Comparison of Sysmex XN-V body fluid mode and deep-learning-based quantification with manual techniques for total nucleated cell count and differential count for equine bronchoalveolar lavage samples.

BACKGROUND: Bronchoalveolar lavage (BAL) is a diagnostic method for the assessment of the lower respiratory airway health status in horses. Differential cell count and sometimes also total nucleated cell count (TNCC) are routinely measured by time-consuming manual methods, while faster automated methods exist. The aims of this study were to compare: 1) the Sysmex XN-V body fluid (BF) mode with the manual techniques for TNCC and two-part differential into mononuclear and polymorphonuclear cells; 2) the Olympus VS200 slide scanner and software generated deep-learning-based algorithm with manual techniques for four-part differential cell count into alveolar macrophages, lymphocytes, neutrophils, and mast cells. The methods were compared in 69 clinical BAL samples. RESULTS: Incorrect gating by the Sysmex BF mode was observed on many scattergrams, therefore all samples were reanalyzed with manually set gates. For the TNCC, a proportional and systematic bias with a correlation of r = 0.79 was seen when comparing the Sysmex BF mode with manual methods. For the two-part differential count, a mild constant and proportional bias and a very small mean difference with moderate limits of agreement with a correlation of r = 0.84 and 0.83 were seen when comparing the Sysmex BF mode with manual methods. The Sysmex BF mode classified significantly more samples as abnormal based on the TNCC and the two-part differential compared to the manual method. When comparing the Olympus VS200 deep-learning-based algorithm with manual methods for the four-part differential cell count, a very small bias in the regression analysis and a very small mean difference in the difference plot, as well as a correlation of r = 0.85 to 0.92 were observed for all four cell categories. The Olympus VS200 deep-learning-based algorithm also showed better precision than manual methods for the four-part differential cell count, especially with an increasing number of analyzed cells. CONCLUSIONS: The Sysmex XN-V BF mode can be used for TNCC and two-part differential count measurements after reanalyzing the samples with manually set gates. The Olympus VS200 deep-learning-based algorithm correlates well with the manual methods, while showing better precision and can be used for a four-part differential cell count.

Validation of the Sysmex XN-V Automated Nucleated Red Blood Cell Enumeration for Canine and Feline EDTA-Anticoagulated Blood.

The enumeration of nRBCs (nucleated red blood cells) by manual counting is time-consuming and imprecise. As the first veterinary hematology analyzer, Sysmex XN-V provides automated nRBC counts. This study aimed to evaluate the performance of Sysmex XN-V in the enumeration of nRBCs for cats and dogs by comparing automated nRBC counts to manual counts from a total of 3810 canine and 2844 feline specimens. Repeatability, reproducibility, stability, carry-over, and linearity were assessed. The repeatability and reproducibility of Sysmex XN-V were good, with mean coefficients of variation (CV) of 4.5% and 5.4%, respectively. Bland-Altman difference analysis revealed mean biases shown as nRBCs/100 WBCs of 0.01 in dogs and 0.11 in cats with low nRBCs (<5/100 WBCs), mean biases of -1.27 in dogs and -0.24 in cats with moderate nRBC counts (5-20 nRBCs/100 WBCs), and mean biases of -7.76 in dogs and -1.31 in cats with high nRBC counts (>20 nRBCs/100 WBCs). The total observable error was below 9% in both species and at all ranges. Overall concordance between methods was high (91% in canine and 93% in feline samples). The automated nRBC count by Sysmex XN-V was found to be accurate and precise and can replace manual counts for cat and dog samples. Non-statistical quality assurance by scattergram evaluation, re-gating, and confirmation by blood smear evaluation is, however, recommended, especially in cases with severe normoblastosis. This advancement will save time, reduce errors, and add prognostic value to hematological results for animal patients.

Acidification is required for calcium and magnesium concentration measurements in equine urine.

BACKGROUND: Acidification of equine urine to promote dissociation of ion complexes is a common practice for urine ion concentration measurements. The objective of this study was to evaluate the effect of acidification and storage after acidification on calcium (Ca), magnesium (Mg) and phosphate (P) concentrations and on fractional excretion (FE) of these electrolytes. Thirty-two fresh equine urine samples were analysed between December 2016 and July 2020. Complete urinalysis (stick and sediment) was performed on all samples. Ca, Mg, P and creatinine concentrations were measured in supernatant of centrifuged native urine, urine directly centrifuged after acidification and urine centrifuged 1 hour after acidification. Urine was acidified with hydrochloric acid to reach a pH of 1-2. Ca, Mg, P and creatinine concentrations were also measured in blood plasma, and fractional excretion of each electrolyte was calculated. Equality of medians was tested with Friedman tests and Bland-Altman bias plots were used to show the agreement between conditions. RESULTS: Acidification had a statistically significant effect on Ca and Mg concentrations, FECa and FEMg. Bland-Altman plot revealed a strong positive proportional bias between Ca concentration in native and acidified urine with a mean bias of 17.6 mmol/l. For Mg concentration, the difference between native and acidified urine was small with a mean bias of 1.8 mmol/l. The increase in FECa was clinically relevant. Storage of acidified urine had no effect on any of the measured ion concentrations. All P concentrations in native urine samples were below the detection limit of the assay and statistical analysis and calculation of FEP was not possible. CONCLUSIONS: Urine acidification is essential for accurate measurement of Ca and Mg concentrations and therefore FE calculations in equine urine. Storage time of 1 hour after acidification does not significantly change Ca and Mg concentrations.

Differences in selected blood parameters between brachycephalic and non-brachycephalic dogs.

Introduction: Cranial and upper-airway anatomy of short-nosed, flat-faced brachycephalic dogs predisposes them to brachycephalic obstructive airway syndrome (BOAS). Periodic apnoea increased inspiratory resistance, and an inability to thermoregulate effectively are characteristic of BOAS, but internationally accepted objective markers of BOAS severity are missing. The objective of this study was to compare the selected blood parameters between non-brachycephalic (NC) and brachycephalic (BC) dogs, exploring the possibility of developing a blood test for BOAS severity grading in the future. Methods: We evaluated blood biochemistry, complete blood cell counts, red blood cell (RBC) indices, reticulocyte counts, a blood-born marker of intermittent hypoxia (glutathione, NO production), RBC hydration, deformability, and blood markers of metabolic changes and stress between BC (n = 18) and NC (meso- and dolichocephalic, n = 22) dogs. Results: Reticulocyte counts and the abundance of middle-fluorescence immature reticulocytes were significantly (p < 0.05) higher in BC dogs compared to NC dogs. BC dogs had significantly more NO-derived NO2-/NO3- in plasma than NC dogs. RBCs of BC dogs were shedding significantly more membrane, as follows from the intensity of eosin maleimide staining, and had a significantly higher mean corpuscular hemoglobin concentration than NC dogs. Intracellular reduced glutathione content in RBCs of BC dogs was significantly lower, while plasma lactate was significantly higher in BC dogs compared to NC dogs. Plasma cholesterol and triglycerides were significantly lower, and cortisol was significantly higher in BC dogs compared to NC dogs. Eosinophil counts were significantly lower and the neutrophil-to-lymphocyte ratio was higher in BC dogs compared to NC dogs. Discussion: Taken together, our findings suggest that the brachycephalic phenotype in dogs is associated with alterations at the level of blood cells and, systemically, with oxidation and metabolic changes. The parameters identified within this study should be further investigated for their potential as objective indicators for BOAS.

Preliminary Investigation of Side Effects of Polymyxin B Administration in Hospitalized Horses.

Neuro- and nephrotoxicity of polymyxins are known but clinical studies in horses are lacking. The aim of this study was to describe neurogenic and nephrogenic side effects of hospitalized horses receiving Polymyxin B (PolyB) as part of their treatment plan. Twenty horses diagnosed with surgical colic (n = 11), peritonitis (n = 5), typhlocolitis (n = 2), pneumonia, and pyometra (each n = 1) were included. Antimicrobial treatment was randomized to GENTA (gentamicin 10 mg/kg bwt q24 h IV, penicillin 30.000 IU/kg q6 h IV) or NO GENTA (marbofloxacin 2 mg/kg bwt q24 h IV, penicillin 30.000 IU/kg q6 h IV). The duration of PolyB treatment ranged from 1 to 4 days. Clinical and neurological examinations were performed, and serum PolyB concentrations were measured daily during and three days following PolyB treatment. Urinary analysis, plasma creatinine, urea and SDMA were assessed every other day. Video recordings of neurological examinations were graded by three blinded observers. All horses showed ataxia during PolyB treatment in both groups (median maximum ataxia score of 3/5, range 1-3/5). Weakness was detected in 15/20 (75%) horses. In 8/14 horses, the urinary γ-glutamyltransferase (GGT)/creatinine ratio was elevated. Plasma creatinine was mildly elevated in 1/16 horses, and SDMA in 2/10 horses. Mixed-model analysis showed a significant effect of time since last PolyB dose (p = 0.0001, proportional odds: 0.94) on the ataxia score. Ataxia and weakness should be considered as reversible adverse effects in hospitalized horses receiving PolyB. Signs of tubular damage occurred in a considerable number of horses; therefore, the nephrotoxic effect of polymyxins should be considered and urinary function monitored.

Methemoglobinemia, Increased Deformability and Reduced Membrane Stability of Red Blood Cells in a Cat with a CYB5R3 Splice Defect.

Methemoglobinemia is an acquired or inherited condition resulting from oxidative stress or dysfunction of the NADH-cytochrome b5 reductase or associated pathways. This study describes the clinical, pathophysiological, and molecular genetic features of a cat with hereditary methemoglobinemia. Whole genome sequencing and mRNA transcript analyses were performed in affected and control cats. Co-oximetry, ektacytometry, Ellman's assay for reduced glutathione concentrations, and CYB5R activity were assessed. A young adult European domestic shorthair cat decompensated at induction of anesthesia and was found to have persistent methemoglobinemia of 39 ± 8% (reference range < 3%) of total hemoglobin which could be reversed upon intravenous methylene blue injection. The erythrocytic CYB5R activity was 20 ± 6% of normal. Genetic analyses revealed a single homozygous base exchange at the beginning of intron 3 of the CYB5R3 gene, c.226+5G>A. Subsequent mRNA studies confirmed a splice defect and demonstrated expression of two mutant CYB5R3 transcripts. Erythrocytic glutathione levels were twice that of controls. Mild microcytosis, echinocytes, and multiple Ca2+-filled vesicles were found in the affected cat. Erythrocytes were unstable at high osmolarities although highly deformable as follows from the changes in elongation index and maximal-tolerated osmolarity. Clinicopathological presentation of this cat was similar to other cats with CYB5R3 deficiency. We found that methemoglobinemia is associated with an increase in red blood cell fragility and deformability, glutathione overload, and morphological alterations typical for stress erythropoiesis.

Findings Related to Cerebrospinal Fluid and Central Nervous System Disorders in Small Ruminants-A Retrospective Study on Sheep and Goats.

BACKGROUND: Small ruminants often suffer from central nervous system (CNS) disorders, and cerebrospinal fluid (CSF) analysis can be used as a diagnostic tool in this regard. In small animals and cattle, specific CSF patterns have been defined for specific disease categories. No data exist regarding CSF results obtained from small ruminants and their association with certain CNS diseases. OBJECTIVES: The objective of this study was to retrospectively investigate CSF findings obtained from sheep and goats and to identify possible CSF patterns associated with disease categories. METHODS: CSF samples and medical records from 44 sheep and 27 goats were included in this study. All animals were presented to the Veterinary Teaching Hospital Zurich of the Veterinary Teaching Hospital Zurich of the Vetsuisse Faculty of the University of Zurich between 2003 and 2016 and had either a confirmed CNS diagnosis or showed CSF changes without a specific CNS diagnosis. RESULTS: Mixed mononuclear pleocytosis was the most common CSF pattern in sheep (25%), followed by monocytic pleocytosis (21%). Lymphocytic pleocytosis was most frequently found in goats (37%). In 75% of sheep and 56% of goats, infectious CNS diseases were diagnosed, with listeriosis being the most common infectious disease in both species, followed by parasitic disorders (nematodiasis and coenurosis). CONCLUSIONS: The cytologic CSF patterns in small ruminants are mainly based on the increased presence of monocytic and lymphocytic cells with variable quantitative expression, whereas neutrophilic pleocytosis and cytoalbuminologic dissociation were rare findings. Infectious diseases of bacterial origin were the most common underlying causes for CSF alterations in sheep and goats, followed by parasitic disorders. The pleocytosis type is not helpful for differentiating disease types.

Evaluation of the Effect of Storage Time on ROTEM S® Parameters in Healthy and Ill Dogs.

Viscoelastic testing as a bedside test to assess global haemostasis has gained popularity in the past decade, with rotational thromboelastometry (ROTEM) and thromboelastography (TEG) being the two commonly used devices. TEG studies suggest analysis 30 min after blood sampling. However, the reproducibility of results over time for ROTEM analysis using lyophilized samples in dogs has not been established. In this study, we investigated the influence of time on viscoelastic testing, using 33 healthy staff-/client-owned dogs for blood sampling and repeated measurements of ROTEM tracings at three different time points after blood collection. Additionally, a group of 21 hospitalized patients with suspected coagulation disorders were included to investigate whether stability over time was comparable between healthy and ill dogs. We demonstrated a significant difference of ROTEM tracings over time, with a tendency towards hypocoagulability over time. These changes do have a clinical relevance as they exceed reference intervals and could therefore lead to erroneous conclusions about a patient's coagulation status. Therefore, time-specific reference intervals are proposed and presented in this publication.

Comparison of Jugular vs. Saphenous Blood Samples, Intrarater and In-Between Device Reliability of Clinically Used ROTEM S Parameters in Dogs.

Rotational Thromboelastometry (ROTEM) allows for the global assessment of hemostasis in whole blood samples. Preanalytical and analytical factors may influence test results, and data about the reliability and reproducibility of lyophilized ROTEM tests are scarce. Therefore, the objective of this study was to evaluate the influence of blood collection site on ROTEM S parameters and to assess intrarater and in-between device variability. A total of thirty, healthy, staff-owned dogs were included. Blood collection and ROTEM analysis were performed by trained staff according to a standardized protocol. Extrinsically activated (tissue factor; Ex-TEM S), with the addition of cytochalasin for platelet inhibition (Fib-TEM S), and intrinsically activated (In-TEM) analyses were performed. Analysis of our data showed significant variability for various Ex-TEM S and Fib-TEM S parameters from different collection sites and intrarater and in-between device measurements. We conclude that serial monitoring with ROTEM should be performed on the same device, with blood always taken from the same collection site using a standardized blood sampling technique. While In-TEM S, apart from maximum lysis, showed very stable and reliable results, we suggest interpreting especially clotting and clot formation parameters from Ex-TEM S and Fib-TEM S tests with caution and using duplicate measurements to detect outliers and to prevent initiation of incorrect therapies.

Adverse effects of polymyxin B administration to healthy horses.

BACKGROUND: Polymyxin B (PolyB) is used to treat endotoxemia in horses; neurologic and nephrogenic adverse effects occur in humans. OBJECTIVES: To describe PolyB adverse effects in horses. ANIMALS: Five healthy horses (ataxia 0/5), 1 horse with cervical osteoarthritis (ataxia 1/5). METHODS: Prospective blinded randomized cross-over trial; 3-weeks wash out. Horses received PolyB (PolyB 6000 IU/kg IV, 7 doses q12h, n = 6) and PolyB/gentamicin (PolyB 6000 IU/kg IV, q12h 7 doses; gentamicin 10 mg/kg IV q24h 4 doses n = 4, or q12-24 h 5 doses because of an additional erroneous dose, n = 2). Daily neurological examinations were video recorded, and ataxia graded by 3 observers. Urine status, urinary GGT/creatinine ratio, plasma creatinine, and urea were assessed every other day, EMG daily. Mixed model analysis was used to evaluate factors associated with ataxia grade and [PolyB]. RESULTS: Median ataxia score increased from 0/5 (range 0-2/5) to 2/5 (range 1-3/5) during administration and declined to 0.5/5 (range 0-2/5) after cessation. Gentamicin co-administration (P < .01, effect size: .8), number of PolyB doses (P < .001, effect size: .6), and time since last PolyB dose (P < .001, effect size: .5) had a significant effect on ataxia grades, while horse, day, [Genta], [PolyB], and [PolyB]CSF did not. Gentamicin co-administration and [Genta] Cpeak had no effect on median [PolyB] Cpeak (4.67 and 4.89 µg/ml for PolyB and PolyB/gentamicin, respectively). Urinary GGT/creatinine ratio was elevated in 3/6 horses receiving PolyB/gentamicin. The EMG remained unchanged. CONCLUSIONS AND CLINICAL IMPORTANCE: PolyB caused transient ataxia, worsening with cumulative PolyB doses and gentamicin co-administration. Nephrotoxicity of PolyB was only evident when gentamicin was co-administered.

Preclinical investigations using [177Lu]Lu-Ibu-DAB-PSMA toward its clinical translation for radioligand therapy of prostate cancer.

[177Lu]Lu-Ibu-DAB-PSMA was previously characterized with moderate albumin-binding properties enabling high tumor accumulation but reasonably low retention in the blood. The aim of this study was to investigate [177Lu]Lu-Ibu-DAB-PSMA in preclinical in vivo experiments and compare its therapeutic efficacy and potential undesired side effects with those of [177Lu]Lu-PSMA-617 and the previously developed [177Lu]Lu-PSMA-ALB-56. BALB/c nude mice without tumors were investigated on Day 10 and 28 after injection of 10 MBq radioligand. It was revealed that most plasma parameters were in the same range for all groups of mice and histopathological examinations of healthy tissue did not show any alternations in treated mice as compared to untreated controls. Based on these results, a therapy study over twelve weeks was conducted with PC-3 PIP tumor-bearing mice for comparison of the radioligands's therapeutic efficacy up to an activity of 10 MBq (1 nmol) per mouse. In agreement with the increased mean absorbed tumor dose, [177Lu]Lu-Ibu-DAB-PSMA (~ 6.6 Gy/MBq) was more effective to inhibit tumor growth than [177Lu]Lu-PSMA-617 (~ 4.5 Gy/MBq) and only moderately less potent than [177Lu]Lu-PSMA-ALB-56 (~ 8.1 Gy/MBq). As a result, the survival of mice treated with 2 MBq of an albumin-binding radioligand was significantly increased (p < 0.05) compared to that of mice injected with [177Lu]Lu-PSMA-617 or untreated controls. The majority of mice treated with 5 MBq or 10 MBq [177Lu]Lu-Ibu-DAB-PSMA or [177Lu]Lu-PSMA-ALB-56 were still alive at study end. Hemograms of immunocompetent mice injected with 30 MBq [177Lu]Lu-Ibu-DAB-PSMA or 30 MBq [177Lu]Lu-PSMA-617 showed values in the same range as untreated controls. This was, however, not the case for mice treated with [177Lu]Lu-PSMA-ALB-56 which revealed a drop in lymphocytes and hemoglobin at Day 10 and Day 28 after injection. The data of this study demonstrated a significant therapeutic advantage of [177Lu]Lu-Ibu-DAB-PSMA over [177Lu]Lu-PSMA-617 and a more favorable safety profile as compared to that of [177Lu]Lu-PSMA-ALB-56. Based on these results, [177Lu]Lu-Ibu-DAB-PSMA may has the potential for a clinical translation.

Iron- and erythropoietin-resistant anemia in a spontaneous breast cancer mouse model.

Anemia of cancer (AoC) with its multifactorial etiology and complex pathology is a poor prognostic indicator for cancer patients. One of the main causes of AoC is cancer-associated inflammation that activates mechanisms, commonly observed in anemia of inflammation, whereby functional iron deficiency and iron-restricted erythropoiesis are induced by increased hepcidin levels in response to raised levels of interleukin-6. So far only a few AoC mouse models have been described, and most of them did not fully recapitulate the interplay of anemia, increased hepcidin levels and functional iron deficiency in human patients. To test if the selection and the complexity of AoC mouse models dictates the pathology or if AoC in mice per se develops independently of iron deficiency, we characterized AoC in Trp53floxWapCre mice that spontaneously develop breast cancer. These mice developed AoC associated with high levels of interleukin-6 and iron deficiency. However, hepcidin levels were not increased and hypoferremia coincided with anemia rather than causing it. Instead, an early shift in the commitment of common myeloid progenitors from the erythroid to the myeloid lineage resulted in increased myelopoiesis and in the excessive production of neutrophils that accumulate in necrotic tumor regions. This process could not be prevented by either iron or erythropoietin treatment. Trp53floxWapCre mice are the first mouse model in which erythropoietin-resistant anemia is described and may serve as a disease model to test therapeutic approaches for a subpopulation of human cancer patients with normal or corrected iron levels who do not respond to erythropoietin.

HEALTH ASSESSMENT OF CAPTIVE AND FREE-LIVING EUROPEAN POND TURTLES (EMYS ORBICULARIS) IN SWITZERLAND.

The highly endangered European pond turtle (Emys orbicularis) was reintroduced in Switzerland in 2010. Up until 2019, no routine medical examinations have been carried out prior to its release or during recapture events. The aim of this study was to assess the health status of captive and free-living Emys orbicularis populations in Switzerland, taking into account the most important and frequently occurring health threats to freshwater turtles. A total of 141 European pond turtles, including captive (n = 89) and free-living (n = 52) individuals, underwent clinical examination (n = 136), choanal and cloacal swab collection for microbiology investigation (n = 140), blood sampling (n = 121), fecal examination for parasitology (n = 92), radiography (n = 84), and ultrasound (n = 46). Microbiology investigation included conventional PCR for herpesvirus, ranavirus, and Mycoplasma spp. Blood was used for the establishment of reference values for hematocrit, leukocyte count, and differential blood count as well as for biochemistry parameters tested with the VetScan VS2. An emydid Mycoplasma was detected in 40% (n = 56/140; 95%CI: 31.82-48.61%) of the turtles, including one individual with upper respiratory signs. Four animals positive for Mycoplasma arrived dead or were euthanized during the study period. Their necropsies revealed no evidence of respiratory disease. No ranavirus or herpesvirus was detected in any of the tested turtles. Two presumptively fatal infections with spirorchiid trematodes were reported during the study period. Endoparasites were detected in only 7.94% of the samples examined. This study provides comprehensive data on the current health status of the largest sample size of captive and free-living populations of Emys orbicularis ever assessed to date and serves as a baseline for future research investigations and management recommendations in this species.

Whole blood platelet impedance aggregometry with the ROTEM platelet device: comparison of 2 anticoagulants and storage times for the establishment of canine reference intervals.

The ROTEM platelet device, a point-of-care whole blood platelet impedance aggregometer, is an add-on to the rotational thromboelastometry ROTEM delta device. The latter has been validated in dogs. We examined whether canine whole blood is suited for analysis with the ROTEM platelet device using adenosine-5'-diphosphate (ADP) and arachidonic acid (ARA) as agonists for platelet activation, and if there are significant differences between sample storage times and anticoagulants used. Subsequently, we determined canine reference intervals (RIs) for the ROTEM platelet device for ADP and ARA. In a pilot study, we examined whole blood from 7 dogs after 15-min and 60-min storage of lithium-heparinized samples and 40-min and 80-min storage of hirudinized samples. Statistical analysis showed no significant differences between ROTEM platelet device results for both ADP and ARA in lithium-heparin and hirudin anticoagulated canine whole blood. Lithium-heparinized blood samples analyzed after 15-min storage had the lowest coefficient of variation. RIs were determined for heparinized whole blood samples from 49 dogs after 15 min of storage.

Modified-Live Feline Calicivirus Vaccination Elicits Cellular Immunity against a Current Feline Calicivirus Field Strain in an Experimental Feline Challenge Study.

Feline calicivirus (FCV) is a common cat virus associated with oral ulcerations and virulent-systemic disease. Efficacious FCV vaccines protect against severe disease but not against infection. The high genetic diversity of FCV poses a challenge in vaccine design. Protection against FCV has been related to humoral and cellular immunity; the latter has not been studied in detail. This study investigates the cellular and humoral immune response of specified pathogen-free (SPF) cats after modified-live FCV F9 vaccinations and two heterologous FCV challenges by the analysis of lymphocyte subsets, cytokine mRNA transcription levels, interferon (IFN)-γ release assays in peripheral blood mononuclear cells (PBMCs), anti-FCV antibodies, and neutralisation activity. Vaccinated cats developed a Th1 cytokine response after vaccination. Vaccination resulted in antibodies with neutralising activity against the vaccine but not the challenge viruses. Remarkably, IFN-γ-releasing PBMCs were detected in vaccinated cats upon stimulation with the vaccine strain and the first heterologous FCV challenge strain. After the first experimental infection, the mRNA transcription levels of perforin, granzyme B, INF-γ, and antiviral factor MX1 and the number of IFN-γ-releasing PBMCs when stimulated with the first challenge virus were higher in vaccinated cats compared to control cats. The first FCV challenge induced crossneutralising antibodies in all cats against the second challenge virus. Before the second challenge, vaccinated cats had a higher number of IFN-γ-releasing PBMCs when stimulated with the second challenge virus than control cats. After the second FCV challenge, there were less significant differences detected between the groups regarding lymphocyte subsets and cytokine mRNA transcription levels. In conclusion, modified-live FCV vaccination induced cellular but not humoral crossimmunity in SPF cats; innate immune mechanisms, secretory and membranolytic pathways, and IFN-γ-releasing PBMCs seem to be important in the host immune defence against FCV.

Modified-Live Feline Calicivirus Vaccination Reduces Viral RNA Loads, Duration of RNAemia, and the Severity of Clinical Signs after Heterologous Feline Calicivirus Challenge.

Feline calicivirus (FCV) is a common cat virus causing clinical signs such as oral ulcerations, fever, reduced general condition, pneumonia, limping and occasionally virulent-systemic disease. Efficacious FCV vaccines protect against severe disease but not against infection. FCV is a highly mutagenic RNA virus whose high genetic diversity poses a challenge in vaccine design. The use of only one modified-live FCV strain over several decades might have driven the viral evolution towards more vaccine-resistant variants. The present study investigated the clinical signs, duration, and amount of FCV shedding, RNAemia, haematological changes and acute phase protein reaction in SPF cats after subcutaneous modified-live single strain FCV vaccination or placebo injection and two subsequent oronasal heterologous FCV challenge infections with two different field strains. Neither clinical signs nor FCV shedding from the oropharynx and FCV RNAemia were detected after vaccination. After the first experimental infection, vaccinated cats had significantly lower clinical scores, less increased body temperature and lower acute phase protein levels than control cats. The viral RNA loads from the oropharynx and duration and amount of RNAemia were significantly lower in the vaccinated animals. No clinical signs were observed in any of the cats after the second experimental infection. In conclusion, FCV vaccination was beneficial for protecting cats from severe clinical signs, reducing viral loads and inflammation after FCV challenge.

Determination of reference intervals for single vial rotational thromboelastometry (ROTEM) parameters and correlation with plasmatic coagulation times in 49 clinically healthy dogs.

The objectives of this prospective study were determination of reference intervals (RI) for rotational thromboelastometry (ROTEM) parameters in single use reagents and to evaluate correlations between plasmatic coagulation times and ROTEM parameters. Blood was sampled from a jugular vein in 49 client-owned healthy dogs and ex-tem S, in-tem S, fib-tem S and ap-tem S parameters, prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen, haematology, blood chemistry and venous blood gas analysis was performed. Determination of RI was performed using Excel add-in Reference Value Advisor and correlations between PT, aPTT and fibrinogen with selected ROTEM parameters were determined by Spearman correlation. Ex-tem S maximum clot firmness (MCF) RI are smaller compared to RI in people and liquid ex-tem in dogs while maximum lysis was comparable to those in people but smaller than previously reported in dogs. A strong correlation was found between fibrinogen measured by Clauss and fib-tem S and in-tem S MCF (r = 0.541, P < .001 and r = 0.610, P < .001, respectively). PT showed a significant but moderate correlation with ex-tem S CT (r = 0.340, P = .030), in-tem S CFT (r = 0.433, P = .003), fib-tem S CT (r = 0.426, P = .009) and ap-tem S CT (r = 0.354, P = .015) while aPTT was not significantly correlated with any of the evaluated parameters. In conclusion, this study provides single use reagent ROTEM parameter RIs that are different from RI determined with liquid reagents. Significant correlations between fibrinogen concentrations measured by Clauss and clot firmness of fib-tem S and in-tem S profiles and between PT and clotting times of all reagents were identified.

Fatal acute babesiosis associated with Babesia venatorum infection (Babesia sp. EU1) in a captive reindeer calf in Switzerland.

Babesia venatorum was isolated from a captive reindeer calf in Switzerland. The clinical signs consistent with acute babesiosis included hemolytic anemia and hemoglobinuria. The diagnosis was made based on visualization of intraerythrocytic parasites in the stained blood smears and confirmed by PCR analysis of the 18S rRNA gene, with subsequent species identification within Babesia confirmed by sequencing. The reindeer calf was initially treated with supportive care and an antiprotozoal drug (imidocarb dipropionate) but died a few days after hospitalization. Babesia venatorum is also known as Babesia sp. EU1 and can infect different mammalian species, including humans. The current case report aims to increase awareness among veterinarians and reindeer owners about the presence and the associated risk of this zoonotic pathogen. Considering the high morbidity and possible mortality associated with acute babesiosis, captive reindeer should receive tick prevention measures and be tested for subclinical infections in endemic area.

Prevalence, Geographic Distribution, Risk Factors and Co-Infections of Feline Gammaherpesvirus Infections in Domestic Cats in Switzerland.

Recently, a gammaherpesvirus was described in domestic cats (FcaGHV1). The goal of the present study was to investigate the presence of FcaGHV1 in Swiss domestic cats and analyze potential risk factors. Blood samples from 881 cats presented to veterinarians in all Swiss cantons and from 91 stray cats and neoplastic tissue samples from 17 cats with lymphoma were evaluated. FcaGHV1 was detected by real-time PCR targeting the glycoprotein B gene, followed by sequencing. Blood samples were also tested for feline hemoplasmas, feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV). The molecular prevalence of FcaGHV1 was 6.0% (95% confidence interval (CI), 4.5-7.8%) in cats presented to veterinarians and 5.5% (95% CI, 1.8-12.4%) in stray cats. FcaGHV1 PCR-positive cats originated from 19/26 Swiss cantons. Factors significantly associated with FcaGHV1 detection included male sex, age >3 years, nonpedigree status and co-infection with FIV and hemoplasmas. Moreover, FeLV viremia tended to be associated with FcaGHV1 detection. High FcaGHV1 blood loads were found more frequently in FeLV-viremic cats and less frequently in hemoplasma-infected cats than in uninfected cats. Clinical information was unavailable for most of the 881 cats, but leukemia, carcinoma and cardiomyopathy were reported in FcaGHV1-positive cats. None of the tissue samples from the 17 cats with lymphoma tested positive for FcaGHV1. Sequence analyses revealed homogeneity among the Swiss isolates and >99.7% identity to published FcaGHV1 sequences. In conclusion, FcaGHV1 is present in Switzerland with a similar prevalence in cats presented to veterinarians and in stray cats. The pathogenic potential of FcaGHV1 needs further evaluation.