Thomas Fett

Purification and characterization of a 43.5 kDa keratinolytic metalloprotease from Microsporum canis.

A keratinolytic protease secreted by a feline clinical isolate of Microsporum canis cultivated in a broth containing feline keratin as the sole nitrogen source was purified from the culture filtrate by affinity chromatography on bacitracin-agarose and by hydrophobic chromatography on octyl-agarose. The enzyme had an apparent molecular mass of 43.5 kDa and the pI was 7.7. It had a significant activity against keratin azure, elastin-Congo red and denatured type I collagen (azocoll). Using the latter substrate, the optimum pH was around 8 and the apparent optimum temperature around 50 degrees C. The protease was strongly inhibited by 1,10-phenanthroline, phosphoramidon and EDTA. The first 13 N-terminal amino acid sequence showed a 61% homology with that of the extracellular metalloprotease of Aspergillus fumigatus and with the neutral protease I of A. oryzae, confirming that this 43.5 kDa keratinase is a metalloprotease. This keratinolytic metalloprotease could be a virulence-related factor involved in pathophysiological mechanisms of M. canis dermatophytosis.

Belgian Wildlife as Potential Zoonotic Reservoir of Hepatitis E Virus

Hepatitis E is an acute human liver disease in healthy individuals but may become chronic in immunocompromised patients. It is caused by the hepatitis E virus (HEV) and can have a zoonotic origin, particularly in high-income countries. In this study, 383 sera from wild boars were selected for serology; for virological analyses, 69 sera and 61 livers from young wild boars were used. A total of 189 and 235 sera of, respectively, red deer and roe deer were collected for serological analysis. For virological analyses, 84 and 68 sera and 29 and 27 livers from, respectively, red and roe deer were sampled. An apparent seroprevalence of 34% (95% CI 29.71-39.46) was found in wild boars, of 1% (95% CI 0-2.4) in red deer and 3% (95% CI 0.8-4.2) in roe deer. To assess the ELISA screening prevalence, Western blot (WB) analyses were carried out, a receiver operating characteristic curve analysis was performed and different scenarios with varying ELISA specificities relative to WB were analysed. Seroprevalence remained high whatever the scenario in the wild boar population. In wild boar, 4 of 69 sera and 4 of 61 livers were detected as positive for HEV RNA. All sequences obtained from sera belonged to genotype HEV-3. HEV RNA, belonging to genotype HEV-3, was detected in one of 29 red deer livers. Wild boar can be considered as a host reservoir of the virus in Belgium. However, in contrast to the epidemiological role played by them in other countries, the low prevalence in deer makes these species an unlikely reservoir. This evidence needs further investigation to determine in which situation deer can serve as reservoir. These results also raise the question of the dynamics of HEV infection between wild fauna, domestic pigs and humans.

Characterization of the caprine (Capra hircus) beta-2 integrin CD18-encoding cDNA and identification of mutations potentially responsible for the ruminant-specific virulence of Mannheimia haemolytica

The leukocyte integrins play a critical role in a great number of cellular adhesive interactions during the immune response. We describe here the isolation and characterization of the caprine β2 (CD18) sub-unit, common to the leukocyte β2-integrin family. The deduced 770-amino-acid sequence reveals a transmembrane protein with 80, 81, 83, 96 and 99% identity with its canine, murine, human, bovine and ovine homologues respectively. Analysis of CD18 sequences emphasizes the functional importance of the β2 sub-unit I-like domain, and included metal ion-dependent adhesion site-like motif and confirms that of the cytoplasmic tail. Moreover, comparisons of ruminant versus non-ruminant CD18 sequences allowed the identification of 16 potential mutation sites that could be held responsible for the unique virulence of Mannheimia haemolytica for ruminants. Mannheimiosis is known to be the major respiratory disease among ruminants, whereas it is not pathogenic for other mammals, an observation that has been attributed to a specific interaction between M. haemolytica leukotoxin and ruminants’ CD18. Therefore, the data provided here offer the possibility to explore new avenues in studies based on the caprine model and provide key information for future studies aimed at elucidating the molecular mechanisms underlying the ruminant-specific virulence of M. haemolytica.

Flow cytometric probing of mitochondrial function in equine peripheral blood mononuclear cells

BackgroundThe morphopathological picture of a subset of equine myopathies is compatible with a primary mitochondrial disease, but functional confirmation in vivo is still pending. The cationic dye JC-1 exhibits potential-dependent accumulation in mitochondria that is detectable by a fluorescence shift from green to orange. As a consequence, mitochondrial membrane potential can be optically measured by the orange/green fluorescence intensity ratio. A flow cytometric standardized analytic procedure of the mitochondrial function of equine peripheral blood mononuclear cells is proposed along with a critical appraisal of the crucial questions of technical aspects, reproducibility, effect of time elapsed between blood sampling and laboratory processing and reference values.ResultsThe JC-1-associated fluorescence orange and green values and their ratio were proved to be stable over time, independent of age and sex and hypersensitive to intoxication with a mitochondrial potential dissipator. Unless time elapsed between blood sampling and laboratory processing does not exceed 5 hours, the values retrieved remain stable. Reference values for clinically normal horses are given.ConclusionWhenever a quantitative measurement of mitochondrial function in a horse is desired, blood samples should be taken in sodium citrate tubes and kept at room temperature for a maximum of 5 hours before the laboratory procedure detailed here is started. The hope is that this new test may help in confirming, studying and preventing equine myopathies that are currently imputed to mitochondrial dysfunction.

Mannheimia haemolytica leukotoxin-induced cytolysis of caprine (Capra hircus) leukocytes is mediated by the CD18 subunit of beta2-integrins

Mannheimiosis is the major respiratory disease among some ruminants, whereas it is not pathogenic for other mammals, an observation that has been attributed to a specific interaction between Mannheimia haemolytica leukotoxin (Lkt) and bovine or ovine CD18 subunit of lymphocyte function-associated antigen-1 (LFA-1) and Mac-1. We therefore hypothesized that Lkt utilizes CD18 as its receptor on caprine leukocytes as well. We have transiently transfected the beta2-integrins-deficient K-562 cell line with cDNAs encoding caprine CD11a and caprine CD18 to determine the susceptibility of the transfectants to Lkt-induced cytolysis. Flow cytometric analysis of the transfectants revealed surface expression of caprine LFA-1 and lysis by Lkt in a concentration-dependent manner whereas the parent cells were not. Moreover, K562 cells expressing caprine CD18 and human or bovine CD11a were also sensitive to Lkt whereas K-562 cells expressing caprine CD11a and human CD18 were not. Taken together, these results indicate that CD18 on caprine leukocytes serves as a receptor for Lkt.

Molecular evidence of Anaplasma phagocytophilum in wild boar (Sus scrofa) in Belgium

BackgroundAnaplasma phagocytophilum is a tick-borne pathogen of veterinary and human importance. Both ticks as vectors and vertebrates as reservoir hosts are essential for the cycle maintenance of this bacterium. Currently, the whole range of animal species reservoirs for A. phagocytophilum in natural environment is still unknown. Therefore, the aim of this study was to estimate the prevalence of infection with A. phagocytophilum in the wild boar population in southern Belgium.ResultsIn the frame of a targeted surveillance program, 513 wild boars were sampled during the hunting season 2011. A nested 16S rRNA PCR was used to screen the presence of A. phagocytophilum DNA in spleen of boars. Within 513 samples, 5 (0,97%) were tested PCR positive and identification was confirmed by sequencing.ConclusionsThis study gives the first insight of presence of A. phagocytophilum in wild boars in southern Belgium.

Porcine CD18 mediates Actinobacillus pleuropneumoniae ApxIII species-specific toxicity

Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, produces Apx toxins that are recognized as major virulence factors. Recently, we showed that ApxIIIA-cytotoxic activity specifically targets Sus scrofa leukocytes. Since both LtxA from Aggregatibacter actinomycetemcomitans (aggressive periodontitis in humans) and LktA from Mannheimia haemolytica (pneumonia in ruminants) share this characteristic, respectively towards human and ruminant leukocytes, and because both use the CD18 subunit to interact with their respective LFA-1, we hypothesized that ApxIIIA was likely to bind porcine CD18 to exercise its deleterious effects on pig leukocytes. A β 2−integrin-deficient ApxIIIA-resistant human erythroleukemic cell line was transfected either with homologous or heterologous CD11a/CD18 heterodimers using a set of plasmids coding for human (ApxIIIA-resistant), bovine (-resistant) and porcine (-susceptible) CD11a and CD18 subunits. Cell preparations that switched from ApxIIIA-resistance to -susceptibility were then sought to identify the LFA-1 subunit involved. The results showed that the ApxIIIA-resistant recipient cell line was rendered susceptible only if the CD18 partner within the LFA-1 heterodimer was that of the pig. It is concluded that porcine CD18 is necessary to mediate A. pleuropneumoniae ApxIIIA toxin-induced leukolysis.

The CD11a partner in Sus scrofa lymphocyte function-associated antigen-1 (LFA-1): mRNA cloning, structure analysis and comparison with mammalian homologues

BackgroundLymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18, alphaLbeta2), the most abundant and widely expressed beta2-integrin, is required for many cellular adhesive interactions during the immune response. Many studies have shown that LFA-1 is centrally involved in the pathogenesis of several diseases caused by Repeats-in-toxin (RTX) -producing bacteria.ResultsThe porcine-LFA-1 CD11a (alpha) subunit coding sequence was cloned, sequenced and compared with the available mammalian homologues in this study. Despite some focal differences, it shares all the main characteristics of these latter. Interestingly, as in sheep and humans, an allelic variant with a triplet insertion resulting in an additional Gln-744 was consistently identified, which suggests an allelic polymorphism that might be biologically relevant.ConclusionTogether with the pig CD18-encoding cDNA, which has been available for a long time, the sequence data provided here will allow the successful expression of porcine CD11a, thus giving the first opportunity to express the Sus scrofa beta2-integrin LFA-1 in vitro as a tool to examine the specificities of inflammation in the porcine species.

Molecular characterisation of the caprine (Capra hircus) lymphocyte function-associated antigen-1 alpha subunit-encoding cDNA

BackgroundLymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18, alpha L beta 2) is required for many cellular adhesive interactions during the immune response.ResultsThe Capra hircus CD11a-encoding cDNA was sequenced and compared with its human, murine, rat, bovine and ovine counterparts. Despite some focal differences, it shares all the main characteristics of its known mammalian homologues.ConclusionTherefore, along with the caprine CD18-encoding cDNA, which has been available for a few months, the sequence data revealed here will allow the Capra hircus LFA-1 expression in vitro as a tool to explore the specificities of inflammation in the caprine species.

Feline panleukopenia virus in cerebral neurons of young and adult cats

BackgroundPerinatal infections with feline panleukopenia virus (FPV) have long been known to be associated with cerebellar hypoplasia in kittens due to productive infection of dividing neuroblasts. FPV, like other parvoviruses, requires dividing cells to replicate which explains the usual tropism of the virus for the digestive tract, lymphoid tissues and bone marrow in older animals.ResultsIn this study, the necropsy and histopathological analyses of a series of 28 cats which died from parvovirus infection in 2013 were performed. Infections were confirmed by real time PCR and immunohistochemistry in several organs. Strikingly, while none of these cats showed cerebellar atrophy or cerebellar positive immunostaining, some of them, including one adult, showed a bright positive immunostaining for viral antigens in cerebral neurons (diencephalon). Furthermore, infected neurons were negative by immunostaining for p27Kip1, a cell cycle regulatory protein, while neighboring, uninfected, neurons were positive, suggesting a possible re-entry of infected neurons into the mitotic cycle. Next-Generation Sequencing and PCR analyses showed that the virus infecting cat brains was FPV and presented a unique substitution in NS1 protein sequence. Given the role played by this protein in the control of cell cycle and apoptosis in other parvoviral species, it is tempting to hypothesize that a cause-to-effect between this NS1 mutation and the capacity of this FPV strain to infect neurons in adult cats might exist.ConclusionsThis study provides the first evidence of infection of cerebral neurons by feline panleukopenia virus in cats, including an adult. A possible re-entry into the cell cycle by infected neurons has been observed. A mutation in the NS1 protein sequence of the FPV strain involved could be related to its unusual cellular tropism. Further research is needed to clarify this point.