Intranasal type I interferon treatment is beneficial only when administered before clinical signs onset in the SARS-CoV-2 hamster model.

Impaired type I interferons (IFNs) production or signaling have been associated with severe COVID-19, further promoting the evaluation of recombinant type I IFNs as therapeutics against SARS-CoV-2 infection. In the Syrian hamster model, we show that intranasal administration of IFN-α starting one day pre-infection or one day post-infection limited weight loss and decreased viral lung titers. By contrast, intranasal administration of IFN-α starting at the onset of symptoms three days post-infection had no impact on the clinical course of SARS-CoV-2 infection. Our results provide evidence that early type I IFN treatment is beneficial, while late interventions are ineffective, although not associated with signs of enhanced disease.

Quality of hematology and clinical chemistry results in laboratory and zoo nonhuman primates: Effects of the preanalytical phase. A review.

Most errors in clinical pathology originate in the preanalytical phase, which includes all steps from the preparation of animals and equipment to the collection of the specimen and its management until analyzed. Blood is the most common specimen collected in nonhuman primates. Other specimens collected include urine, saliva, feces, and hair. The primary concern is the variability of blood hematology and biochemistry results due to sampling conditions with the effects of capture, restraint, and/or anesthesia. Housing and diet have fewer effects, with the exception of food restriction to reduce obesity. There has been less investigation regarding the impact of sampling conditions of nonblood specimens.

Effect of needle gauge on pain and specimen quality of ultrasound-guided fine needle sampling without aspiration of the canine spleen.

Modifications of splenic parenchyma are common ultrasonographic findings in dogs. Splenic fine needle aspiration (FNA) is a rapid, safe procedure, routinely performed in veterinary institutions. However, 22-gauge (G) needle usually reported is selected according to general practice and the most appropriate needle size to be used remains unclear. The aim of this prospective, single-center, methods comparison study was to assess the effect of needle size on cytologic specimens' evaluation and animal welfare during the procedure. Dogs underwent ultrasound-guided splenic FNA using 23, 25, and 27G needles. Needles were compared based on initial and then detailed cytologic evaluation. The initial evaluation assessed overall cellularity, cell preservation, hemodilution, and detailed cytologic evaluation referred to exhaustive splenic components. Welfare evaluation was performed based on a scoring system. A total of 54 dogs were included in this study with 54 of 54 welfare evaluations and 35 of 54 cytologic evaluations by one or two European College of Veterinary Clinical Pathology-certified cytologists. The final cytologic diagnosis was unchanged regardless of the needle size. For the initial evaluation, 23G needles provided significantly higher cellularity than the 27G needles. For detailed cytologic evaluation, only the richness in mesothelial cells and stroma was affected by needle size. Pain induced by the procedures was considered low using 23, 25, and 27G needles with the 27G needle producing the least adverse reactions. Findings from the current study supported using needle gauges smaller than the previously published standard 22G needle for spleen ultrasound-guided fine needle nonaspiration in dogs. Due to higher cellularity and lower pain scores, authors recommend the use of 23G needles with a nonaspiration technique.

Fibrinogen deficiency in a dog - a case report.

BACKGROUND: Among coagulation disorders, primary fibrinogen deficiency is very rare in dogs. It is divided into hypofibrinogenemia, afibrinogenemia and dysfibrinogenemia. Afibrinogenemia has been described in three dogs. There are, however, no published case reports of primary hypofibrinogenemia in dogs. CASE PRESENTATION: A 1.5 year-old male German Pointer dog was evaluated for a locked-jaw syndrome associated with eye protrusion which appeared after a minor head trauma. Three months before the trauma, a persistent increase in coagulation times was detected by the referring veterinarian after a strong suspicion of snake envenomation. Apart for the primary complaint, physical examination was normal. A complete hemostatic profile revealed a moderately increased prothrombin time, activated partial thromboplastin times and a dramatically decreased fibrinogen concentration (0.34 g/L, reference interval [1.3-4.8 g/L]). Platelet count, plasma D-dimers and antithrombin, were all within the reference intervals and not consistent with a disseminated intravascular coagulation. Other possible causes of hypofibrinogenemia such as chronic hemorrhage and liver failure were excluded by laboratory work-up and imaging studies. Finally, antifibrinogen circulating anticoagulants were excluded using a dilution of citrated plasma from the pooled plasma of healthy dogs. These results supported a diagnosis of congenital fibrinogen deficiency and secondary retrobulbar hematoma and/or cellulitis. The dog's condition improved rapidly after symptomatic treatment with corticosteroids and antibiotics. At the 1 year follow-up, the dog was clinically normal but a persistent hypofibrinogenemia (≤ 0.8 g/L) remained. CONCLUSIONS: Various clinical presentations may occur in canine primary hypofibrinogenemia which should be included in the list of coagulation disorders. Diagnosis should include fibrinogen determination by coagulometric and non-coagulometric methods to differentiate from dysfibrinogenemia. There is no specific treatment but care should be taken to prevent bleeding and trauma. Emergency management of bleeding episodes with cryoprecipitate is the treatment of choice.

Alloantibodies against MHC class I: a novel mechanism of neonatal pancytopenia linked to vaccination.

Fetal/neonatal alloimmune thrombocytopenia is a frequent disease in humans where alloantibodies against platelet Ags lead to platelet destruction and hemorrhage. Although a role in the disease for Abs against MHC has been suspected, this has not been formally demonstrated. Since 2007, a hemorrhagic syndrome due to thrombocytopenia and designated as bovine neonatal pancytopenia (BNP) has been recognized in calves in several European countries. An inactivated antiviral vaccine is strongly suspected to be involved in this syndrome because of its highly frequent use in the dams of affected calves. In this study, we show that BNP is an alloimmune disease, as we reproduced the signs by transferring serum Abs from vaccinated BNP dams into healthy neonatal calves. Ab specificity was strongly associated with the presence of allogeneic MHC class I Abs in the dams. MHC class I staining was also observed on Madin-Darby bovine kidney cells, a cell line related to the one used to produce the vaccine Ag. Our report emphatically demonstrates that alloimmunization against MHC class I is associated with a substantial risk of developing cytopenia-associated syndromes in neonates when a cell line of the same species is used to produce an inactivated vaccine injected into the mother.

Waldenström's Macroglobulinemia in a Normoproteinemic Dog with Atypical Bimorphic Plasmacytoid Differentiation and Monoclonal Gammopathy.

A 2-year-old neutered female Small Munsterlander dog was presented for an insect bite. Physical examination revealed a poor body condition, a peripheral lymphadenomegaly, and suspected splenomegaly. A complete blood count (Sysmex XN-V) revealed marked leukocytosis with lymphocytosis and abnormal dot plots. An abnormal monomorphic lymphoid population and marked rouleaux formation were noted on the blood smear. Lymph node aspirates contained an atypical bimorphic population of lymphocytes, either with a plasmacytoid or a blastic appearance. This double population was also found in the spleen, liver, bone marrow, tonsils, and other tissues. Peripheral blood and lymph node clonality assays revealed clonal BCR gene rearrangement. Flow cytometry revealed a mixed population of small-sized B-cells (CD79a+ CD21+ MHCII+) and medium-sized B-cells (CD79a+ CD21- MHCII-) in lymph nodes and a dominant population of small-sized mature B-cells (CD21+ MHCII+) in peripheral blood. Though normoproteinemic, serum protein electrophoresis revealed an increased α2-globulin fraction with an atypical restricted peak, identified as monoclonal IgM by immunofixation. Urine protein immunofixation revealed a Bence-Jones proteinuria. A diagnosis of Waldenström's macroglobulinemia was made. Chemotherapy was initiated, but the dog was euthanized 12 months after the initial presentation due to marked clinical degradation.

Multifocal cutaneous phaeohyphomycosis caused by Exophiala spinifera with clinical resolution in an immunocompromised cat.

Case summary: A 3-year-old neutered domestic shorthair cat with a long history of idiopathic immune-mediated haemolytic anaemia and thrombocytopenia treated with ciclosporin and prednisolone was referred 2 months after the appearance of nodular dermatitis. A single pigmented nodule was present in the lateral carpal region of the right foreleg. The lesion was 7 mm in diameter, non-exudative and cutaneous to subcutaneous. Fine-needle aspiration of the mass revealed the presence of pigmented fungal elements. Excisional surgery was planned; in the meantime, a plaque-like lesion developed in the interorbital region. Histopathological examination confirmed the presumptive diagnosis of phaeohyphomycosis, and Exophiala spinifera was identified as the aetiological agent. Itraconazole, given orally at a dose of 10 mg/kg for 8 weeks following surgery, enabled clinical resolution despite continued use of immunosuppressants. The follow-up was carried out over 14 weeks. Relevance and novel information: This case report provides the first evidence of multifocal cutaneous phaeohyphomycosis caused by E spinifera with clinical resolution after combined surgical and itraconazole treatment in an immunocompromised cat.

Improving the Quality of EDTA-treated Blood Specimens from Mice.

Nonterminal blood sampling in laboratory mice is a very common procedure. With the goal of improving animal welfare, different sampling sites and methods have been compared but have not achieved a consensus. Moreover, most of these studies overlooked the quality of blood specimens collected. The main preanalytical concern with EDTA-treated blood specimens for hematology analyses is platelet aggregation, which is known to cause analytical errors. Our objective was to find a nonterminal blood sampling method with minimal adverse effects on mice and few or no platelet aggregates. We tested and compared 2 collection sites, 4 sampling methods, and 3 antithrombotic drugs in 80 C57BL6/j male and female mice by evaluating platelet aggregates on blood smears and platelet, WBC, and RBC counts. In addition, the blood collection process was carefully evaluated, and adverse effects were recorded. Platelet aggregation was lower in specimens collected from the jugular vein than from the facial vein, with no effect of the sampling device or the presence of an antithrombotic additive. Highly aggregated specimens were significantly associated with lower platelet counts, whereas aggregation had no effect on WBC or RBC counts. Adverse events during sampling were significantly associated with more numerous platelet aggregates. The jugular vein is thus a satisfactory sampling site in mice in terms of both animal welfare and low platelet aggregation. Using antithrombotic agents appears to be unnecessary, whereas improving sampling conditions remains a key requirement to ensure the quality of EDTA-treated blood specimens from mice.

Validation of the Sysmex XN-V hematology analyzer for canine specimens.

BACKGROUND: The Sysmex XN-V is derived from the new Sysmex XN series of human hematology analyzers. The main changes from the previously validated XT-2000iV analyzer include an optic-fluorescent analysis for platelets and nucleated RBC count. OBJECTIVE: We aimed to validate the Sysmex XN-V for canine blood according to American College for Veterinary Clinical Pathology and International Council for Standardization in Hematology recommendations. MATERIALS AND METHODS: Canine EDTA blood specimens and quality control material were analyzed on the Sysmex XN-V to evaluate imprecision, bias, linearity, a comparison with the XT-2000iV analyzer, interference effects, carry-over, and stability. We also verified previously established Sysmex XT-2000iV reference intervals (RIs). RESULTS: Imprecision and bias were low (<5%) for most variables. Observed total error was lower than allowable total error for most measured variables except lymphocytes and monocytes. Visually determined linearity was excellent for all variables, except for lymphocytes. The correlation between the XN-V and XT-2000iV analyzers was high (>0.93) for all variables except MCHC and reticulocyte indices. Correlations between the Sysmex XN-V and manual differential counts were good for neutrophils and eosinophils, acceptable for lymphocytes, and fair for monocytes. Hemolysis, lipemia, and to a lesser extent icterus, had significant effects on measured hemoglobin concentration and associated variables. Carry-over was not visually observed for any variable. Changes in the Sysmex XN-V measurements after storage at 4℃ and 24℃ were similar to those described for the Sysmex XT-2000iV analyzer. The previously established Sysmex XT-2000iV RIs can be used to interpret results from the Sysmex XN-V analyzer for most variables except red blood cell distribution width and mean platelet volume. CONCLUSIONS: The performance of the Sysmex XN-V analyzer was excellent and compared favorably with the Sysmex XT-2000iV analyzer.

Detection of circulating microfilariae in canine EDTA blood using lens-free technology: preliminary results.

Dirofilaria immitis causes life-threatening heart disease in dogs, thus screening of dog populations is important. Lens-free technology (LFT) is a low-cost imaging technique based on light diffraction that allows computerized recognition of small objects in holographic images. We evaluated an algorithm capable of recognizing microfilariae in canine whole blood using the LFT. We examined 3 groups of 10 EDTA blood specimens, from dogs with microfilaremia (group A), healthy dogs (B), and dogs with hematologic modifications other than microfilaremia (C). The LFT analyzer photographed repeated series of 5 images of all samples. The algorithm declared a sample positive if a microfilaria was detected on ≥1, ≥2, or ≥3 of the 5 images of a series. Microfilariae were detected visually in the images in 9 of 10 cases in group A; no microfilariae were seen in the images from groups B and C. Of the 30 cases, there were 14, 4, and only 3 false-positives with the 1 of 5, 2 of 5, and 3 of 5 image cutoffs, respectively. There were no false-negatives, regardless of cutoff. LFT seems useful for detecting microfilaria and could have application in clinical pathology.

Reference Values for Hematology, Plasma Biochemistry, Bone Marrow Cytology and Bone Histology of NOD.Cg-Prkdcscid Il2rgtm1Wjl/ SzJ Immunodeficient Mice.

Highly immunodeficient NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) are commonly used as a models in preclinical studies for patient-derived engraftment. However, despite the frequency of their use, reference values for their clinical pathology markers have not been determined. In accordance with the American Society of Veterinary Clinical Pathology (ASVCP) recommendations, we established de novo reference values for hematologic and biochemical variables and evaluated bone marrow cytology and histology in forty 9-wk-old male and female NSG mice. Hematologic analyses were performed using 2 separate analyzers (IDEXX ProCyte Dx, Sysmex XT-2000iV) and biochemical values were measured using a Scil VetScan2. The primary hematologic characteristic seen in NSG mice was a very low white blood cell (WBC) count (below 1.6 109/L). Lymphocyte and monocyte counts were respectively over- and under-estimated by the analyzers, as compared with manual counts, likely due to misidentification of the very low concentrations of these cell types by the analyzers. This analytical bias highlights the need for confirmatory microscopic observation of blood smears from these mice for WBC differential identification. Results for all other hematology and biochemistry variables were similar to those previously reported in inbred mice, except for MPV and an unexpectedly high glucose concentration (11.5 to 19.0 mmol/L), potentially due to the nonfasting status of the animals. The differential bone marrow cell count and Myeloid:Erythroid ratio (median 1.76) were also established. Megakaryocyte and adipocyte count differed significantly between the femoral diaphysis and metaphysis and between genders. These results provide a reliable resource of baseline data for hematologic variables for researchers monitoring graft rejection studies in NSG mice.

Canine reference intervals for coagulation markers using the STA Satellite(R) and the STA-R Evolution(R) analyzers.

The aim of the current study was to determine canine reference intervals for prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen, and antithrombin (AT) according to international recommendations. The STA Satellite(R) coefficients of variation of within-laboratory imprecision were 3.9%, 1.3%, 6.9%, and 5.1% for PT, APTT, fibrinogen, and AT, respectively. At 4 degrees C, citrated specimens were stable up to 8 hr for whole blood and 36 hr for plasma, except for APTT, which increased slightly (<1 sec). Nonparametric reference intervals determined in citrated plasma from 139 healthy fasting purebred dogs were 6.9-8.8 sec, 13.1-17.2 sec, 1.24-4.30 g/l, and 104-188% for PT, APTT, fibrinogen, and AT, respectively. Based on Passing-Bablok comparison between STA Satellite and STA-R Evolution(R) using 60 frozen specimens from a canine plasma bank, the corresponding reference intervals were transferred to the STA-R Evolution: 7.1-9.2 sec, 12.9-17.3 sec, 1.20-4.43 g/l, and 94-159% for PT, APTT, fibrinogen, and AT, respectively.

Multinodular Malignant Cutaneous Mast Cell Tumor in a Horse With Generalized Pruritus and Reactive Fibrosis: A Case Report.

Mast cell tumor (MCT) has long been considered as an uncommon neoplasm in horses. Cytological and behavioral evidence of its malignancy is usually lacking, and only a few reports have described MCT displaying malignant behavior. An 18-year-old Friesian stallion presented with a one-year history of intermittent and progressive skin lesions on the left forelimb associated with intense, generalized pruritus and apathy temporarily responsive to glucocorticoids and antibiotics. The horse was alert and responsive with poor body condition and marked generalized pruritus. The left forelimb was markedly enlarged and surrounded by numerous firm 2- to 20-cm masses that were ulcerated and focally necrotic. A 7-cm round firm mass was observed on the left dorsal neck. Dermatological examination revealed generalized moth-eaten alopecia and scaling with erosions and ulcers secondary to pruritus. A direct skin smear from the affected leg showed severe eosinophilic inflammation and neutrophilic inflammation with pleomorphic bacteria. Histopathology of the skin and biopsies of the underlying tissues revealed an abundant population of atypical mast cells consistent with a malignant MCT. The horse was euthanized and necropsy revealed a marked fibrous reaction on longitudinal sections of the affected limb, and the tumor could be detected on only a few histological slides. Diagnosis of equine MCT can be challenging because of the massive accompanying fibrous reaction. Mast cell tumor should be suspected in the presence of eosinophilic infiltration of the affected tissue and in cases of generalized pruritus not attributable to other causes.

Validation of the Medonic CA620/530 Vet 20-microl microcapillary sampler system for hematology testing of feline blood.

The aim of the current study was to compare feline hematologic variables in blood collected in microcapillary tubes (20 microl) and conventional blood tubes with the Medonic CA620/530 Vet in-house hematologic analyzer. A comparison of results obtained in 60 cats presented at the clinics of the veterinary school showed that the correlations between the 2 methods were 0.97 for white blood cell, 0.95 for red blood cell, and 0.93 for platelet counts; 0.92 for hemoglobin concentration; and 0.99 for mean corpuscular volume. No clinically relevant differences between the 2 blood sampling techniques were observed for any variable, which suggests that both techniques are interchangeable in cats. Moreover, microcapillary tubes would allow easier repeated sampling in the same cat and would likely be useful in other small species.

Reference values: a review.

Reference values are used to describe the dispersion of variables in healthy individuals. They are usually reported as population-based reference intervals (RIs) comprising 95% of the healthy population. International recommendations state the preferred method as a priori nonparametric determination from at least 120 reference individuals, but acceptable alternative methods include transference or validation from previously established RIs. The most critical steps in the determination of reference values are the selection of reference individuals based on extensively documented inclusion and exclusion criteria and the use of quality-controlled analytical procedures. When only small numbers of values are available, RIs can be estimated by new methods, but reference limits thus obtained may be highly imprecise. These recommendations are a challenge in veterinary clinical pathology, especially when only small numbers of reference individuals are available.

Abnormal Sysmex XT-2000iV DIFF scattergram in a cat with a prominent mastocytemia.

A 2-year-old neutered male domestic shorthair cat was presented to the emergency service of the National Veterinary School of Toulouse (France) for acute vomiting and diarrhea with lethargy, inappetence, and adypsia for the past 48 hours. Complete blood counts were performed with the ProCyte DX at the emergency department and with the Sysmex XT-2000iV at the laboratory 2 weeks later. The scattergrams from the two analyzers revealed similar unusual and abnormal dot plots. The Sysmex XT-2000iV DIFF scattergram also showed no clear separation between different leukocyte populations. The eosinophil cluster was in an abnormal location compared with that of the "typical" location in a normal cat. A blood smear evaluation revealed the presence of numerous mast cells. Thus, we hypothesized that the Sysmex XT-2000iV had detected the mast cell population, and this led to errors in the differential counts. To explore this hypothesis, we manually gated on the DIFF scattergram and performed a manual differential on the blood smear. With this new gating strategy, the Sysmex XT-2000iV and manual differentials were similar. Thus, in the case of systemic mastocytosis, mast cells can be located between the lymphocyte, monocyte, and eosinophil clusters on scattergrams.

The effects of storage at 4°C and 20°C on the hemograms of C57BL/6 mice and Wistar rats using the IDEXX ProCyte Dx and blood smear evaluations.

BACKGROUND: Delayed blood analysis might be unavoidable in laboratory practice, but little is known about rodent blood stability, especially cell morphology and scattergram results. OBJECTIVES: This study aimed to evaluate the stability of rodent blood cell counts and morphologies at different temperatures using the ProCyte Dx analyzer and performing manual observations. METHODS: Ten Wistar rats and 10 C57bl/6 mice were sampled on EDTA tubes and aliquoted for storage (4°C, 20°C). Hematologic analyses were performed immediately and at T6h, T24h, T48h (rats and mice), and T72h (rats only) after storage. RESULTS: In rats, at any temperature, red blood cell counts, hemoglobin concentrations (HGB), mean corpuscular hemoglobin (MCH) levels, and reticulocyte, white blood cell (WBC), eosinophil, and impedance platelet counts remained stable over time. The main changes were observed at 20°C for hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), and WBC differential counts. Optical platelet counts (PLT-O) and platelet variables underwent changes at both temperatures from T24h. In mice, red blood cell counts by impedance (RBC-I), MCH, and WBC, lymphocyte, eosinophil, and platelet counts, and plateletcrit (PCT) were stable over time and at all temperatures. As in rats, the most significant changes were observed at 20°C and concerned the optical RBC (RBC-O) counts, HCTs, MCVs, MCHCs, and reticulocyte, neutrophil, and monocyte counts. For both species, blood cell morphologies were altered from T24h at all temperatures, and platelet clumps were more numerous at 4°C. CONCLUSIONS: When rodent blood analyses need to be delayed, storage at 4°C is preferred and should not exceed 24 hours. PLT counts should be interpreted cautiously in refrigerated specimens with mandatory blood smear evaluations when abnormal scattergrams are observed.

Urinalysis and determination of the urine protein-to-creatinine ratio reference interval in healthy cows.

BACKGROUND: There are no reference intervals for urinalysis in cattle. HYPOTHESIS/OBJECTIVES: Characterize the urine of healthy cows, establish urine protein-to-creatinine ratio (UPC) reference intervals, and test possible differences among dairy and beef cattle, age groups, or stage of lactation. ANIMALS: Seventy-seven dairy and 74 beef 2.5 to 17 year-old cows of different breeds housed mainly in free stall. METHODS: In this prospective study, urine specimens were collected by catheterization. Complete urinalysis was performed within 1 hour including specific gravity, dipstick evaluation, visual urine pH evaluation with 0.3 pH unit graded strips, and microscopic evaluation of the sediment. Urinary protein and creatinine concentrations and protein electrophoresis were determined on frozen aliquots. RESULTS: Overall reference intervals were 1.020 to 1.045 for USG, 7.0 to 8.7 for pH, and 0.04 to 0.25 for UPC; because of differences in creatinine concentration, UPC was lower in beef (0.04-0.14) than in dairy (0.05-0.25) cows and in the latter in dry than lactating cows. With dipstick evaluation, most analytes were absent except for blood, ketone, and protein in 24.7, 16.0, and 64.7% of cases, respectively. Microscopic evaluation revealed less than 3 red blood cells, leukocytes, and epithelial cells in 84, 99.3, and 100% cows, respectively. No band was observed at electrophoresis, except in 1 case at MW ~66 000. CONCLUSIONS AND CLINICAL IMPORTANCE: Creatininuria is higher in beef than dairy cows and proteinuria is likely more efficiently characterized by protein concentration than by UPC.

Fractional excretion tests: a critical review of methods and applications in domestic animals.

The fractional excretion (FE) of a constituent by the kidney is the fraction of the amount filtered by the glomerulus, which is excreted into urine. It is mostly determined for electrolytes, and is expressed as the ratio of the clearance of a given electrolyte to creatinine clearance. The main physiologic factors affecting FE variation are species, age, and the alimentary supply of electrolytes. The value of FE tests in the diagnosis of kidney disease is limited, except in canine Fanconi's syndrome. FEs of many constituents often are increased in chronic kidney disease, but their diagnostic value is no greater than that of plasma creatinine concentration. FEs also are altered in nonrenal diseases such as diabetes mellitus and rhabdomyolysis, and during treatment with xylazine, rehydration fluids, and diuretics. FEs, especially of calcium, phosphates, and magnesium, are useful in clinical nutrition to assess mineral balance. FE is difficult to measure, so its use should be limited to nutritional investigations and nephrology research.