Clinical bleeding diathesis, laboratory haemostatic aberrations and survival in dogs infected with Angiostrongylus vasorum: 180 cases (2005-2019).

OBJECTIVES: Bleeding diathesis is a complication in dogs infected with Angiostrongylus vasorum. This retrospective study investigated clinical and laboratory haemostatic differences in A. vasorum-positive dogs with and without signs of bleeding and impact of bleeding on survival. MATERIALS AND METHODS: Demographics, type of clinical bleeding, haematocrit and a range of haemostatic tests, including thromboelastography and derived velocity curves were retrospectively registered from A. vasorum-positive dogs. All parameters were compared between dogs with and without signs of bleeding using univariable analyses. Binomial and multinomial regression models were applied to examine specific indicators in the bleeding dogs. P-values were false discovery rate adjusted, and adjusted P<0.05 was considered significant. RESULTS: One hundred and eighty dogs entered the study, including 65 dogs (36.1%) presenting with bleeding diathesis. Different types of cutaneous and mucosal bleeding were the most common clinical findings. Twenty dogs presented with neurological signs associated with intracranial and intra-spinal bleeding. One hundred and thirty-seven dogs had haematological and/or haemostatic laboratory analyses performed. Haematocrit, platelet count, thromboelastographic angle, maximum amplitude, global clot strength, maximum rate of thrombin generation and total thrombin generation were decreased, while prothrombin time was prolonged in bleeding dogs. Survival rate of bleeding dogs was lower at hospital discharge (76.9%) and 1 month after diagnosis (66.0%) than in dogs without signs of bleeding (94.8% and 90.1% at discharge and at 1 month, respectively). CLINICAL SIGNIFICANCE: Several haemostatic aberrations were detected in A. vasorum-positive dogs with bleeding diathesis. Bleeding was identified as an important negative prognostic indicator in A. vasorum-positive dogs.

Presence of nucleosomes in plasma and increased thrombin generation in dogs with acute and chronic gastroenteropathies.

Neutrophil extracellular traps (NETs) which contain nucleosomes protect the host by eliminating extracellular pathogens. However, any inflammatory stimuli can activate NETs and eventually lead to an immune overreaction leading to autoimmune diseases and thrombosis. Acute/chronic gastroenteropathies(aGE/cGE) are prevalent in dogs, and are associated with a strong inflammatory component. The aim of this study was to investigate if dogs with aGE and cGE have increased concentrations of nucleosomes indicative of NETs formation, and whether increased concentrations of nucleosomes are associated with hypercoagulability determined by increased thrombin generation. Twenty-six dogs were enrolled. The dogs were healthy (n = 11), or presented with aGE(n = 7) or cGE(n = 8). Minimum database including CRP, APTT, PT and fibrinogen, was obtained from all dogs. Citrated plasma was batched and used for subsequent analyses. Nucleosome concentration was analysed using a Cell-Death Detection ELISA-kit and thrombin generation by a calibrated automated thrombogram assay. No statistical differences in nucleosome concentrations were present between the groups. Although a numerically increased concentration of nucleosomes where seen in dogs with aGE(median;range) (0.019 AU;0.003-0.088) and cGE(0.023 AU;0.011-0.256) compared to controls(0.007 AU;0.003-0.042). One dog with GI-lymphoma demonstrated a markedly increased concentration of nucleosomes (0.256 AU). Dogs with aGE showed increased thrombin generation by increased peak (p = 0.03) and endogenous thrombin potential (p = 0.03); and increased CRP (p = 0.001), fibrinogen (p = 0.0002) and prolonged APTT (p = 0.03) compared to controls. This proof of concept study demonstrates that dogs with aGE and cGE have presence of nucleosomes with marked increase in one dog with GI-lymphoma. Nucleosomes might be linked to haemostatic alterations in dogs with inflammatory and neoplastic diseases.

Circulating let-7g is down-regulated in Bernese Mountain dogs with disseminated histiocytic sarcoma and carcinomas - a prospective study.

Cancer is a prevalent cause of mortality in Bernese mountain dogs (BMDs). Circulating microRNAs (miRNAs) are found in blood and have been identified as promising biomarkers in various neoplastic diseases in humans. In the current study, the expression profile of different types of miRNAs was investigated in healthy BMDs and BMDs with cancer. Seven healthy and six non-treated BMDs with cancer [four with disseminated histiocytic sarcomas (DHS)] were enrolled in this study. Clinical evaluations including physical examination, blood analysis, urinalysis and diagnostic imaging were performed on all dogs. Twenty-four different miRNAs were profiled from RNA isolated from whole blood preserved in PAXgene® tubes using quantitative real-time PCR (qPCR). The miRNA let-7g was significantly down-regulated in dogs with cancer (P = 0.002) and dogs with DHS (P = 0.011) compared with healthy controls. This miRNA is a known tumour suppressor and further analyses are warranted to assess its value as a non-invasive biomarker for early detection of different types of cancer in BMDs.

Visualization of haemophilic arthropathy in F8-/- rats by ultrasonography and micro-computed tomography.

INTRODUCTION: A major complication of haemophilia is haemophilic arthropathy (HA), a debilitating disorder with an incompletely defined pathobiology. High-resolution imaging may provide new knowledge about onset and progression of HA, and thereby support identification of new treatment opportunities. Recently, a F8-/- rat model of HA was developed. The size of the rat allows for convenient and high resolution imaging of the joints, which could enable in vivo studies of HA development. AIM: To determine whether HA in the F8-/- rat can be visualized using ultrasonography (US) and micro-computed tomography (µCT). METHODS: Sixty F8-/- and 20 wild-type rats were subjected to a single or two induced knee bleeds. F8-/- rats were treated with either recombinant human FVIII (rhFVIII) or vehicle before the induction of knee bleeds. Haemophilic arthropathy was visualized using in vivo US and ex vivo µCT, and the observations correlated with histological evaluation. RESULTS: US and µCT detected pathologies in the knee related to HA. There was a strong correlation between disease severity determined by µCT and histopathology. rhFVIII treatment reduced the pathology identified with both imaging techniques. CONCLUSION: US and µCT are suitable imaging techniques for detection of blood-induced joint disease in F8-/- rats and may be used for longitudinal studies of disease progression.

Altered collagen turnover in factor VIII-deficient rats with hemophilic arthropathy identifies potential novel serological biomarkers in hemophilia.

Essentials Joint bleeding in hemophilia may induce significant remodeling of the extracellular matrix. Biomarkers of collagen turnover were investigated in a F8-/- rat model of hemophilic arthropathy. Biomarkers of cartilage degradation increased significantly during development of arthropathy. Basement membrane and interstitial matrix turnover changed significantly following hemarthrosis. SUMMARY: Background Hemophilic arthropathy is a severe complication of hemophilia. It is caused by recurrent bleeding into joint cavities, which leads to synovial inflammation, fibrosis, cartilage degradation and bone remodeling. Extracellular matrix remodeling of affected tissues is a hallmark of these pathological processes. Objectives The aim of this study was to use serological biomarkers of collagen turnover to evaluate extracellular matrix remodeling in a factor VIII-deficient rat model of hemophilic arthropathy. Methods F8-/- rats and wild-type littermate controls were subjected to repeated knee bleeds induced by needle puncture on days 0 and 14. Development of arthropathy was confirmed by histology after termination on day 28. Serum samples were collected at baseline and throughout the study and analyzed for biomarkers of collagen turnover, including collagens of the basement membrane (type IV collagen), the interstitial matrix (collagen types III, V and VI) and cartilage (type II collagen). Results In F8-/- rats, induced knee bleeding and subsequent development of arthropathy caused significant alterations in collagen turnover, measured as changes in serological biomarkers of basement membrane turnover, interstitial matrix turnover and cartilage degradation. Biomarkers of type II collagen degradation correlated significantly with cartilage degradation and degree of arthropathy. Hemophilic rats had a 50% higher turnover of the basement membrane than wild-type littermates at baseline. Conclusions Joint bleeding and hemophilic arthropathy cause changes in turnover of extracellular matrix collagens in hemophilic rats. Biomarkers of collagen turnover may be used to monitor joint bleeding and development of blood-induced joint disease in hemophilia.

The F8(-/-) rat as a model of hemophilic arthropathy.

UNLABELLED: Essentials Validating the F8 rat as a new intermediate-size animal model of hemophilic arthropathy. Factor VIII (FVIII) treated F8(-/-) rats suffered induced hemarthrosis analyzed by histopathology. F8 (-/-) animals develop hemophilic arthropathy upon hemarthrosis, preventable by FVIII treatment. The F8 (-/-) rat presents as a new pharmacologic model of hemophilic arthropathy. SUMMARY: Background Translational animal models of hemophilia are valuable for determining the pathobiology of the disease and its co-morbidities (e.g. hemophilic arthropathy, HA). The biologic mechanisms behind the development of HA, a painful and debilitating condition, are not completely understood. We recently characterized a F8(-/-) rat, which could be a new preclinical model of HA. Objectives To establish the F8(-/-) rat as a model of HA by determining if the F8(-/-) rat develops HA resembling human HA after an induced joint bleed and whether a second joint bleed causes further disease progression. Methods Wild-type and F8(-/-) rats were treated with vehicle or recombinant human factor VIII (rhFVIII) prior to a needle-induced joint bleed. Joint swelling was measured prior to injury, the following 7 days and upon euthanasia. Histologic sections of the joint were stained, and athropathic changes identified and scored with regard to synovitis, bone remodelling, cartilage degradation and hemosiderin deposition. Results Vehicle-treated F8(-/-) rats experienced marked joint swelling and developed chronic degenerative joint changes (i.e. fibrosis of the subsynovial membrane, chondrocyte loss and excessive bone remodeling). Treatment with rhFVIII reduced or prevented swelling and degenerative joint changes, returning the F8(-/-) animals to a wild-type phenotype. Conclusion The hemophilic phenotype of the F8(-/-) rat resulted in a persistent hemarthrosis following an induced joint bleed. This caused development of HA resembling human HA, which was prevented by rhFVIII treatment, confirming the potential of the F8(-/-) rat as a model of HA.

Prevalence, Antimicrobial Resistance and Risk Factors for Thermophilic Campylobacter Infections in Symptomatic and Asymptomatic Humans in Tanzania.

The genus Campylobacter comprises members known to be a leading cause of foodborne gastrointestinal illness worldwide. A study was conducted to determine the epidemiology and antimicrobial resistance of Campylobacter in humans in Morogoro, Eastern Tanzania. Isolation of Campylobacter from stool specimens adopted the Cape Town protocol. Campylobacter isolates were preliminarily identified by conventional phenotypic tests and subsequently confirmed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry and polymerase chain reaction. Antimicrobial resistance testing employed the disc diffusion method. A small proportion of the test isolates was also subjected to agar dilution method. Risk factors for human illness were determined in an unmatched case-control study. Thermophilic Campylobacter were isolated from 11.4% of the screened individuals (n = 1195). The agreement between PCR and MALDI-TOF was perfect (κ = 1.0). Symptomatics and young individuals were infected with higher numbers than asymptomatic and adults, respectively. The majority (84.6%) of the isolates were C. jejuni and the remaining were C. coli. Isolates had highest resistance (95.6%) for colistin sulphate and lowest for ciprofloxacin (22.1%). The rates of resistance for other antibiotics (azithromycin, erythromycin, tetracycline, cephalothin, gentamycin, nalidixic acid, ampicillin, amoxycillin, norfloxacin, chloramphenicol) ranged from 44.1% to 89%. Comparison between disc diffusion and agar dilution methods indicated a good correlation, and the tests were in agreement to each other (κ ≥ 0.75). Human illness was found to be associated with young age and consumption of chicken meat and pre-prepared salad. Our data indicate the presence of antibiotic-resistant thermophilic Campylobacter in humans in the study area. There is a need for routine investigation of the presence of the organisms in gastroenteritis aetiology, including determination of their antibiotic susceptibilities.

A novel F8 -/- rat as a translational model of human hemophilia A.

BACKGROUND: In preclinical hemophilia research, an animal model that reflects both the phenotype and the pathology of the disease is needed. OBJECTIVES: Here, we describe the generation and characterization of a novel genetically engineered F8(-/-) rat model. METHODS: The rats were produced on a Sprague Dawley background with the zinc finger nuclease technique. A founder with a 13-bp deletion in exon 16 causing a premature translational stop in the C-terminal part of the A3 domain of factor VIII was selected, and a breeding colony was established. RESULTS: Seventy per cent of the homozygous rats had clinically manifest spontaneous hemorrhagic episodes that needed treatment. The F8(-/-) rats had no detectable FVIII activity, and had a significantly prolonged activated partial thromboplastin time (APTT) and clot formation time as compared with wild-type (WT)/WT rats. In vitro spiking of rat plasma with human recombinant FVIII resulted in dose-dependent normalization of the APTT. CONCLUSION: On the basis of the targeted deletion in F8, and the distinct physical and analytic characteristics of the rat, we conclude that an FVIII-deficient rat strain has been generated that has the potential to contribute greatly to translational research.

Investigation of a screening programme and the possible identification of biomarkers for early disseminated histiocytic sarcoma in Bernese Mountain dogs.

The aim of the study was to construct a screening programme for disseminated histiocytic sarcoma (DHS) in Bernese Mountain dogs using diagnostic imaging and blood analysis and evaluate blood borne biomarkers as early disease detection biomarkers. Healthy Bernese Mountain dogs were screened on four occasions in an attempt to detect early disease. Eleven blood borne biomarkers were examined for their worth as early tumour biomarkers. During 2.5 years, five dogs with early DHS were identified; four of these by diagnostic imaging. No dogs developed symptomatic DHS without being detected within 6 months of the screening programme. Only serum ferritin showed potential as a blood borne marker of the disease. Median survival times for the dogs with early DHS were 226 days. Screening programmes every 6 months for Bernese Mountain dogs over 4 years of age including diagnostic imaging and ferritin measurements may identify early DHS.

The presence of antiphospholipid antibodies in healthy Bernese Mountain Dogs.

BACKGROUND: The role of antiphospholipid antibodies in the prolonged activated partial thromboplastin time (aPTT) previously identified in healthy Bernese Mountain Dogs remains unknown. In people, an isolated prolonged aPTT without evidence of bleeding might be because of a thrombophilic condition caused by antiphospholipid antibodies. OBJECTIVE: To examine if prolonged aPTT in healthy Bernese Mountain Dogs is because of antiphospholipid antibodies. ANIMALS: Twenty-two healthy Bernese Mountain Dogs and 10 healthy adult dogs of various breeds. METHODS: Prospective case control study. Healthy Bernese Moutain Dogs were examined twice over 6 months. Dogs were investigated for the presence of lupus anticoagulants and anticardiolipin (aCL) antibodies by the use of multiple aPTT tests with low and high lupus anticoagulant sensitivities, a mixing study, and an ELISA test for aCL antibody optical density to detect solid phase antiphospholipid antibodies. RESULTS: In all, 15 of 22 healthy Bernese Mountain Dogs were positive for lupus anticoagulants. The Bernese Mountain Dogs had markedly higher levels of aCL antibodies compared with the control dogs (P = .006). In all, 7 of 21 of the Bernese Mountain Dogs were positive for both lupus anticoagulants and aCL antibodies, whereas 4 of 21 Bernese Mountain Dogs were negative for both. CONCLUSIONS AND CLINICAL IMPORTANCE: Lupus anticoagulants and aCL antibodies could be the cause of prolonged aPTT in healthy Bernese Mountain Dogs. The importance of the antiphospholipid antibodies in the dogs remains unknown.

MLST clustering of Campylobacter jejuni isolates from patients with gastroenteritis, reactive arthritis and Guillain-Barré syndrome.

AIMS: To determine the diversity and population structure of Campylobacter jejuni (C. jejuni) isolates from Danish patients and to examine the association between multilocus sequence typing types and different clinical symptoms including gastroenteritis (GI), Guillain-Barré syndrome (GBS) and reactive arthritis (RA). METHODS AND RESULTS: Multilocus sequence typing (MLST) was used to characterize 122 isolates, including 18 from patients with RA and 8 from patients with GBS. The GI and RA isolates were collected in Denmark during 2002-2003 and the GBS isolates were obtained from other countries. In overall, 51 sequence types (STs) were identified within 18 clonal complexes (CCs). Of these three CCs, ST-21, ST-45 and ST-22 clonal complexes accounted for 64 percent of all isolates. The GBS isolates in this study significantly grouped into the ST-22 clonal complex, consistent with the PubMLST database isolates. There was no significant clustering of the RA isolates. CONCLUSIONS: Isolates from Denmark were found to be highly genetically diverse. GBS isolates grouped significantly with clonal complex ST-22, but the absence of clustering of RA isolates indicated that the phylogenetic background for this sequela could not be reconstructed using variation in MLST loci. Possibly, putative RA-associated genes may vary, by recombination or expression differences, independent of MLST loci. SIGNIFICANCE AND IMPACT OF THE STUDY: MLST typing of C. jejuni isolates from Danish patients with gastroenteritis confirmed that the diversity of clones in Denmark is comparable to that in other European countries. Furthermore, a verification of the grouping of GBS isolates compared to RA isolates provides information about evolution of the bacterial population resulting in this important sequela.

Imported schistosomiasis in Europe: sentinel surveillance data from TropNetEurop.

BACKGROUND: Schistosomiasis is a major parasitic disease, increasingly imported into temperate climates by immigrants from and travelers to endemic areas. METHOD: To generate valid data on imported infectious diseases to Europe and to recognize trends over time, the European Network on Imported Infectious Diseases Surveillance (TropNetEurop) was founded in 1999. Three hundred and thirty-three reports of schistosomiasis were analyzed for epidemiologic and clinical features. RESULTS: Male patients accounted for 64% of all cases. The average age of all patients was 29.5 years. The majority of patients were of European origin (53%). Europeans traveled predominantly for tourism (52%). Main reasons for travel for people from endemic areas were immigration and refuge (51%) and visits to relatives and friends (28%). The majority of infections were acquired in Africa; 92 infections were clearly attributable to Schistosoma haematobium, 130 to Schistosoma mansoni, and 4 to Schistosoma intercalatum. Praziquantel was the only treatment used. No deaths were recorded. CONCLUSION: TropNetEurop sentinel provides valuable epidemiologic and clinical data on imported schistosomiasis to Europe.

Epidemiology and clinical features of imported dengue fever in Europe: sentinel surveillance data from TropNetEurop.

Travelers have the potential both to acquire and to spread dengue virus infection. The incidence of dengue fever (DF) among European travelers certainly is underestimated, because few centers use standardized diagnostic procedures for febrile patients. In addition, DF is currently not reported in most European public health systems. Surveillance has commenced within the framework of a European Network on Imported Infectious Disease Surveillance (TropNetEurop) to gain information on the quantity and severity of cases of dengue imported into Europe. Descriptions of 294 patients with DF were analyzed for epidemiological information and clinical features. By far the most infections were imported from Asia, which suggests a high risk of DF for travelers to that region. Dengue hemorrhagic fever occurred in 7 patients (2.4%) all of whom recovered. Data reported by member sites of the TropNetEurop can contribute to understanding the epidemiology and clinical characteristics of imported DF.

Imported Falciparum malaria in Europe: sentinel surveillance data from the European network on surveillance of imported infectious diseases.

Malaria continues to have a high morbidity rate associated among European travelers. Thorough recording of epidemiological and clinical aspects of imported malaria has been helpful in the detection of new outbreaks and areas of developing drug resistance. Sentinel surveillance of data collected prospectively since 1999 has begun within TropNetEurop, a European network focusing on imported infectious diseases. TropNetEurop appears to cover approximately 10% of all patients with malaria seen in Europe. Reports of 1659 immigrants and European patients with Plasmodium falciparum malaria were analyzed for epidemiological information and data on clinical features. Regional data were quite diverse, reflecting local patterns of immigration and international travel. By far, the most infections were imported from West Africa. Europeans had more clinical complications; consequently, all deaths occurred in this group. Compared with European standards, the mortality rate was low (0.6% in Europeans). Data from TropNetEurop member sites can contribute to our understanding of the epidemiological and clinical findings regarding imported falciparum malaria.

Apical expression of herpes simplex virus type 2 glycoproteins in human neuroblastoma cells.

The expression of herpes simplex virus type 2 (HSV-2) glycoproteins on the surface of human neuroblastoma cells has been investigated using Millipore Millicell culture plate inserts. Utilizing a modified radioimmunoassay, we learned that glycoproteins B, C, D, E, and I were expressed predominantly on the apical membrane domain of the infected neuroblastoma cells. The unidirectional transport of HSV-2 glycoproteins was substantiated by the analysis of extracellular glycoproteins released from neuroblastoma cells. The results suggest that the evaluated HSV-2 glycoproteins were transported primarily to the apical plasma membrane domain of human neuroblastoma cells.

Cytokeratin intermediate filament pattern in uterine cervical biopsies.

Twenty-seven uterine cervical biopsies with histological diagnoses ranging from normal through dysplasia to invasive carcinoma were analysed for cytokeratin pattern in two-dimensional gel electrophoresis. No direct correlation between histological diagnosis and cytokeratin pattern was observed.

Cytokeratin intermediate filament pattern and human papillomavirus type in uterine cervical biopsies with different histological diagnosis.

The cytokeratin pattern and the presence of human papillomavirus (HPV) were analyzed in 53 uterine cervical biopsies. The biopsies were histologically characterized and the diagnosis ranged from normal through dysplasia to carcinoma. The cytokeratins were identified by their immunological reactivity with the monoclonal antibodies AE1 and AE3. The tissue was typed for the presence of HPV types 11, 16 and 18. We have previously shown that there was no correlation between the expression of cytokeratins No. 14, 15, 16 and 19 (K14, K15, K16 and K19) and the histological diagnosis of cervical biopsies. The present study shows that the cytokeratin pattern cannot be correlated to HPV infection of the cervical tissue either.