Molecular Evidence for Association of Chlamydiales Bacteria with Epitheliocystis in Leafy Seadragon (Phycodurus eques), Silver Perch (Bidyanus bidyanus), and Barramundi (Lates calcarifer)

Epitheliocystis in leafy seadragon (Phycodurus eques), silver perch (Bidyanus bidyanus), and barramundi (Lates calcarifer), previously associated with chlamydial bacterial infection using ultrastructural analysis, was further investigated by using molecular and immunocytochemical methods. Morphologically, all three species showed epitheliocystis cysts in the gills, and barramundi also showed lymphocystis cysts in the skin. From gill cysts of all three species and from skin cysts of barramundi 16S rRNA gene fragments were amplified by PCR and sequenced, which clustered by phylogenetic analysis together with other chlamydia-like organisms in the order Chlamydiales in a lineage separate from the family Chlamydiaceae. By using in situ RNA hybridization, 16S rRNA Chlamydiales-specific sequences were detected in gill cysts of silver perch and in gill and skin cysts of barramundi. By applying immunocytochemistry, chlamydial antigens (lipopolysaccharide and/or membrane protein) were detected in gill cysts of leafy seadragon and in gill and skin cysts of barramundi, but not in gill cysts of silver perch. In conclusion, this is the first time epitheliocystis agents of leafy seadragon, silver perch and barramundi have been undoubtedly identified as belonging to bacteria of the order Chlamydiales by molecular methods. In addition, the results suggested that lymphocystis cysts, known to be caused by iridovirus infection, could be coinfected with the epitheliocystis agent. Epitheliocystis is an infection of the gills and skin of many fish species. Sometimes it can be grossly visible as cyst-like lesions, and sometimes the cysts can be seen microscopically in gill squashes, but often the only way to detect it is through histology. Epitheliocystis has been reported worldwide, both from freshwater and marine species (12). This condition is usually benign; however, sometimes it can be associated with a high mortality, particularly in cultured fish (3, 4, 26). Due to swelling of the cells of the gills and the increase in mucus around heavily infected gills, fish can become lethargic and show respiratory distress. The causative agent of epitheliocystis replicates intracellularly in the cysts and, since 1969 epitheliocystis has been associated with chlamydia-like bacteria based on the ultrastructural characteristics of the content of the cysts (4). Attempts to identify the causative agent by using monoclonal antibodies have not been successful, and their results are often inconsistent. In 1999 we discovered many new chlamydia-like sequences by using a universal Chlamydiales 16S rRNA gene PCR (30). Because of the unconfirmed ultrastructural association of epitheliocystis with chlamydia-like organisms we started investigation of archived epitheliocystis material of leafy seadragon (Phycodurus eques) and silver perch (Bidyanus bidyanus) and a new case of epitheliocystis in barramundi (Lates calcarifer) using this Chlamydiales-specific PCR. Ultrastructural analysis of epitheliocystis in these fish species was described previously, in leafy seadragon by Langdon et al. (19), in silver perch by Frances et al. (10), and in barramundi by Anderson and Prior (2). After we communicated our first preliminary positive findings during the Tenth International Symposium on Human Chlamydial Infections in Antalya, Turkey, in 2002 (25), Draghi et al. (6) undoubtedly identified a chlamydia-like bacterium as the cause of epitheliocystis in farmed Atlantic salmon (Salmo salar) using DNA sequence analysis and in situ hybridization (ISH). They proposed the name “Candidatus Piscichlamydia salmonis” for this bacterium. We describe here the characterization of the epitheliocystis agents of leafy seadragon, silver perch, and barramundi by molecular and immunocytochemical methods

Chlamydia pneumoniae in abdominal aortic aneurysms; abundance of membrane components in the absence of heat shock protein 60 and DNA

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Detection of microorganisms in vessel waal specimens of the abdominal aorta: development of a PCR-assay in the absence of a gold standard.

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Vitamin A deficiency potentiates carbon tetrachloride-induced liver fibrosis in rats

Earlier studies have shown that retinoid administration suppresses the generation of hepatic fibrosis and stimulates its regression in normal (i.e., vitamin A-sufficient) carbon tetrachloride-treated rats. This study focuses on the possible role of a marginal or deficient vitamin A status on carbon tetrachloride-induced fibrosis. This experimental study in rats shows that vitamin A status, reflected by hepatic retinoid content (retinol and retinyl esters), modulates the development of hepatic fibrosis induced by carbon tetrachloride. In rats with low hepatic retinoid levels (12 ± 0.9 μg/gm liver), carbon tetrachloride-induced liver fibrosis was more pronounced than in rats with sufficient hepatic retinoid levels (1,065 ± 327 μg/gm liver). Enhanced liver fibrogenesis was confirmed both morphologically and by a higher hydroxyproline content of the liver. It was associated with a reduced liver weight and the development of parenchymal regeneration nodules. Furthermore, carbon tetrachloride treatment itself reduced the hepatic retinoid content in rats independently of the liver vitamin A status before treatment and increased serum retinol levels in vitamin A-sufficient rats. The results show that the vitamin A status of the liver plays an important role in hepatic fibrogenesis. Low hepatic vitamin A levels, which can be the result not only of low dietary intake but also of interference with vitamin A metabolism by agents such as ethanol and carbon tetrachloride, may be a risk factor for the development of liver fibrosis. We suggest that retinoids modulate collagen synthesis and deposition irrespective of the degree of hepatocellular necrosis induced by carbon tetrachloride. The reduction of retinoid levels in fat-storing cells and fibroblast-like cells by an enhanced secretion of retinol from the liver into the circulation during carbon tetrachloride treatment may stimulate the transformation of these cells to fibroblasts and, in this way, contribute to fibrogenesis of the liver. Chemicals/CAS: Carbon Tetrachloride, 56-23-5; Collagen, 9007-34-5; Hydroxyproline, 51-35-4; Retinoids; Vitamin A, 11103-57-

A macaque model for hantavirus infection.

Cynomolgus macaques (Macaca fascicularis) were experimentally infected with Puumala virus (strain Hallnas), which causes nephropathia epidemica in humans in western Europe. During the first week after intratracheal inoculation, the monkeys exhibited signs of lethargy followed by mild proteinuria and microhematuria. Histopathologic changes during the first 7 weeks after infection were largely confined to abnormalities in medullary tubular cells of the kidneys, which coincided with the demonstration of viral antigen and viral RNA. The development of different classes of virus-specific plasma antibodies to the respective viral antigens were similar to those observed in humans with nephropathia epidemica. This first description of a nonhuman primate model for hantavirus infection shows that the cynomolgus macaque provides a suitable model with which to study the pathogenesis of Puumala virus infections and to evaluate new diagnostic methods, immunization strategies, and therapies

Acholeplasma vituli sp. nov., from bovine serum and cell cultures

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