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.

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.

Cloning and characterisation of the primary structure of the sheep lymphocyte function-associated antigen-1 alpha subunit.

The leukocyte integrins play a critical role in a number of cellular adhesive interactions during the immune response. The ovine cDNA encoding CD11a, the predominant alpha subunit of the beta2-integrin family, was sequenced and compared with the human, bovine and murine sequences. Despite some focal differences, it shares all the main characteristics of its known mammalian homologues. Along with the ovine CD18-encoding cDNA, which is available for a few months, the sequence data provided here will allow the Ovis aries beta2-integrin CD11a/CD18 (LFA-1, alpha(L)beta2) expression in vitro as a tool to examine the specificities of inflammation in the ovine species.

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 beta(2) (CD18) sub-unit, common to the leukocyte beta(2)-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 beta(2) 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.

Molecular cloning and characterisation of the CD18 partner in ovine (Ovis aries) beta2-integrins.

The leukocyte integrins play a critical role in a number of cellular adhesive interactions during the immune response. We describe here the isolation and characterization of the ovine beta(2) (CD18) subunit, common to the leukocyte beta(2)-integrin family. The deduced 770-amino-acid sequence reveals a transmembrane protein with 81%, 83% and 95% identity with its murine, human and bovine homologues, respectively. Comparisons of CD18 sequences emphasize the functional importance of the beta(2) subunit I-like domain and included metal ion-dependent adhesion site (MIDAS)-like motif and confirm that of the cytoplasmic tail. The data provided here will offer the possibility to explore new avenues in studies based on the ovine model.

The bovine (Bos taurus) CD11a-encoding cDNA: molecular cloning, characterisation and comparison with the human and murine glycoproteins.

The bovine cDNA encoding CD11a, a cell-surface glycoprotein involved in multiple leukocyte functions, was sequenced and compared with the human and murine sequences. Despite some focal differences, it shares all the main characteristics of its known mammalian homologs. Along with the bovine CD18-encoding cDNA, which is available for a long time, the sequence data provided here will allow the successful expression of bovine CD11a, thus giving the first opportunity to express the Bos taurus beta(2)-integrin CD11a/CD18 (LFA-1, alpha(L)beta(2)) in vitro as a tool to examine the specificities of inflammation in the bovine species.

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.