The pathogen of frogs Amphibiocystidium ranae is a member of the order dermocystida in the class mesomycetozoea.

The pathogen of frogs Amphibiocystidium ranae was recently described as a new genus. Due to its spherical shape, containing hundred of endospores, it was thought to be closely related to the pathogens of fish, mammals, and birds known as Dermocystidium spp., Rhinosporidium seeberi, and Sphaerothecum destruens in the Mesomycetozoea, but further studies were not conducted to confirm this relationship. To investigate its phylogenetic affinities, total genomic DNA was extracted from samples collected from infected frogs containing multiple cysts (sporangia) and endospores. The universal primers NS1 and NS8, used to amplify the 18S small-subunit rRNA by PCR, yielded approximately 1,770-bp amplicons. Sequencing and basic local alignment search tool analyses indicated that the 18S small-subunit rRNA of A. ranae from both Rana esculenta and Rana lessonae was closely related to all of the above organisms. Our phylogenetic analysis placed this pathogen of frogs as the sister group to the genus Dermocystidium and closely related to Rhinosporidium. These data strongly supported the placement of the genus Amphibiocystidium within the mesomycetozoeans, which is in agreement with the phenotypic features that A. ranae shares with the other members of this class. Interestingly, during this study Dermocystidium percae did not group within the Dermocystidium spp. from fish; rather, it was found to be the sister group to Sphaerothecum destruens. This finding suggests that D. percae could well be a member of the genus Sphaerothecum or perhaps represents a new genus.

Experimental infections of Rana esculenta with Aeromonas hydrophila: a molecular mechanism for the control of the normal flora.

Frogs can be useful models for studying the mechanisms that may regulate their natural microbial flora. Their skin glands produce a secretion containing 20-30 different peptides, some antimicrobial some neurotrophic. As they often live in soil or silt that is rich in microbes, they can be expected to be able to prevent or eliminate infections in very short periods of time. The bacterium Aeromonas hydrophila is widely distributed in nature and is considered as part of the natural flora of frogs and many animals, including humans. From an alternative frog strain of A. hydrophila, Bo-3, we isolated a spontaneous and stable mutant (Bo-3N), resistant to nalidixic acid, here used to follow the host-microbe interactions in experimental infection of mouth and skin of Rana esculenta. The skin peptides had been previously isolated, sequenced and cloned. We showed that skin treatment with a glucocorticoid (GC) cream blocked de novo synthesis of these peptides and, simultaneously, prepropeptide mRNAs disappeared while IkappaBalpha was up-regulated. Experimental mouth infections with 20 million cells of A. hydrophila Bo-3N showed that a normal wild frog can eliminate the bacteria from the mouth within 15 min, while a frog pretreated with GC cream for 1 h could not reduce Bo-3N below 3500 colony-forming units (CFU)/5 microl 'saliva'. An in vitro comparison showed that frog blood or serum allowed bacteria to grow, while the skin secretion killed the bacteria within 10 min. Using different enzyme-linked immunosorbent assays (ELISAs) with rabbit anti-Bo-3 serum as a positive control, we were able to rule out immunoglobulin G (IgG) binding to A. hydrophila. An assay for immunoglobulin M (IgM) (or some other serum component) in frog serum showed binding to A. hydrophila only corresponding to a few per cent of the positive control. For skin infections we bathed the frogs for 10 min in an overnight culture of Bo-3N diluted to about 10(7) CFU/ml. Electrical stimulation after the bath showed, for the total secretion, a two to fourfold increase in the antibacterial activity, while a pretreatment with GC cream reduced the activity to about one-third of that of the non-bathed control frog. HPLC analysis of the peptide pattern confirmed these findings. The survival value of antimicrobial peptides have earlier been demonstrated in vivo and in vitro only in Drosophila. The present experiments are the first combined in vivo and in vitro demonstrations of the function of peptide antibiotics in a vertebrate. One such function is involved in the control of the natural flora.

The morphological features of Aegyptianella bacterifera: an intraerythrocytic rickettsia of frogs from Corsica.

Study of ultrathin sections and freeze-fracture replicas of erythrocytes containing Aegyptianella bacterifera (Rickettsiales; Anaplasmataceae) revealed that the organisms develop within a membrane bound vacuole in the erythrocyte cytoplasm. The organisms divide by binary fission to produce about 12 rickettsiae in a mature inclusion. The organisms have gram-negative cell envelopes. The distribution of intramembranous particles within the outer and plasma membranes of A. bacterifera is similar to that of other rickettsiae and gram-negative bacteria such as Escherichia coli. The definition of the genus Aegyptianella should be broadened to include rickettsiae measuring up to 5 microns in length prior to division which develop within membrane bound inclusions in erythrocytes of their hosts.

Viral particles in the hepatocytes of Rana esculenta (L).

The hepatocytes of Rana esculenta exhibit hexagonal, probably icosahedral, viral particles disseminated in the cytoplasm among cellular organelles (mitochondria, cisternae of the endoplasmic reticulum, Golgi complex). The particles consist of an electron-dense core (nucleoid) and a less electron-dense envelope (capsid). Empty capsids (without nucleoid) or semilunar capsid fragments can be sometimes observed. The virus seems to be a cytoplasmic one, as it has not been found in the nucleus of hepatocytes.