C-reactive protein concentration has limited value in the diagnosis of meningoencephalitis of unknown origin in dogs.

OBJECTIVES: To evaluate blood and cerebrospinal fluid (CSF) concentrations of C-reactive protein (CRP) in dogs with meningoencephalitis of unknown origin (MUO); to evaluate whether blood CRP concentration is associated with epidemiological, clinicopathologic, and MRI findings; and to investigate blood CRP predictive power in survival. ANIMALS: 30 client-owned dogs with MUO, 15 client-owned dogs with steroid-responsive meningitis arteritis (SRMA; positive control group), and 15 healthy dogs (negative control group). METHODS: Blood CRP concentration was measured in each group, while it was performed in CSF only in the MUO and SRMA groups. The analysis of epidemiological data included breed, age, sex, duration of clinical signs, and history of seizures. Blinded analysis of MRI was performed based on a classification grid, and traditional CSF analysis parameters were assessed. The predictive power of blood CRP concentration regarding survival at 6 months was investigated. RESULTS: Of the 30 dogs with MUO, 9 (30%) had an increased CRP concentration in blood, and 3 (10%) showed a measurable CRP in CSF. Median blood CRP concentration in dogs with MUO was 0.1 mg/L (range, 0.1 to 102 mg/L), which was not statistically different from the healthy dog group but significantly lower than the SRMA control group. Only the duration of clinical signs was positively associated with an increased blood CRP level. Blood CRP concentration was not associated with survival at 6 months. CLINICAL RELEVANCE: Blood CRP concentration is of limited value for the diagnosis and prognosis of dogs with MUO. Chronicity of the disease may be associated with an increased concentration of blood CRP.

Suppurative spinal meningomyelitis in a dog with intra-neutrophilic cerebrospinal fluid cells Ehrlichia canis morulae.

A 2-year-old castrated male Creole Shepherd mixed dog was presented for non-ambulatory paraparesis of the pelvic limbs. The magnetic resonance imaging and cerebrospinal fluid (CSF) analysis were consistent with meningomyelitis. Positive serology for Ehrlichia canis/Ehrlichia ewingii suggested exposure to a pathogen; qPCR on the serum and the CSF confirmed active infection. Ehrlichial morulae were observed within CSF and peripheral blood polymorphonuclear neutrophils; a species-specific PCR confirmed E. canis infection. This is an interesting report of E. canis infection in a dog with morulae observed in neutrophils both in the peripheral blood and CSF.

First Evidence of Natural SARS-CoV-2 Infection in Domestic Rabbits.

We tested 144 pet rabbits sampled in France between November 2020 and June 2021 for antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by microsphere immunoassay. We reported the first evidence of a natural SARS-CoV-2 infection in rabbits with a low observed seroprevalence between 0.7% and 1.4%.

BCL-2 Inhibitor ABT-737 Effectively Targets Leukemia-Initiating Cells with Differential Regulation of Relevant Genes Leading to Extended Survival in a NRAS/BCL-2 Mouse Model of High Risk-Myelodysplastic Syndrome.

During transformation, myelodysplastic syndromes (MDS) are characterized by reducing apoptosis of bone marrow (BM) precursors. Mouse models of high risk (HR)-MDS and acute myelogenous leukemia (AML) post-MDS using mutant NRAS and overexpression of human BCL-2, known to be poor prognostic indicators of the human diseases, were created. We have reported the efficacy of the BCL-2 inhibitor, ABT-737, on the AML post-MDS model; here, we report that this BCL-2 inhibitor also significantly extended survival of the HR-MDS mouse model, with reductions of BM blasts and lineage negative/Sca1+/KIT+ (LSK) cells. Secondary transplants showed increased survival in treated compared to untreated mice. Unlike the AML model, BCL-2 expression and RAS activity decreased following treatment and the RAS:BCL-2 complex remained in the plasma membrane. Exon-specific gene expression profiling (GEP) of HR-MDS mice showed 1952 differentially regulated genes upon treatment, including genes important for the regulation of stem cells, differentiation, proliferation, oxidative phosphorylation, mitochondrial function, and apoptosis; relevant in human disease. Spliceosome genes, found to be abnormal in MDS patients and downregulated in our HR-MDS model, such as Rsrc1 and Wbp4, were upregulated by the treatment, as were genes involved in epigenetic regulation, such as DNMT3A and B, upregulated upon disease progression and downregulated upon treatment.

Comparison of STA-NeoPTimal (Stago) and STA-Neoplastine CI Plus (Stago) thromboplastin reagents using a STA Satellite Max analyzer to measure prothrombin times in dogs.

BACKGROUND: Different thromboplastins are available to measure prothrombin time (PT). Stago coagulation analyzers and reagents are currently used in veterinary laboratories and enable PT measurements to explore the coagulation cascade (extrinsic pathway). OBJECTIVES: The main objective was to compare PT measurements obtained with the STA-NeoPTimal reagent with the commonly used STA-Neoplastine CI Plus reagent. The secondary objective was to compare the PT ratio with the international normalized ratio (INR) calculated from our derived clotting times. METHODS: Analytical performance was evaluated with intra-assay and inter-assay precision. Seventy-two individual canine plasma samples were collected. Each sample was tested with both thromboplastins, using an STA Satellite Max analyzer. The PT, PT ratio, and INR values obtained with the two reagents were compared using Passing-Bablok regression for correlations and Bland-Altman plots for method agreements. RESULTS: The analytical performance of STA-NeoPTimal reagent was acceptable. Compared with the STA-Neoplastine CI Plus reagent, the STA-NeoPTimal reagent showed a positive proportional bias for PT values. Narrow range analyses showed good agreement for normal PT values (less than 9.5 seconds, internal reference cutoff with STA-Neoplastine CI Plus), and clinical concordance was achieved. When PT was prolonged (more than 9.5 seconds), PT increases were more marked with the STA-NeoPTimal reagent. Agreement was good for INR values across the whole range of PT results. CONCLUSION: STA-NeoPTimal can be reliably implemented in veterinary laboratories for canine PT measurements, as agreement between the PT results measured with the two reagents was clinically acceptable.

Pathogenic Escherichia coli in Dogs Reveals the Predominance of ST372 and the Human-Associated ST73 Extra-Intestinal Lineages.

Escherichia coli is a ubiquitous commensal and pathogen that has also been recognized as a multi-sectoral indicator of antimicrobial resistance (AMR). Given that latter focus, such as on resistances to extended-spectrum cephalosporins (ESC) and carbapenems, the reported population structure of E. coli is generally biased toward resistant isolates, with sequence type (ST)131 being widely reported in humans, and ST410 and ST648 being reported in animals. In this study, we characterized 618 non-duplicate E. coli isolates collected throughout France independently of their resistance phenotype. The B2 phylogroup was over-represented (79.6%) and positively associated with the presence of numerous virulence factors (VFs), including those defining the extra-intestinal pathogenic E. coli isolates (presence of ≥2 VFs: papA, sfaS, focG, afaD, iutA, and kpsMTII) and those more specifically related to uropathogenic E. coli (cnf1, hlyD). The major STs associated with clinical isolates from dogs were by far the dog-associated ST372 (20.7%) and ST73 (20.1%), a lineage that had commonly been considered until now as human-associated. Resistance to ESC was found in 33 isolates (5.3%), along with one carbapenemase-producing isolate, and was mostly restricted to non-B2 isolates. In conclusion, the presence of virulent E. coli lineages may be the issue, rather than the presence of ESC-resistant isolates, and the risk of transmission of such virulent isolates to humans needs to be further studied.

European multicenter study on antimicrobial resistance in bacteria isolated from companion animal urinary tract infections.

BACKGROUND: There is a growing concern regarding the increase of antimicrobial resistant bacteria in companion animals. Yet, there are no studies comparing the resistance levels of these organisms in European countries. The aim of this study was to investigate geographical and temporal trends of antimicrobial resistant bacteria causing urinary tract infection (UTI) in companion animals in Europe. The antimicrobial susceptibility of 22 256 bacteria isolated from dogs and cats with UTI was determined. Samples were collected between 2008 and 2013 from 16 laboratories of 14 European countries. The prevalence of antimicrobial resistance of the most common bacteria was determined for each country individually in the years 2012-2013 and temporal trends of bacteria resistance were established by logistic regression. RESULTS: The aetiology of uropathogenic bacteria differed between dogs and cats. For all bacterial species, Southern countries generally presented higher levels of antimicrobial resistance compared to Northern countries. Multidrug-resistant Escherichia coli were found to be more prevalent in Southern countries. During the study period, the level of fluoroquinolone-resistant E. coli isolated in Belgium, Denmark, France and the Netherlands decreased significantly. A temporal increase in resistance to amoxicillin-clavulanate and gentamicin was observed among E. coli isolates from the Netherlands and Switzerland, respectively. Other country-specific temporal increases were observed for fluoroquinolone-resistant Proteus spp. isolated from companion animals from Belgium. CONCLUSIONS: This work brings new insights into the current status of antimicrobial resistance in bacteria isolated from companion animals with UTI in Europe and reinforces the need for strategies aiming to reduce resistance.

GEP analysis validates high risk MDS and acute myeloid leukemia post MDS mice models and highlights novel dysregulated pathways.

BACKGROUND: In spite of the recent discovery of genetic mutations in most myelodysplasic (MDS) patients, the pathophysiology of these disorders still remains poorly understood, and only few in vivo models are available to help unravel the disease. METHODS: We performed global specific gene expression profiling and functional pathway analysis in purified Sca1+ cells of two MDS transgenic mouse models that mimic human high-risk MDS (HR-MDS) and acute myeloid leukemia (AML) post MDS, with NRASD12 and BCL2 transgenes under the control of different promoters MRP8NRASD12/tethBCL-2 or MRP8[NRASD12/hBCL-2], respectively. RESULTS: Analysis of dysregulated genes that were unique to the diseased HR-MDS and AML post MDS mice and not their founder mice pointed first to pathways that had previously been reported in MDS patients, including DNA replication/damage/repair, cell cycle, apoptosis, immune responses, and canonical Wnt pathways, further validating these models at the gene expression level. Interestingly, pathways not previously reported in MDS were discovered. These included dysregulated genes of noncanonical Wnt pathways and energy and lipid metabolisms. These dysregulated genes were not only confirmed in a different independent set of BM and spleen Sca1+ cells from the MDS mice but also in MDS CD34+ BM patient samples. CONCLUSIONS: These two MDS models may thus provide useful preclinical models to target pathways previously identified in MDS patients and to unravel novel pathways highlighted by this study.

Primary bronchial carcinoma associated with bone marrow metastasis and paraneoplastic monoclonal gammopathy in a cat.

CASE SUMMARY: Herein we describe an unusual metastatic pattern and paraneoplastic manifestation of a bronchial carcinoma in a cat. An 8 year-old cat presented with a diminished appetite, dysphagia, weight loss, lethargy and coughing. Thoracic radiographs revealed a lung mass. Bronchial carcinoma was diagnosed on the basis of histology and was associated with a lymphoplasmocytic infiltration of the fibrovascular stroma. Biochemistry showed hyperproteinaemia. Serum protein electrophoresis showed a narrow spike in the gamma region. Bone marrow cytology revealed an infiltrate with numerous clustered epithelial cells. The cat was euthanased 2 months later because of anorexia and poor general condition. RELEVANCE AND NOVEL INFORMATION: To the best of our knowledge, this is the first clinical description of primary bronchial carcinoma associated with bone marrow metastases and paraneoplastic monoclonal gammopathy in a cat.

Determination of morphological, biometric and biochemical susceptibilities in healthy Eurasier dogs with suspected inherited glaucoma.

In both humans and dogs, the primary risk factor for glaucoma is high intraocular pressure (IOP), which may be caused by iridocorneal angle (ICA) abnormalities. Oxidative stress has also been implicated in retinal ganglion cell damage associated with glaucoma. A suspected inherited form of glaucoma was recently identified in Eurasier dogs (EDs), a breed for which pedigrees are readily available. Because of difficulties in assessing ICA morphology in dogs with advanced glaucoma, we selected a cohort of apparently healthy dogsfor the investigation of ICA morphological status, IOP and plasma concentrations of oxidative stress biomarkers. We aimed to establish correlations between these factors, to identify predictive markers of glaucoma in this dog breed. A cohort of 28 subjects, volunteered for inclusion by their owners, was selected by veterinary surgeons. These dogs were assigned to four groups: young males, young females (1-3 years old), adult males and adult females (4-8 years old). Ocular examination included ophthalmoscopy, tonometry, gonioscopy, biometry and ultrasound biomicroscopy (UBM), and the evaluation of oxidative stress biomarkers consisting of measurements of plasma glutathione peroxidase (GP) activity and taurine and metabolic precursor (methionine and cysteine) concentrations in plasma. The prevalence of pectinate ligament abnormalities was significantly higher in adult EDs than in young dogs. Moreover, in adult females, high IOP was significantly correlated with a short axial globe length, and a particularly large distance between Schwalbe's line and the anterior lens capsule. GP activity levels were significantly lower in EDs than in a randomized control group of dogs, and plasma taurine concentrations were higher. Hence, ICA abnormalities were associated with weaker antioxidant defenses in EDs, potentially counteracted by higher plasma taurine concentrations. This study suggests that EDs may constitute an appropriate canine model for the development of glaucoma. This cohort will be used as a sentinel for longitudinal monitoring.

BCL-2 inhibition with ABT-737 prolongs survival in an NRAS/BCL-2 mouse model of AML by targeting primitive LSK and progenitor cells.

Myelodysplastic syndrome (MDS) transforms into an acute myelogenous leukemia (AML) with associated increased bone marrow (BM) blast infiltration. Using a transgenic mouse model, MRP8[NRASD12/hBCL-2], in which the NRAS:BCL-2 complex at the mitochondria induces MDS progressing to AML with dysplastic features, we studied the therapeutic potential of a BCL-2 homology domain 3 mimetic inhibitor, ABT-737. Treatment significantly extended lifespan, increased survival of lethally irradiated secondary recipients transplanted with cells from treated mice compared with cells from untreated mice, with a reduction of BM blasts, Lin-/Sca-1(+)/c-Kit(+), and progenitor populations by increased apoptosis of infiltrating blasts of diseased mice assessed in vivo by technicium-labeled annexin V single photon emission computed tomography and ex vivo by annexin V/7-amino actinomycin D flow cytometry, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, caspase 3 cleavage, and re-localization of the NRAS:BCL-2 complex from mitochondria to plasma membrane. Phosphoprotein analysis showed restoration of wild-type (WT) AKT or protein kinase B, extracellular signal-regulated kinase 1/2 and mitogen-activated protein kinase patterns in spleen cells after treatment, which showed reduced mitochondrial membrane potential. Exon specific gene expression profiling corroborates the reduction of leukemic cells, with an increase in expression of genes coding for stem cell development and maintenance, myeloid differentiation, and apoptosis. Myelodysplastic features persist underscoring targeting of BCL-2-mediated effects on MDS-AML transformation and survival of leukemic cells.

Engineering mouse models with myelodysplastic syndrome human candidate genes; how relevant are they?

Myelodysplastic syndromes represent particularly challenging hematologic malignancies that arise from a large spectrum of genetic events resulting in a disease characterized by a range of different presentations and outcomes. Despite efforts to classify and identify the key genetic events, little improvement has been made in therapies that will increase patient survival. Animal models represent powerful tools to model and study human diseases and are useful pre-clinical platforms. In addition to enforced expression of candidate oncogenes, gene inactivation has allowed the consequences of the genetic effects of human myelodysplastic syndrome to be studied in mice. This review aims to examine the animal models expressing myelodysplastic syndrome-associated genes that are currently available and to highlight the most appropriate model to phenocopy myelodysplastic syndrome disease and its risk of transformation to acute myelogenous leukemia.

Localization of the NRAS:BCL-2 complex determines anti-apoptotic features associated with progressive disease in myelodysplastic syndromes.

We have previously demonstrated that two prognostic features of myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML), mutant NRAS and over-expressing BCL-2, cooperate physically and functionally in vivo. Screening of MDS patient bone marrow (BM) identified NRAS:BCL-2 co-localization in 64% cases, correlating with percentage BM blasts, apoptotic features and disease status (p<0.0001). Localization of the complex at the plasma membrane or the mitochondria correlated with disease and apoptosis features in MDS patients, whilst caspase-9 mediated mechanism was elucidated in vivo and in vitro. The intensity and localization of the RAS:BCL-2 complex merits further evaluation as a novel biomarker of MDS.

Identification of JAK2 mutations in canine primary polycythemia.

OBJECTIVE: Primary polycythemia in dogs is classified as a myeloproliferative syndrome with a chronic progressive course and unspecific symptoms. Diagnosis is based on exclusion criteria. In humans, the presence of an acquired recurrent mutation within the JAK2 gene has recently been identified in 90% of the patients with polycythemia vera. This mutation (V617F) is located in the pseudokinase domain of JAK2, leading to constitutive activation of the kinase responsible for the polycythemia. Detection of the mutation has now become a major diagnostic tool in humans for polycythemia vera diagnosis. As the canine JAK2 gene shares strong homology with its human counterpart, we looked for the presence of JAK2 mutations in dogs with an elevated hematocrit. MATERIALS AND METHODS: Direct sequencing of the JAK2 exon 14 was performed on DNA extracted from the peripheral blood of five dogs suspected of primary polycythemia. Mutant subclones were expressed in interleukin-3-dependent BaF3 cells and tested for cytokine independency. RESULTS: One dog presented with a three-base change in codons 617 and 618 of JAK2 giving rise to V617F and C618L mutations. By polymerase chain reaction product subcloning, we demonstrated the coexistence of the wild-type sequence and a triple mutant sequence, while DNA from buccal swab contained the wild-type sequence only. Transfection of BaF3 cells with the triple mutant cDNA, but not with the wild-type complementary DNA, resulted in cytokine-independent growth and constitutive signal transducer and activation of transcription 5 phosphorylation. CONCLUSIONS: Identical mutations of the JAK2 gene occur in humans and dogs, giving rise to a constitutively active JAK2 kinase, suggesting a common mechanism for human and canine diseases. Thus, common diagnostic tools and therapeutic approaches may be relevant.

BCL-2 and mutant NRAS interact physically and functionally in a mouse model of progressive myelodysplasia.

Myelodysplastic syndromes (MDS) are clonal stem cell hematologic disorders that evolve to acute myeloid leukemia (AML) and thus model multistep leukemogenesis. Activating RAS mutations and overexpression of BCL-2 are prognostic features of MDS/AML transformation. Using NRASD12 and BCL-2, we created two distinct models of MDS and AML, where human (h)BCL-2 is conditionally or constitutively expressed. Our novel transplantable in vivo models show that expression of hBCL-2 in a primitive compartment by mouse mammary tumor virus-long terminal repeat results in a disease resembling human MDS, whereas the myeloid MRP8 promoter induces a disease with characteristics of human AML. Expanded leukemic stem cell (Lin(-)/Sca-1(+)/c-Kit(+)) populations and hBCL-2 in the increased RAS-GTP complex within the expanded Sca-1(+) compartment are described in both MDS/AML-like diseases. Furthermore, the oncogenic compartmentalizations provide the proapoptotic versus antiapoptotic mechanisms, by activating extracellular signal-regulated kinase and AKT signaling, in determination of the neoplastic phenotype. When hBCL-2 is switched off with doxycycline in the MDS mice, partial reversal of the phenotype was observed with persistence of bone marrow blasts and tissue infiltration as RAS recruits endogenous mouse (m)BCL-2 to remain active, thus demonstrating the role of the complex in the disease. This represents the first in vivo progression model of MDS/AML dependent on the formation of a BCL-2:RAS-GTP complex. The colocalization of BCL-2 and RAS in the bone marrow of MDS/AML patients offers targeting either oncogene as a therapeutic strategy.

Reactive oxygen species, DNA damage, and error-prone repair: a model for genomic instability with progression in myeloid leukemia?

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of disorders characterized by ineffective hematopoiesis, with an increased propensity to develop acute myelogenous leukemia (AML). The molecular basis for MDS progression is unknown, but a key element in MDS disease progression is loss of chromosomal material (genomic instability). Using our two-step mouse model for myeloid leukemic disease progression involving overexpression of human mutant NRAS and BCL2 genes, we show that there is a stepwise increase in the frequency of DNA damage leading to an increased frequency of error-prone repair of double-strand breaks (DSB) by nonhomologous end-joining. There is a concomitant increase in reactive oxygen species (ROS) in these transgenic mice with disease progression. Importantly, RAC1, an essential component of the ROS-producing NADPH oxidase, is downstream of RAS, and we show that ROS production in NRAS/BCL2 mice is in part dependent on RAC1 activity. DNA damage and error-prone repair can be decreased or reversed in vivo by N-acetyl cysteine antioxidant treatment. Our data link gene abnormalities to constitutive DNA damage and increased DSB repair errors in vivo and provide a mechanism for an increase in the error rate of DNA repair with MDS disease progression. These data suggest treatment strategies that target RAS/RAC pathways and ROS production in human MDS/AML.