oxygen metabolites (dROM), basal and stimulant-induced intraerythrocytic reactive oxygen species (ROS) concentrations, and stimulant-induced neutrophil ROS production, and increase plasma antioxidant capacity (PAC) in a dose-dependent manner. ANIMALS: Eight healthy horses. METHODS: Randomized placebo-controlled crossover study. Each horse received 4 single-dose IV treatments including AA at 25, 50, and 100 mg/kg and saline (placebo) with each treatment separated by ≥1 week. Blood was collected at baseline, 2 and 6 hours for assessment of plasma dROM and PAC via photometer, intraerythrocytic ROS by flow cytometry, and stimulant-induced neutrophil ROS by a fluorometric assay. Plasma AA concentrations were measured by high-performance liquid chromatography/electrochemical detection. RESULTS: Ascorbic acid at 100 mg/kg resulted in decreased dROM 2 hours after treatment (P = .03, 95% CI 5.51-121.2, point estimate 63.3). There was no effect of AA on basal or stimulant-induced intraerythrocytic ROS (P = .88, 95% CI -0.156 to 0.081, point estimate -0.037; P = .93, 95% CI -0.123 to 0.112, point estimate -0.006, respectively), basal or stimulant-induced neutrophil ROS (P ≥ .12, 95% CI -644.9 to 56.2, point estimate -294.4), or PAC (P ≥ .64, 95% CI -1567 to 463.4, point estimate -552.0) at any dose or timepoint. Plasma AA concentrations increased in a dose-dependent manner. CONCLUSIONS AND CLINICAL IMPORTANCE: High-dose administration of AA might provide antioxidant benefits in horses.

Marked paraneoplastic basophilia in a cat with alimentary T-cell lymphoma.

An 8-year-old, spayed female domestic shorthair cat was presented for acute weight loss, hyporexia, intermittent vomiting, and loose stools. A caudal abdominal mass and thickened intestinal loops were palpated on initial examination. An abdominal ultrasound identified a circumferential intramural jejunal mass with complete loss of wall layering, diffuse thickening of the jejunal muscularis, and jejunal and ileocecal lymphadenomegaly. Initial routine bloodwork revealed mild monocytosis and minimal lymphopenia with reactive lymphocytes. Cytologic evaluation of the jejunal mass and enlarged lymph nodes was consistent with lymphoma (intermediate cell size), and PCR for antigen receptor rearrangement revealed a clonal T-cell receptor rearrangement consistent with T-cell lymphoma. Chemotherapy (CHOP protocol) was initiated, but despite initial improvement of clinical signs, a repeat ultrasound examination 5 weeks after initiation of treatment revealed no improvement in the lymphadenomegaly or reduction in the size of the jejunal mass. At this visit, the cat also developed a marked basophilia (basophils 12.28 × 103 /µL, RI 0.00-0.10) with low numbers of circulating atypical lymphocytes; no concurrent eosinophilia was noted. Heartworm disease, ectoparasites, and allergic diseases were evaluated for and considered unlikely. The chemotherapy protocol was changed to L-asparaginase, followed by lomustine. The basophilia was significantly reduced 2 days after the initial dose of L-asparaginase and remained within the reference interval for 40 days before an eventual decline in the cat's health. To the authors' knowledge, this is the first report of paraneoplastic basophilia without concurrent eosinophilia in a cat with T-cell lymphoma.

miRNome expression analysis in canine diffuse large B-cell lymphoma.

Introduction: Lymphoma is a common canine cancer with translational relevance to human disease. Diffuse large B-cell lymphoma (DLBCL) is the most frequent subtype, contributing to almost fifty percent of clinically recognized lymphoma cases. Identifying new biomarkers capable of early diagnosis and monitoring DLBCL is crucial for enhancing remission rates. This research seeks to advance our knowledge of the molecular biology of DLBCL by analyzing the expression of microRNAs, which regulate gene expression by negatively impacting gene expression via targeted RNA degradation or translational repression. The stability and accessibility of microRNAs make them appropriate biomarkers for the diagnosis, prognosis, and monitoring of diseases. Methods: We extracted and sequenced microRNAs from ten fresh-frozen lymph node tissue samples (six DLBCL and four non-neoplastic). Results: Small RNA sequencing data analysis revealed 35 differently expressed miRNAs (DEMs) compared to controls. RT-qPCR confirmed that 23/35 DEMs in DLBCL were significantly upregulated (n = 14) or downregulated (n = 9). Statistical significance was determined by comparing each miRNA's average expression fold-change (2-Cq) between the DLCBL and healthy groups by applying the unpaired parametric Welch's 2-sample t-test and false discovery rate (FDR). The predicted target genes of the DEMs were mainly enriched in the PI3K-Akt-MAPK pathway. Discussion: Our data point to the potential value of miRNA signatures as diagnostic biomarkers and serve as a guideline for subsequent experimental studies to determine the targets and functions of these altered miRNAs in canine DLBCL.

Impact of an autophagy-inducing peptide on immunogenicity and protection efficacy of an adenovirus-vectored SARS-CoV-2 vaccine.

Because of continual generation of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is critical to design the next generation of vaccines to combat the threat posed by SARS-CoV-2 variants. We developed human adenovirus (HAd) vector-based vaccines (HAd-Spike/C5 and HAd-Spike) that express the whole Spike (S) protein of SARS-CoV-2 with or without autophagy-inducing peptide C5 (AIP-C5), respectively. Mice or golden Syrian hamsters immunized intranasally (i.n.) with HAd-Spike/C5 induced similar levels of S-specific humoral immune responses and significantly higher levels of S-specific cell-mediated immune (CMI) responses compared with HAd-Spike vaccinated groups. These results indicated that inclusion of AIP-C5 induced enhanced S-specific CMI responses and similar levels of virus-neutralizing titers against SARS-CoV-2 variants. To investigate the protection efficacy, golden Syrian hamsters immunized i.n. either with HAd-Spike/C5 or HAd-Spike were challenged with SARS-CoV-2. The lungs and nasal turbinates were collected 3, 5, 7, and 14 days post challenge. Significant reductions in morbidity, virus titers, and lung histopathological scores were observed in immunized groups compared with the mock- or empty vector-inoculated groups. Overall, slightly better protection was seen in the HAd-Spike/C5 group compared with the HAd-Spike group.

Impact of Rehydration Following Systemic Dehydration on Vocal Fold Gene Expression.

OBJECTIVE: Biological data on the beneficial effects of vocal fold rehydration are lacking. This study aimed to examine the effects of acute systemic dehydration on vocal fold gene expression and determine whether rehydration would reverse these changes. METHODS: Male New Zealand White rabbits (N = 24, n = 8/group) provided the animal model. Systemic dehydration was induced by 5 days of water volume restriction. Rehydration was provided by ad-lib water for 3 days following dehydration. Euhydrated rabbits were used as the control group. Vocal fold tissue was dissected. Seventeen genes were selected based on physiological function and role in supporting vocal fold structure, oxidative stress, hemodynamics, and extracellular matrix turnover. Relative gene expression was assessed by RT-qPCR. RESULTS: Rehydration following systemic dehydration can modulate gene expression, with expression patterns suggesting that rehydration reverses dehydration-induced changes in over half of the tested genes. CLIC5 (chloride intracellular channel 5) and EFEMP1 (EGF containing fibulin extracellular matrix protein 1) genes were significantly upregulated in the dehydration group compared with the euhydrated control. A1BG (alpha-1B-glycoprotein) and IL1RAP (interleukin 1 receptor accessory protein) were downregulated by rehydration compared with the dehydration group. CONCLUSION: This study provides molecular evidence for a transcriptional response to rehydration following acute systemic dehydration in the vocal folds. These data are the first to study gene expression following realistic dehydration and rehydration paradigms and provide biological data to support clinical recommendations to increase water intake after acute dehydration. LEVEL OF EVIDENCE: NA Laryngoscope, 133:3499-3505, 2023.

Hemophagocytic syndrome in a cat with Mycoplasma haemofelis infection.

A six-year-old, castrated male domestic shorthair cat was presented for a week-long history of lethargy, acute anorexia, and adipsia. On presentation, the cat was weak with pale mucous membranes, open-mouth breathing, and mild popliteal lymphadenomegaly. Routine bloodwork revealed bicytopenia due to marked non-regenerative anemia and moderate thrombocytopenia; erythrocyte clumping was apparent on the blood smear, but no agglutination was noted on a saline dispersion test. Abdominal and thoracic imaging showed marked splenomegaly and multiple mildly enlarged lymph nodes. Aspirates from the bone marrow and spleen contained many erythrophagocytic macrophages and occasional lymphocytes containing engulfed erythrocytes. The macrophages also occasionally contained phagocytosed erythroid precursors, platelets, and leukocytes. A diagnosis of hemophagocytic syndrome was made based on the presence of bicytopenia and increased numbers of hemophagocytic macrophages in the spleen and bone marrow. Though no organisms were observed, Mycoplasma spp. infection was suspected and confirmed via PCR. To the authors' knowledge, this is the first report of a hemophagocytic syndrome in a cat with Mycoplasma haemofelis. Lymphocyte engulfment of erythrocytes has been previously reported in a cat with M. haemofelis infection. Both hemophagocytic syndrome and engulfment of erythrocytes by lymphocytes should prompt testing for Mycoplasma spp. even with a lack of evident parasitemia.

Proteomic analysis reveals that aging rabbit vocal folds are more vulnerable to changes caused by systemic dehydration.

BACKGROUND: Older adults are more prone to develop systemic dehydration. Systemic dehydration has implications for vocal fold biology by affecting gene and protein expression. The objective of this study was to quantify vocal fold protein changes between two age groups and hydration status, and to investigate the interaction of age and hydration status on protein expression, which has not been investigated in the context of vocal folds before. Comparative proteomics was used to analyze the vocal fold proteome of 6.5-month-old and > 3-year-old rabbits subjected to water ad libitum or water volume restriction protocol. RESULTS: Young and older adult rabbits (n = 22) were either euhydrated (water ad libitum) or dehydrated by water volume restriction. Dehydration was confirmed by body weight loss of - 5.4% and - 4.6% in young and older groups, respectively, and a 1.7-fold increase of kidney renin gene expression in the young rabbits. LC-MS/MS identified 2286 proteins in the rabbit vocal folds of young and older adult rabbits combined. Of these, 177, 169, and 81 proteins were significantly (p ≤ 0.05) affected by age, hydration status, or the interaction of both factors, respectively. Analysis of the interaction effect revealed 32 proteins with opposite change patterns after dehydration between older and young rabbit vocal folds, while 31 proteins were differentially regulated only in the older adult rabbits and ten only in the young rabbits in response to systemic dehydration. The magnitude of changes for either up or downregulated proteins was higher in the older rabbits. These proteins are predominantly related to structural components of the extracellular matrix and muscle layer, suggesting a disturbance in the viscoelastic properties of aging vocal fold tissue, especially when subjected to systemic dehydration. CONCLUSIONS: Water restriction is a laboratory protocol to assess systemic dehydration-related changes in the vocal fold tissue that is translatable to human subjects. Our findings showed a higher number of proteins differentially regulated with a greater magnitude of change in the vocal folds of older adult rabbits in the presence of systemic dehydration compared to younger rabbits. The association of these proteins with vocal fold structure and biomechanical properties suggests that older human subjects may be more vulnerable to the effects of systemic dehydration on vocal function. The clinical implications of these protein changes warrant more investigation, but age should be taken into consideration when evaluating vocal treatment recommendations that interfere with body fluid balance.

The miRNome of canine invasive urothelial carcinoma.

Urothelial carcinoma (UC) comprises up to 2% of all naturally occurring neoplasia in dogs and can be challenging to diagnose. MicroRNAs (miRNAs) have been reported to be dysregulated in numerous diseases, including neoplasia. MiRNA expression has been evaluated in human UC, but there is limited information regarding the miRNA transcriptome of UC in dogs. Our study aimed to evaluate differential miRNA expression in bladder tissue collected from normal canine urothelium and canine invasive UC (iUC) to elucidate the dysregulated pathways in canine UC. Next-Generation RNA sequencing (RNA-Seq) was performed for dogs with UC (n = 29) and normal canine urothelium (n = 4). Raw RNA data were subjected to normalization, and pairwise comparison was performed using EdgeR with Benjamini-Hochberg FDR multiple testing correction (p < 0.05; >2-fold change) comparing tissue samples of normal urothelium to canine iUC samples. Principal component analysis and hierarchical cluster analysis were performed. MiRNA of FFPE tissue samples of separate iUC (n = 5) and normal urothelium (n = 5) were used to evaluate five miRNAs using RT-qPCR. Pathway analysis was performed utilizing miRWalk, STRING database, and Metascape utilizing KEGG pathways and GO terms databases. Twenty-eight miRNAs were differentially expressed (DE) by RNA-Seq. RT-qPCR confirmed that four miRNAs are significantly downregulated in UC compared to healthy urothelial samples (miR-105a, miR-143, miR-181a, and miR-214). Principal component analysis and hierarchical cluster analysis showed separation between miRNAs in iUC and the control group. The DE miRNAs are most often associated with gene silencing by miRNA, miRNAs in cancer, and miRNAs involved in DNA damage responses. Proteins involved include HRAS, KRAS, ARAF, RAF1, MAPK1, MAP2K1, MAPK3, FGFR3, EGFR, HBEGF, RASSF1, E2F2, E2F3, ERBB2, SRC, MMP1, and UP3KA. The differential expression of miRNAs in canine iUC compared to normal canine urothelial tissue indicates that these markers should be further evaluated for their potential role as diagnostic and therapeutic targets.

Nested PCR Detection of Pythium sp. from Formalin-Fixed, Paraffin-Embedded Canine Tissue Sections.

Pythium insidiosum is an infectious oomycete affecting dogs that develop the cutaneous or gastrointestinal form of pythiosis with a poor prognosis. If left untreated, pythiosis may be fatal. This organism is not a true fungus because its cell wall and cell membrane lack chitin and ergosterol, respectively, requiring specific treatment. Identifying the organism is challenging, as a hematoxylin and eosin (H&E) stain poorly stain the P. insidiosum hyphae and cannot be differentiated conclusively from other fungal or fungal-like organisms (such as Lagenidium sp.) morphologically. Our study aimed to develop a nested PCR to detect P. insidiosum and compare it with the traditional histopathologic detection of hyphae. Formalin-fixed, paraffin-embedded (FFPE) tissue scrolls from 26 dogs with lesions suggesting the P. insidiosum infection were assessed histologically, and DNA was extracted from the FFPE tissue sections for nested PCR. Agreement between the histologic stains, (H&E), periodic acid-Schiff (PAS), and/or Grocott methenamine silver (GMS) and the nested PCR occurred in 18/26 cases. Hyphae consistent with Pythium sp. were identified via histopathology in 57.7% of the samples, whereas the nested PCR detected P. insidiosum in 76.9% of samples, aiding in the sensitivity of the diagnosis of pythiosis in dogs. Using this combination of techniques, we report 20 canine cases of pythiosis over 18 years in Indiana and Kentucky, an unexpectedly high incidence for temperate climatic regions. Using a combination of histopathology evaluation and nested PCR is recommended to aid in the accurate diagnosis of pythiosis.

Effects of intravenous administration of peripheral blood-derived mesenchymal stromal cells after infusion of lipopolysaccharide in horses.

BACKGROUND: A systemic and dysregulated immune response to infection contributes to morbidity and mortality associated with sepsis. Peripheral blood-derived mesenchymal stromal cells (PB-MSC) mitigate inflammation in animal models of sepsis. Allogeneic PB-MSC administered IV to horses is well-tolerated but therapeutic benefits are unknown. HYPOTHESIS: After IV lipopolysaccharide (LPS) infusion, horses treated with PB-MSC would have less severe clinical signs, clinicopathological abnormalities, inflammatory cytokine gene expression, and oxidative stress compared to controls administered a placebo. ANIMALS: Sixteen horses were included in this study. METHODS: A randomized placebo-controlled experimental trial was performed. Sixteen healthy horses were assigned to 1 of 2 treatment groups (1 × 109 PB-MSC or saline placebo). Treatments were administered 30 minutes after completion of LPS infusion of approximately 30 ng/kg. Clinical signs, clinicopathological variables, inflammatory cytokine gene expression, and oxidative stress markers were assessed at various time points over a 24-hour period. RESULTS: A predictable response to IV LPS infusion was observed in all horses. At the dose administered, there was no significant effect of PB-MSC on clinical signs, clinicopathological variables, or inflammatory cytokine gene expression at any time point. Antioxidant potential was not different between treatment groups, but intracellular ROS increased over time in the placebo group. Other variables that changed over time were likely due to effects of IV LPS infusion. CONCLUSIONS AND CLINICAL IMPORTANCE: Administration of allogeneic PB-MSC did not cause clinically detectable adverse effects in healthy horses. The dose of PB-MSC used here is unlikely to exert a beneficial effect in endotoxemic horses.

A Rare Case of Myelin Oligodendrocyte Glycoprotein Antibody-Associated Transverse Myelitis in a 40-Year-Old Patient With COVID-19.

The coronavirus disease 2019 (COVID-19) includes an extensive spectrum of clinical manifestations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Previous studies have shown that SARS-CoV-2 often exhibits central nervous system (CNS) manifestations, including encephalitis, meningitis, and spinal cord pathologies. To date, few cases of COVID-19-associated transverse myelitis (TM) have been described. A 40-year-old unvaccinated man with no significant medical history presented to the emergency department complaining of fever, worsening burning sensation in his lower extremities, unsteady gait, and difficulty initiating urination for five days. Twelve days before presentation, the patient had tested positive for SARS-CoV-2 infection. Physical examination revealed hyperesthesia, starting around the nipple line (T4) and extending distally, involving the lower extremities, accompanied by symmetric weakness in the lower extremities. Magnetic resonance imaging of the thoracic spine with and without contrast revealed mild intramedullary signal abnormality at T3-T4 and T6-T8, confirming the suspicion of TM. Further laboratory testing revealed a C-reactive protein level of 67 mg/L, lactate dehydrogenase level of 181 mg/L, serum B12 level of 781 pg/mL, methylmalonic acid level of 165 nmol/L, folate of >24.5 ng/mL, and thyroid-stimulating hormone level of 0.481 µIU/L. Lumbar puncture was performed, and cerebrospinal fluid analysis revealed a cell count of 14 cells/µL, with 69% lymphocytes, glucose level of 81 mg/dL, protein level of 32 mg/dL, and negative cultures. Human immunodeficiency virus, antinuclear antibody screening, anti-DNA, rapid plasma reagin, Lyme serology, anti-SSA, and anti-SSB antibodies were unremarkable. Serum aquaporin-4 immunoglobulin G was negative, and myelin oligodendrocyte glycoprotein (MOG) antibodies were positive. The patient was treated with intravenous methylprednisolone and oral gabapentin and was discharged after five days when his urinary retention improved. Most previously reported cases of COVID-19-related TM were negative for autoimmune workup. Although the exact pathophysiology of COVID-19-related TM remains unclear, one hypothesis suggests that it is a consequence of the direct viral invasion. However, our patient had MOG antibodies, suggesting the possible involvement of a different mechanism. In MOG-associated TM, it has been suggested that MOG antibodies gain access to the CNS through disruption of the blood-brain barrier. This unique presentation demonstrates that further studies are needed to understand the effects of SARS-CoV-2 infection on the immune and nervous systems. It also highlights that young and otherwise healthy patients are at risk of severe COVID-19-related complications, including CNS disorders.

First reported cases of SARS-CoV-2 infection in pets in São Paulo, Brazil.

Background and Aim: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the global coronavirus pandemic (COVID-19) in humans in 2019. Although SARS-CoV-2 infection is primarily asymptomatic and transitory in companion animals, the role of these animals in the life cycle of the virus remains unclear. This study aimed to survey the first SARS-CoV-2 infection cases in pets, including a dog and three cats in São Paulo, Brazil. Materials and Methods: We invited COVID-19-positive pet owners to participate in the survey and obtained nasal, oropharyngeal, and rectal swab samples from their pets. These samples were placed in vials and subjected to a real-time quantitative polymerase chain reaction. In addition, the owners answered an epidemiological questionnaire, and the pets underwent clinical examination and monitoring. Results: Out of 49 sampled pets, 3/19 (15.8%) cats and 1/30 (3.3%) dogs tested positive, with wide variations in viral loads. Despite the limitations of size and non-randomized sampling, our results showed that cats are more susceptible than dogs to SARS-CoV-2 infection, presenting a cat: dog ratio of 4.8: 1. Only one cat presented mild and transitory respiratory symptoms. Conclusion: Although SARS-CoV-2 infection was detected in pets in the largest South American city and the COVID-19 epicenter at the time, these first detected pet cases displayed either none or mild clinical signs.

Incidental diagnosis of a spindle cell type gastrointestinal stromal tumor in a dog with ethylene glycol intoxication.

A 6-year-old castrated male American Pit Bull Terrier dog was presented for evaluation of acute onset of tonic-clonic seizures, anorexia, and vomiting. On physical examination, neurologic signs, such as generalized proprioceptive ataxia, salivation, circling to the right, and absent patellar reflexes bilaterally, were noted. A complete blood cell count revealed mild hemoconcentration and an inflammatory leukogram, while a chemistry panel showed severe azotemia, marked hypochloremia, and a severe titrational metabolic acidosis, suggesting possible ethylene glycol intoxication. However, an irregularly round, small mass was identified in the large intestine on abdominal ultrasound. Additionally, bilateral hyperechoic renal cortices with medullary rim sign were suggestive of acute nephritis or tubular necrosis. The cytologic evaluation of a fine-needle aspiration biopsy of the abdominal mass revealed a large population of mesenchymal cells, suggesting the presence of neoplasia. Due to the worsening of symptoms, the dog was humanely euthanized. Necropsy confirmed ethylene glycol intoxication, and the incidental finding of a neoplastic intestinal mass was diagnosed as spindle cell sarcoma. Immunohistochemical staining showed strong, diffuse positivity for CD117, smooth muscle actin, and S-100, indicating the final diagnosis of a spindle cell type gastrointestinal stromal tumor (GIST). This report briefly discusses the classifications of nonlymphoid, nonangiogenic intestinal mesenchymal tumors, characteristics of GISTs, and the importance of the immunohistochemical classification of mesenchymal tumors of the gastrointestinal tract.

Development and validation of a flow cytometric assay for detecting reactive oxygen species in the erythrocytes of healthy dogs.

OBJECTIVE: To validate the use of a flow cytometric assay that uses 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) to measure reactive oxygen species in the erythrocytes of healthy dogs. ANIMALS: 50 healthy adult dogs. PROCEDURES: Erythrocytes were incubated with DCFH-DA or a vehicle control (dimethyl sulfoxide), then incubated with (stimulated) or without (unstimulated) hydrogen peroxide. The flow cytometric assay was evaluated for specificity with increasing concentrations of DCFH-DA and hydrogen peroxide, and a polynomial regression line was applied to determine optimal concentrations. For precision, samples were analyzed 5 consecutive times for determination of intra- and interassay variability. Stability of samples stored at 4°C for up to 48 hours after blood collection was determined with flow cytometric analysis. Coefficient of variation (CV) was considered acceptable at 20%. Baseline measurements were used to determine an expected range of median fluorescence intensity for unstimulated erythrocytes incubated with DCFH-DA. RESULTS: Erythrocytes were successfully isolated, and stimulated samples demonstrated higher median fluorescence intensity, compared with unstimulated samples. The intra-assay CV was 11.9% and 8.9% and interassay CV was 11.9% and 9.1% for unstimulated and stimulated samples, respectively. Unstimulated samples were stable for up to 24 hours, whereas stimulated samples were stable for up to 48 hours. CONCLUSIONS AND CLINICAL RELEVANCE: Flow cytometry for the measurement of reactive oxygen species in the erythrocytes of healthy dogs by use of DCFH-DA had acceptable specificity, precision, and stability. Flow cytometry is a promising technique for evaluating intraerythrocytic oxidative stress for healthy dogs.

Leptospira spp. Antibody in Wild Boars (Sus scrofa), Hunting Dogs (Canis lupus familiaris), and Hunters of Brazil.

Hunting activities are a potential risk factor for human infection with Leptospira spp. and, although wild boar seroprevalence has been studied, there are no concurrent serosurveys of wild boars (Sus scrofa), hunting dogs (Canis lupus familiaris), and hunters. The aim of our study was to assess the seroprevalence of Leptospira spp. antibodies in free-ranging wild boars, hunting dogs, and hunters, and risk factors associated with exposure in southern and central-western Brazil. Leptospira spp. antibodies were serologically detected using the microscopic agglutination test, with a total 30 serovars. Overall, 12.2% (9/74) of wild boars and 10.6% (16/170) of hunting dogs were seropositive for at least one serovar and all hunters 0.0% (0/49) were seronegative for Leptospira spp. Seropositivity was statistically higher in 42.1% (8/19) wild boars from natural areas when compared to 2.4% (1/41) from anthropized areas (P<0.001), with prevalence ratio of 17.14 (95% confidence interval: 2.29-128.36). Despite the limited sample size, our findings showed that hunters may be less exposed to Leptospira spp. than are wild boars, particularly in natural areas where Leptospira spp. may be maintained by wild reservoirs. In addition to acting as sentinels, hunting dogs may play a role in disease transmission of sylvatic leptospiral serovars.