Transcytosis of Streptococcus iniae through skin epithelial barriers: an in vitro study.

By constructing a biological model based on in vitro culture of polarized rainbow trout primary skin epithelial cell monolayers, the series of early events that precede Streptococcus iniae infection, particularly colonization and translocation through external barriers, were analyzed. Streptococcus iniae promptly invades skin epithelial cells, but the rapid decline of viable intracellular bacteria points out the limited capability of intracellular survival for this bacterium. Translocation assays, supported by electron microscopy microphotographs, demonstrate that following successful in vitro invasion of skin epithelial cell, the bacterium exists free in the cytoplasm after release from the endosome, and translocates through the skin barrier. Bacterial invasion and transcytosis is not accompanied by apparent cell-line damages or disruption of host cells' tight junctions. It is hypothesized that the phenomenon of epithelial invasion coupled to the rapid translocation through the barrier plays a crucial role in Streptococcus iniae infection.

Re-establishment of Carabus (Cathoplius) aliai Escalera, 1944 as a separate valid species (Coleoptera, Carabidae).

Carabus (Cathoplius) aliai was described as a separate species by Escalera in 1944 but since the 1950-60s it has been considered as a subspecies of Carabus (Cathoplius) stenocephalus Lucas, 1866. This downgrading was adopted after examining only a few specimens, due to their rarity in collections. In recent years, an important population of this taxon was rediscovered in the Tan-Tan area in southern Morocco. By combining field observations with laboratory breeding experiments including hybridization trials, and through the morphological examination of a representative number of individuals, it is confirmed that Carabusaliai is indeed a valid species. Despite close geographic distribution, the morphological and biological characteristics of Carabusaliai and Carabusstenocephalusifniensis Zarco, 1941, its northern substitutive taxon, are very different. Carabusaliai adults are characterized by a smaller size, a slender silhouette, a more brilliant aspect, a narrower pronotum, a coarser elytral sculpture, longer legs, and a wider and a little more curved apex of the median lobe of the aedeagus. Carabusaliai larvae are also characterized by a much smaller size and the Carabusaliai pupa has a narrower thoracic area and a different chaetotaxy compared to that of Carabusstenocephalusifniensis. Contrary to this, Carabusaliai has a life cycle belonging to the annual univoltine winter semelparous type. Moreover, the duration of its development cycle is shorter. Carabusaliai is a sabulicolous steppe-wandering species with an intensive running activity, while Carabusstenocephalusifniensis is a more sedentary taxon. Crossbreeding experiments showed a marked reproductive isolation between Carabusaliai and Carabusstenocephalusifniensis. When F1 hybrids were crossed with one another, a very high mortality rate during embryonic, larval and pupal development was evident and no vital F2 neo-adults were obtained. Morphological and biological differences, together with the reproductive failure in Carabusaliai × Carabusstenocephalusifniensis hybrids, clearly indicate that Carabusaliai is a separate Cathoplius species that is distributed in an area south of the Anti-Atlas chain, from Plage Blanche (Guelmim) to Lemsid and Bou Kra (south of Laâyoune). Carabusaliai is therefore both a Saharan desert endemic and an Atlantic resident. Moreover, it is the southernmost Carabus species of the western Palaearctic region.

Carabus of Subgenus Cathoplius C.G. Thomson, 1875, with description of their life-way, life-cycle and pre-imaginal morphology (Coleoptera: Carabidae) .

According to current taxonomy, Subgenus Cathoplius C.G. Thomson, 1875, within the Genus Carabus Linnaeus, 1758 (in the broad sense), includes two species: C. (Cathoplius) asperatus (Dejean, 1826), monotypic with a northern distribution, and the southern polytypic substitutive species C. (Cathoplius) stenocephalus Lucas, 1866. The authors describe the life-way, life-cycle and pre-imaginal characters of the taxa currently ascribed to Subgenus Cathoplius, with details never provided before. Cathoplius are ground beetles adapted to live in arid environments and extreme habitats such as sub-desert areas. All of them are strictly helicophagous, both during the pre-imaginal stages and as adults, and are localized in a narrow fringe along the Atlantic coast of northwestern Africa. Several data and observations on the eco-ethology of the different taxa, obtained both in field and in laboratory, are reported. The life-cycle of Cathoplius belongs to the winter breeding type, with an extremely high fecundity rate concentrated in a very short period of time, that has no similarity to any other Carabus species. Eggs, larvae and pupae of the different species and subspecies of Cathoplius are described and illustrated. Larval characters clearly place Subgenus Cathoplius into the lineage of Neocarabi, confirming it as a monophyletic and homogeneous assemblage. Hybridization trials between some taxa led to a reduced survival rate of the progeny, thus confirming their specific or subspecific differentiation as proposed by classical taxonomy. Furthermore, hybridization results suggest that C. (Cathoplius) stenocephalus aliai could be considered as a distinct species. Notes about the origin, biogeography and phylogeny of Cathoplius are also provided.

Trojan horse effect: phagocyte-mediated Streptococcus iniae infection of fish.

The salmonid macrophage-like cell line RTS-11 and purified trout pronephros phagocytes were used to analyze in vitro entry and survival of two Streptococcus iniae serotypes. Efficient invasion by S. iniae occurred in both cells, but only the type II strain persisted in pronephros phagocytes for at least 48 h. Ex vivo models of opsonin-dependent phagocytosis by pronephros phagocytes demonstrated increased phagocytosis efficacy. Analysis of phagocytes collected from diseased fish demonstrated that approximately 70% of the bacteria contained in the blood during the septic phase of the disease were located within phagocytes, suggesting an in vivo intracellular lifestyle. In addition to the augmented levels of bacteremia and enhanced survival within phagocytes, S. iniae type II induces considerable apoptosis of phagocytes. These variabilities in intramacrophage lifestyle might explain differences in the outcomes of infections caused by different serotypes. The generalized septic disease associated with serotype II strains is linked not only to the ability to enter and multiply within macrophages but also to the ability to cause considerable death of macrophages via apoptotic processes, leading to a highly virulent infection. We assume that the phenomenon of survival within phagocytes coupled to their apoptosis plays a crucial role in S. iniae infection. In addition, it may provide the pathogen an efficient mechanism of translocation into the central nervous system.

Clonality and diversity of the fish pathogen Lactococcus garvieae in Mediterranean countries.

Infection with Lactococcus garvieae is considered the most important risk factor for the European trout industry, and the losses are approximately 50% of the total production. To improve our understanding of the genetic links among strains originating from different countries, we examined the population structure of L. garvieae by comparing 81 strains isolated from different sources and ecosystems (41 farms in six countries) in which the bacterium is commonly found. Genetic similarities (as assessed with molecular tools, including restriction fragment length polymorphism ribotyping with two endonucleases) were compared with serological data. The combined results reveal that in endemic sites the bacterial population displays a clonal structure, whereas bacterial diversity characterizes sites where the infection is sporadic.