Three amoeba species were isolated from 3 out of 193 farmed tilapias, Oreochromis niloticus (L.), screened for the presence of free-living amoebae in parenchymatous organs. Hartmannella vermiformis Page, 1967 and Rosculus ithacus Hawes, 1963 were isolated from the kidney tissue. The third strain isolated from the liver shared morphological features of Mayorella and Platyamoeha spp. and therefore its taxonomic position has not been determined as yet. Pathogenicity of cloned strain of H. vermiformis was proved in two fish hosts.
Ninety four aquarium fishes were screened for the presence of amoebae in their internal organs. Five specimens of Ca-rassius auratus (L.) and one specimen of Xiphophorus hetleri Heckel were positive. Of the three strains which were isolated from C. auratus, successfully cloned and cultivated, one was identified as Vannella platypodia (Gläser, 1912) Page, 1976 and two strains as Rosculus ithacus Hawes, 1963. Both species are reported for the first time from organs of fish. None of them could be identified with the amoeba-like agent of goldfish granulomas described here.
Using degenerative primers designed on the basis of known sequences of lectin genes from different sources a fragment of genomic DNA of Borrelia burgdorferi ( strain B31) that contained a lectin-like sequence was isolated, cloned and sequenced. The presence of an open reading frame of 268 amino acids (position 1501-2304 bp) and the computer analysis of the predicted amino acid sequence showed 37% of identity and 75% of homology over region of 25 amino acids with the legume lectin proteins, including erythroagglutinating phytohcmagglutinin (РНЛ-Е) and leucoagglutinating phylohemagglutinin (PHA-L). The further analysis of the predicted amino acid sequence showed the presence of another two domains (positions 198-211 and 215-226 aa) consisting of the characteristic conserved sequence motifs for legume lectin proteins. Hemagglutinating activity was detected in lysate of В burgdorferi (strain B31) spirochete and the affinity to fetuin was determined in a hemagglutination inhibition test. Hemagglutinating activity was also found in a crude lysate of the recombinant clones carrying the fragment of B. burgdorferi genomic DNA. The inhibition of agglutinating activity by fetuin, D-galactosamine and D-mannosamine was determined using the standard procedure of hemagglutination inhibition test with native rabbit red blood cells (RBC).
Investigations on the epizoic fauna of Gadus morhua (L.), Piatichthys flesus (L.) and Oncorhynchus mykiss (Walbaum) from the Kiel Fjord and Kiel Bight were carried out from September 1996 to March 1997. Smears from 120 G. morhua and 92 P. flesus caught using fish traps and trammel nets, and of 35 O. mykiss obtained from a local fish farm in the Kiel Fjord revealed the presence of three species of trichodinid ciliatcs, Trichodina claviformis sp. n., Trichodina jadranica Haider, 1964 and Trichodina raahei Loin, 1962. The new species can be distinguished from other trichodinids by its large size in combination with the characteristically shaped adhesive disc containing denticles with club-like formed thorns. The thorns are directed anteriorly and not towards the centre of the adhesive disc. As the Kiel Bight and Kiel Fjord are new locality records for T. jadranica and T. raabei, morphological data are provided for both species. Trichodina claviformis is the first record of a pcrilrichous mobiline ciliate from Atlantic cod of the Baltic Sea. An identification Icey for 16 Trichodina species occurring on Baltic Sea fishes is provided based on the morphology of the adhesive disc and other well-established features The occurrence of trichodinid ciliates on G. morhua and P. flesus in the Baltic Sea is discussed, especially considering the biology of the host and a possible host specificity of the species.
This paper summarizes work done in this laboratory over the last two years on the cloning of microsporidian rRNA by homology PCR and its subsequent use in diagnostic tests and phylogenetic studies. Using highly conserved primers in the 16S or small subunit rRNA (SSU-rRNA) these genes were cloned from human intestinal biopsies with transmission electron microscopy proven Enterocytozoon bieneusi and Septata intestinalis. The SSU-rRNA genes were then used to design and test several primer pairs for the diagnosis of microsporidian infection. Utilizing the polymerase chain reaction and primers V1 and EB45Ü Ent. bieneusi infected duodenal aspirates or intestinal biopsies could be detected. Using V I and SI500 infection with S. intestinalis could be detected. In addition to diagnostic tests, phylogenetic relationships were examined using sequence data from the fragment amplified by PCR by primer 530f in the SSU-rRNA and primer 580r in the large subunit rRNA. This data supported the placement of S. intestinalis in the family Encephalitozoonidae. In addition, it confirmed that Encephalitozoon cuniculi, E. hellem and S. intestinalis are distinct organisms. These techniques have broad applications to the study of other microsporidia and the development of a molecular phylogeny.
The Leishmania metalloproteinase GP63 has been reported to play important roles mainly in resistance of promastigotes to complement-mediated lysis and in interaction with macrophage receptors. On the other hand, its function in insect vectors is still unclear. We compared the structure and dosage of gp63 genes and the activity of GP63 in Leishmania major Yakimoff et Schokhor strains and lines differing in virulence for mice and ability to develop in sand flies. The results demonstrate considerable variability in amount and proteolytical activity of GP63 among L. major strains although genomic changes in the gp63 locus were not found. Attenuated LV561/AV line showed low amount and low enzymatic activity of GP63. Serial passages of attenuated parasites through either Phlebotomus duboscqi Neveu-Lemaire or through mice led to a recovery of GP63 proteolytical activity to the level present in virulent LV561/V line. Overexpression of GP63 was found in two L. major strains (L119, Neal) with defective lipophosphoglycan (LPG); both these strains were capable to cause mice infection but unable to survive and multiply in sand flies. Differences were found also in karyotypes and in amount of minichromosomes amplified in some lines of the LV561 strain. The results suggest that parasite virulence is not simply correlated with the activity of GP63; however, this enzyme plays a significant role in association with other surface molecules, especially LPG. Overexpression of GP63 can compensate LPG defect in the vertebrate host but in sand flies both molecules fulfil quite different functions and the defect in LPG is lethal for the parasite. On the other hand, linear minichromosomes of about 200 kb found in some lines of the LV561 strain are associated with development in vitro and in the vector but they are not essential for the infection of the vertebrate host.