It is quite clear nowadays that the pathogenesis of infectious disease, although determined by pathogenic features of their causative agents, cannot be fully comprehended without structural (morphologic) analysis of immediate interaction between these agents and cells, tissues, and defense systems of the host. In diseases caused by agents adapted to certain cellular targets, the pathogenesis is, in addition, influenced by histophysiologic features of these targets wich are utilized or distorted or both by the pathogen.
Acute diarrheal diseases caused by Gram-negative bacteria:
The morphologic method either produced an impetus to bacteriologic studies or served as a tool in the evaluation of bacterial pathogenicity, including that of genetically altered microorganisms. In particular, morphologic studies have revealed that enteric bacteria either colonize enterocytes while remaining epicellular ( i.e., bound to the cell surface) with or whithout affecting cellular architecture, or they invade the cell with or whithout its destruction. On the other hand, biochemical studies have shown that prevalence of the secretory or destructive inflammatory disturbances in the gut is, for the most part, determined by bacterial exototoxins. Although exotoxins affect enterocytes by a variety of mechanisms, they can be combined into two groups that are referred to as cytotonic and cytotoxic or respectively, as enterotoxins and cytotoxins. It has also been established that each step in host-pathogen interaction is governed by multiple determinants encoded in bacterial plasmid and chromosome genes. [1,2,3]
The clinical manifestations of diarrhea vary from autolimited symptoms to severe cases with presence of mucus and blood in faeces, suggesting that, as in
The aim of this chapter, is to show that with the development of an
2. Interaction host/bacterial pathogen
In this regard, the development of an
The present focuses in the analysis of the mechanisms causing the pathogenicity of
3. Materials and methods
3.1. Bacterial strains and growth conditions
Two strains of
3.2. Tissue culture procedure
Segments of the small intestine were removed from the abdominal cavity of young adult NMRI mice to prepare small intestine cylinders of ~3cm in length that were sterilized with a 10% chlorine solution. One end of each intestinal cylinder was tied with sterile surgical thread and the cavity was filled with 1.5 X 108 CFU/ml of a given one of the two
The intestinal cylinders were cut in small sections of approximately 3mm3, washed in 0.1M cacodylate buffer, pH 7.2, and postfixed for 24h in 1% osmium tetroxide prepared in the same buffer. The tissue was then dehydrated in ascending concentration ethanol solutions, followed by propylene oxide and finally embedded in Epon 812. Sections of 1μm were stained with 1% toluidin blue and observed under a high resolution light microscope. Sections of 90 nm were contrasted with uranyl acetate  and lead citrate  using a modification of this classic method  and were analyzed using a Hitachi-7000 transmission electron microscope. [19,20].
4. Effects of
Aeromonas caviaeco-cultured in mouse small intestine
The experimental desing herein presented offers appropriate conditions for the physiopathologic study of the interrelationship between the intestinal wall and bacteria under
On the other hand, this experimental model employed using co-cultures of mouse intestinal mucosa with
The damage produced was demonstrated by the alterations of the integrity of intestinal microvilli, disruption of the epithelium and presence of mucosal microulcerations, which were brought about by the fact that this strain is an high cytotoxin producer and may possess other virulence factors [4,21]
These strains belong to the
Light microscopical observations revealed a varying degree of histological alterations of the intestinal wall, according to the severity of the damage. The major tissue damage is shown in Figure 1, where a large bacterial cluster can be seen occupying the crypts between intestinal folds. In most cases, the intestinal wall showed a high degree of generalized cellular atrophy with tissue lysis when the diarrhea producing strain (D) was used. These images were seen at 24h as well as at the 48h samples of incubation.
In cases of moderate damage, the basic cytoarchitecture of the intestinal mucosa was preserved and it was possible to recognize enterocytes, mucous cells and germinative or mother cells. The villi as well as the majority of the crypts were seen, althought some of them were shorter and thicker (Figures 2 and 3).
These mild damages were caused when the strain A from the asymptomatic species was employed for the co-culture. Microvilli were seen forming part of the apical surface of the enterocyte as well as cytoplasmatic protrusion that come from the apical portion of the enterocytes (Figure 4).
Some segments of the intestinal cylinder co-cultured with
Transmission electron microscopy revealed minor alterations of the intestinal mucosa when the strain from asymptomatic patients (A) was co-cultured. Most enterocytes were seen with seen with typical ultrastructural characteristics, however, they showed numerous apical protrusion detachments (Figures 2 and 4). In more damaged regions, there was a progressive atrophy of the epithelial cells showing loss of microvilli and large cellular vacuoles loaded with cellular detritus (Figure 5).
In the intestinal regions having intermediate epithelial alterations, globular cells identified as blood and lymphatic cells were observed in the gut lumen associated to
Cells identified as lymphocytes (Figure 7) and others as plasma cells (Figure 9) were also observed as part of the clusters of globular cells found in the small intestine cylinder lumen after two days of culture.
In Figure 10 an image is shown of a cylinder of small intestinal segment incubated for 48h using the same culture conditions but without
The cylinders of small intestinal cultures incubated for 24h and 48h revealed good preservation. The glycocalyx was clearly observed covering the microvilli (Figure 11). No vesicular chains were observed in or between the microvilli, nor was any type of bacteria observed in the intestinal lumen.
After 24h de incubation, the
The small intestine tissue cultivated with
When the segments of the small intestine were cultivated with
The pathogenicity of
When the intestinal epithelium lesions were observed no bacteriae adhered to the cells were seen, indicating that direct bacteria-epithelial cell contact was nor required for tissue alterations: on the contrary, it seems that
The lymphatic submucous plaques and the autonomous defensive structures are able to act in culture conditions even in the absence of the circulatory blood elements, bone marrow cells or other lymphatic organs. Among the defense elements that migrate in answer to the chemotactic stimulus are the eosinophilic granulocytes, which have been reported to participate only in parasitic diseases. In this investigation their capability to defend intestinal cells against bacterial aggressions [23,24] is ultrastructurally documented.
Under the conditions of the experiment, no alteration of the intestinal epithelium was observed in the cultures from the small intestine with
On the other hand, in cultures with 48h incubation,
The vesicles produced on the surface of the bacterial outer membrane are also constituted by a double membrane unit. The production of these vesicles was observed in all the cultures analyzed. However, the more important vesicular formation was detected in the cultures incubated for 24h, whereas in the samples incubated for 48h, the vesicles were found where the intestinal damage was more severe.
The ultrastructural analysis suggest that
The vesicles do not appear to be intestinal exocytic vesicles because they are formed on the surface of the bacterial outer membrane. Moreover, no clathrin-like outer skeleton is seen, which suggest that this vesicular system is a part of the pathogenic mechanism of action that induces the migration of eosinophils and macrophages.
In the cultures with a 24h incubation period, the vesicular chains were found adhered to the bacterial outer membrane as well as occupying the free spaces between microvilli. In cultures incubated for 48h, the vesicles were absent, whereas some regions of the enterocytic epithelial surface lost their organization, leading to tissue lysis. The vesicles could constitute a carrier tool that facilitates the approximation and the interchange of enterotoxins, inmunologicals material or any other natural element responsible for the virulence and pathogenicity of
The strain isolated from the patient with diarrhea produced important alterations in the intestinal mucosa, indicating its enteropathogenic potential. This strain is a cytotoxin producer and, as has been pointed out for
In conclusion the genus