RTX toxins are bacterial pore-forming toxins that are particularly abundant among pathogenic species of
Pasteurellaceae, in which they play a major role in virulence. RTX toxins of several primary ...pathogens of the family of
Pasteurellaceae are directly involved in causing necrotic lesions in the target organs. Many RTX toxins are known as haemolysins because they lyse erythrocytes
in vitro, an effect that is non-specific, but which serves as a useful marker in bacteriological identification and as an easily measurable signal
in vitro in experimental studies. More recent studies have shown that the specific targets of most RTX toxins are leukocytes, with RTX toxins binding to the corresponding β-subunit (CD18) of β2 integrins and then exerting cytotoxic activity. After uptake by the target cell, at sub-lytic concentrations, some RTX toxins are transported to mitochondria and induce apoptosis. For several RTX toxins the binding to CD18 has been shown to be host specific and this seems to be the basis for the host range specificity of these RTX toxins. Observations on two very closely related species of the
Pasteurellaceae family,
Actinobacillus suis, a porcine pathogen particularly affecting suckling pigs, and
Actinobacillus equuli subsp.
haemolytica, which causes pyosepticaemia in new-born foals (sleepy foal disease), have revealed that they express different RTX toxins, named ApxI/II and Aqx, respectively. These RTX toxins are specifically cytotoxic for porcine and equine leukocytes, respectively. Furthermore, the ApxI and Aqx toxins of these species, when expressed in an isogenetic background in
Escherichia coli, are specifically cytotoxic for leukocytes of their respective hosts. These data indicate the determinative role of RTX toxins in host specificity of pathogenic species of
Pasteurellaceae.
Abstract Live vaccines possess the advantage of having access to induce cell-mediated and antibody-mediated immunity; thus in certain cases they are able to prevent infection, and not only disease. ...Furthermore, live vaccines, particularly bacterial live vaccines, are relatively cheap to produce and easy to apply. Hence they are suitable to immunize large communities or herds. The induction of both cell-mediated immunity as well as antibody-mediated immunity, which is particularly beneficial in inducing mucosal immune responses, is obtained by the vaccine-strain's ability to colonize and multiply in the host without causing disease. For this reason, live vaccines require attenuation of virulence of the bacterium to which immunity must be induced. Traditionally attenuation was achieved simply by multiple passages of the microorganism on growth medium, in animals, eggs or cell cultures or by chemical or physical mutagenesis, which resulted in random mutations that lead to attenuation. In contrast, novel molecular methods enable the development of genetically modified organisms (GMOs) targeted to specific genes that are particularly suited to induce attenuation or to reduce undesirable effects in the tissue in which the vaccine strains can multiply and survive. Since live vaccine strains (attenuated by natural selection or genetic engineering) are potentially released into the environment by the vaccinees, safety issues concerning the medical as well as environmental aspects must be considered. These involve (i) changes in cell, tissue and host tropism, (ii) virulence of the carrier through the incorporation of foreign genes, (iii) reversion to virulence by acquisition of complementation genes, (iv) exchange of genetic information with other vaccine or wild-type strains of the carrier organism and (v) spread of undesired genes such as antibiotic resistance genes. Before live vaccines are applied, the safety issues must be thoroughly evaluated case-by-case. Safety assessment includes knowledge of the precise function and genetic location of the genes to be mutated, their genetic stability, potential reversion mechanisms, possible recombination events with dormant genes, gene transfer to other organisms as well as gene acquisition from other organisms by phage transduction, transposition or plasmid transfer and cis - or trans -complementation. For this, GMOs that are constructed with modern techniques of genetic engineering display a significant advantage over random mutagenesis derived live organisms. The selection of suitable GMO candidate strains can be made under in vitro conditions using basic knowledge on molecular mechanisms of pathogenicity of the corresponding bacterial species rather than by in vivo testing of large numbers of random mutants. This leads to a more targeted safety testing on volunteers and to a reduction in the use of animal experimentation.
General wildlife health surveillance is a valuable source of information on the causes of mortality, disease susceptibility and pathology of the investigated hosts and it is considered to be an ...essential component of early warning systems. However, the representativeness of data from such surveillance programs is known to be limited by numerous biases. The roe deer (Capreolus capreolus capreolus) is the most abundant ungulate and a major game species all over Europe. Yet, internationally available literature on roe deer pathology is scarce. The aims of this study were (1) to provide an overview of the causes of mortality or morbidity observed in roe deer in Switzerland and to assess potential changes in the disease pattern over time; and (2) to evaluate the value and limitations of a long term dataset originating from general wildlife health surveillance. We compiled 1571 necropsy reports of free ranging roe deer examined at the Centre for Fish and Wildlife Health in Switzerland from 1958 to 2014. Descriptive data analysis was performed considering animal metadata, submitter, pathologist in charge, laboratory methods, morphological diagnoses and etiologies. Recurrent causes of mortality and disease pictures included pneumonia, diarrhea, meningoencephalitis, actinomycosis, blunt trauma, predation, neoplasms and anomalies. By contrast, other diagnoses such as fatal parasitic gastritis, suspected alimentary intoxication and reproductive disorders appeared only in earlier time periods. Diseases potentially relevant for other animals or humans such as caseous lymphadenitis (or pseudotuberculosis), salmonellosis, paratuberculosis and listeriosis were sporadically observed. The disease pattern in roe deer from Switzerland was largely in accordance with previous reports. The observed fluctuations were consistent with methodical and/or personnel changes and varying disease awareness. Nevertheless, despite such limitations, the compiled data provide a valuable baseline. To facilitate comparison among studies, we recommend systematically archiving all case documents and fixed tissues and to perform data analyses more regularly and in a harmonized way.
Bacteria of the genus Pseudomonas occupy diverse environments. The Pseudomonas fluorescens group is particularly well-known for its plant-beneficial properties including pathogen suppression. Recent ...observations that some strains of this group also cause lethal infections in insect larvae, however, point to a more versatile ecology of these bacteria. We show that 26 P. fluorescens group strains, isolated from three continents and covering three phylogenetically distinct sub-clades, exhibited different activities toward lepidopteran larvae, ranging from lethal to avirulent. All strains of sub-clade 1, which includes Pseudomonas chlororaphis and Pseudomonas protegens, were highly insecticidal regardless of their origin (animals, plants). Comparative genomics revealed that strains in this sub-clade possess specific traits allowing a switch between plant- and insect-associated lifestyles. We identified 90 genes unique to all highly insecticidal strains (sub-clade 1) and 117 genes common to all strains of sub-clade 1 and present in some moderately insecticidal strains of sub-clade 3. Mutational analysis of selected genes revealed the importance of chitinase C and phospholipase C in insect pathogenicity. The study provides insight into the genetic basis and phylogenetic distribution of traits defining insecticidal activity in plant-beneficial pseudomonads. Strains with potent dual activity against plant pathogens and herbivorous insects have great potential for use in integrated pest management for crops.
•Identification of A. pleuropneumoniae subtype K4b of serovar 4 type II capsule locus.•Subtype K4b isolate is biovar 1, and harbors apxIICA, apxIBD and apxIVA genes.•Subtype K4b isolate has a ...serogroup 3/6/8/15/19 O-Ag locus.
The aim of this study was to investigate an isolate of Actinobacillus pleuropneumoniae, named 14–760, which was serologically not classifiable among the recognised serovars of A. pleuropneumoniae. It reacted with the antisera raised against serovars 3, 6, 8, 15 and 17 in the agar gel precipitation (AGP) test, and was positive in the capsular serovar 4-specific PCR (cps4B PCR) assay. The isolate contains a type II capsule locus similar to serovar 4 but with variations in the length of four intergeneric regions (modF-cpxA, cpxD-cpsA, cpsC-a 114 bp orf, and lysA-ydeN), and three gene sequences (modF, cpsC and ydeN). The main difference found between the K4 and K4b cps genes is the additional 35 AAs found in type 4b due to a 4 bp insert in cps4bC. The LPS O-Ag locus is highly similar to that of reference strains of serovars 3, 6, 8, 15, 17 and 19. Isolate 14–760 is biovar 1 and contains solely the structural genes required for toxin ApxII production (apxIICA), and the type I secretion system (apxIBD) for the export of ApxII. Antiserum against isolate 14–760 adsorbed with antigen prepared from serovars 8, 15 or 17 reference strains remained reactive with isolate 14–760, but not with antigens prepared from serovars 1–18. Taken together, our results indicate the existence of a subtype of A. pleuropneumoniae, serovar 4, that we called “K4b:O3″, and we propose isolate 14–760 as the reference strain.
A disposable microarray was developed for detection of up to 90 antibiotic resistance genes in gram-positive bacteria by hybridization. Each antibiotic resistance gene is represented by two specific ...oligonucleotides chosen from consensus sequences of gene families, except for nine genes for which only one specific oligonucleotide could be developed. A total of 137 oligonucleotides (26 to 33 nucleotides in length with similar physicochemical parameters) were spotted onto the microarray. The microarrays (ArrayTubes) were hybridized with 36 strains carrying specific antibiotic resistance genes that allowed testing of the sensitivity and specificity of 125 oligonucleotides. Among these were well-characterized multidrug-resistant strains of Enterococcus faecalis, Enterococcus faecium, and Lactococcus lactis and an avirulent strain of Bacillus anthracis harboring the broad-host-range resistance plasmid pRE25. Analysis of two multidrug-resistant field strains allowed the detection of 12 different antibiotic resistance genes in a Staphylococcus haemolyticus strain isolated from mastitis milk and 6 resistance genes in a Clostridium perfringens strain isolated from a calf. In both cases, the microarray genotyping corresponded to the phenotype of the strains. The ArrayTube platform presents the advantage of rapidly screening bacteria for the presence of antibiotic resistance genes known in gram-positive bacteria. This technology has a large potential for applications in basic research, food safety, and surveillance programs for antimicrobial resistance.
Exotoxins play a central role in the pathologies caused by most major bacterial animal pathogens. The large variety of vertebrate and invertebrate hosts in the animal kingdom is reflected by a large ...variety of bacterial pathogens and toxins. The group of repeats in the structural toxin (RTX) toxins is particularly abundant among bacterial pathogens of animals. Many of these toxins are described as hemolysins due to their capacity to lyse erythrocytes in vitro. Hemolysis by RTX toxins is due to the formation of cation-selective pores in the cell membrane and serves as an important marker for virulence in bacterial diagnostics. However, their physiologic relevant targets are leukocytes expressing β2 integrins, which act as specific receptors for RTX toxins. For various RTX toxins, the binding to the CD18 moiety of β
integrins has been shown to be host specific, reflecting the molecular basis of the host range of RTX toxins expressed by bacterial pathogens. Due to the key role of RTX toxins in the pathogenesis of many bacteria, antibodies directed against specific RTX toxins protect against disease, hence, making RTX toxins valuable targets in vaccine research and development. Due to their specificity, several structural genes encoding for RTX toxins have proven to be essential in modern diagnostic applications in veterinary medicine.
Mycoplasma bovis is a wall-less bacterium causing bovine mycoplasmosis, a disease showing a broad range of clinical manifestations in cattle. It leads to enormous economic losses to the beef and ...dairy industries. Antibiotic treatments are not efficacious and currently no efficient vaccine is available. Moreover, mechanisms of pathogenicity of this bacterium are not clear, as few virulence attributes are known. Microscopic observations of necropsy material suggest the possibility of an intracellular stage of M. bovis. We used a combination of a gentamicin protection assay, a variety of chemical treatments to block mycoplasmas entry in eukaryotic cells, and fluorescence and transmission electron microscopy to investigate the intracellular life of M. bovis in calf turbinate cells. Our findings indicate that M. bovis invades and persists in primary embryonic calf turbinate cells. Moreover, M. bovis can multiply within these cells. The intracellular phase of M. bovis may represent a protective niche for this pathogen and contribute to its escape from the host's immune defense as well as avoidance of antimicrobial agents.
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