Anaemia in advanced chronic fasciolosis.

The association between fasciolosis-induced anaemia and related factors has been quantified in a rodent model. Haematological parameters were analysed in Wistar rats at 20 and 60 weeks post-infection (p.i.). Pigment stones and bile specimens were collected. Serum IgG1, IgG2a and IgE were determined in rat serum samples. Cytokine levels have been correlated with haematological parameters. The screening for gastrointestinal bleeding was carried out. Bacteriological bile cultures revealed viable bacteria in 53.8% of specimens at 60 weeks p.i. The results show that the type of anaemia in fasciolosis might be considered a biomarker of the chronicity period of the disease, changing from normocytic to macrocytic in the early chronic period (20 weeks p.i.) and to microcytic in the advanced chronic period (60 weeks p.i.). Likewise, changing from normochromic in the early chronic period to hypochromic in the advanced chronic period. Multivariate analysis suggested an association between anaemia and the following factors: fluke burden, eggs per gram of faeces, body area of parasite, presence of blood in faeces, IgG1 and eosinophil levels, and % of splenic weight. Of all variables analysed, the fluke burden is the one which presents the highest anaemia risk, even exceeding the variable presence of blood in faeces. The development of anaemia appears to be complex and may involve multiple mechanisms. However, to the mechanisms that until now explain Fascioliosis-related anaemia (compensatory increase in erythrocyte production and a continuous drain on iron stores resulting from the parasites' blood-sucking activities) the following causes ought to be added: haemolysis of red blood cells, the general effects of inflammation on erythropoiesis, concomitant parasitic and bacterial infections and pre-morbid nutritional abnormalities. Extrapolation to human fasciolosis is discussed. The results of the rodent model lead to the assumption that a high risk of anaemia in subjects with a heavy parasitic burden in human hyperendemic areas of fasciolosis is to be expected.

[Foodborne Listeria monocytogenes: are all the isolates equally virulent?]. / Listeria monocytogenes en alimentos: son todos los aislamientos igual de virulentos?

Listeria monocytogenes is a foodborne human pathogen responsible for invasive infections presenting overall a high mortality. Despite the ubiquity of the microorganism, the actual disease rate is quite low and the disease is most often associated with an underlying predisposition. Foodborne and environmental isolates were traditionally considered of similar pathogenicity compared to clinical isolates. But the analysis of mutations in the genes encoding specific virulence factors (internalin, hemolysin, phospholipases, surface protein ActA and regulator protein PrfA), quantitative studies with cell cultures and population genetics have raised considerable concerns about virulence differences among L. monocytogenes strains. Despite this great step forward, there is not a single marker available to test the virulence of field isolates of this species. In the future, the combination of different molecular markers will probably allow the screening of food contamination by only the virulent clones of L. monocytogenes, thus improving the prevention of foodborne human listeriosis.

A role for ActA in epithelial cell invasion by Listeria monocytogenes.

We assessed the role of the actin-polymerizing protein, ActA, in host cell invasion by Listeria monocytogenes. An in frame DeltaactA mutant was constructed in a hyperinvasive strain of prfA* genotype, in which all genes of the PrfA-dependent virulence regulon, including actA, are highly expressed in vitro. Loss of ActA production in prfA* bacteria reduced entry into Caco-2, HeLa, MDCK and Vero epithelial cells to basal levels. Reintroduction of actA into the DeltaactA prfA* mutant fully restored invasiveness, demonstrating that ActA is involved in epithelial cell invasion. ActA did not contribute to internalization by COS-1 fibroblasts and Hepa 1-6 hepatocytes. Expression of actA in Listeria innocua was sufficient to promote entry of this non-invasive species into epithelial cell lines, but not into COS-1 and Hepa 1-6 cells, indicating that ActA directs an internalization pathway specific for epithelial cells. Scanning electron microscopy of infected Caco-2 human enterocytes suggested that this pathway involves microvilli. prfA* bacteria, but not wild-type bacteria (which express PrfA-dependent genes very weakly in vitro) or prfA* DeltaactA bacteria, efficiently invaded differentiated Caco-2 cells via their apical surface. Microvilli played an active role in the phagocytosis of the prfA* strain, and actA was required for their remodelling into pseudopods mediating bacterial uptake. Thus, ActA appears to be a multifunctional virulence factor involved in two important aspects of Listeria pathogenesis: actin-based motility and host cell tropism and invasion.

Microinjection and growth of bacteria in the cytosol of mammalian host cells.

Most facultative intracellular bacteria replicate in specialized phagosomes after being taken up by mammalian cells. Relatively few intracellular bacteria escape the phagosomal compartment with the help of cytolytic (pore-forming) proteins and replicate in the host cell cytosol. Without such toxins, intracellular bacteria cannot reach this cellular compartment. To circumvent the requirement of an "escape" step, we developed a procedure allowing the efficient direct injection of bacteria into the cytosol of mammalian cells. With this technique, we show that most bacteria, including extracellular bacteria and intracellular pathogens that normally reside in a vacuole, are unable to replicate in the cytosol of the mammalian cells. In contrast, microorganisms that replicate in the cytosol, such as Listeria monocytogenes, Shigella flexneri, and, to some extent, enteroinvasive Escherichia coli, are able to multiply in this cellular compartment after microinjection. Further L. monocytogenes with deletion in its PrfA-regulated hpt gene was found to be impaired in replication when injected into the cytosol. Complementation of the hpt mutation with a plasmid carrying the wild-type hpt gene restored the replication ability in the cytosol. These data indicate that cytosolic intracellular pathogens have evolved specific mechanisms to grow in this compartment of mammalian cells.