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Viral isolates were obtained in 1998, 1999 and 2000 from the lung, liver and intestine of two bearded dragons (Pogona vitticeps) and a chameleon (Chamaeleo quadricornis) and from the skin of a frill-necked lizard (Chamydosaurus kingii) by using viper heart cells (VH2) at 28 degrees C. Electron microscopic examination of infected VH2 cells revealed the assembly of icosahedral iridovirus-like particles measuring 139 nm (side to side) and 151 nm (apex to apex). Negatively stained virus particles had dimensions of 149 nm (side to side) and 170 nm (apex to apex). Polymerase chain reaction (PCR) amplification of purified viral DNA with primers corresponding to the partial gene encoding the major capsid protein (MCP) of Frog viris-3 (FV-3), the type species of the genus Ranavirus, was unsuccessful. In contrast, primers corresponding to the partial MCP gene of Chilo iridescent virus (CIV; genus Iridovirus) amplified 500-bp products with 97% identity to the nucleotide sequence of CIV and 100% identity to the nucleotide sequence of Gryllus bimaculatus iridescent virus (GbIV), an invertebrate iridescent virus. Virus protein profiles analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and restriction fragment length profiles of purified viral DNA treated with the endonucleases EcoRI, HindIII and HpaII were identical to those of GbIV.

We have reinvestigated the morphology of the salivary glands in the cockroach, Periplaneta americana, by light, electron, and confocal laser scanning microscopy. All secretory acini have a uniform structural layout. They consist of three cell types: peripheral cells, central cells, and duct cells. One pair of peripheral cells forms the base of each acinus. The central cells, arranged concentrically in a fourfold symmetry around the most proximal part of the acinar ducts, lie next downstream. In every acinus, duct cells accompany the central cells and form a thin sheet on the apical surface of the latter. This apical lining of the duct cells is regularly fenestrated, and the central cells secrete the contents of their secretory vesicles only through these openings into the lumen of the ducts. Peripheral cells and central cells are never in direct physical contact, because, apically, extensions of the inner acinar duct cells intervene between the cells. Basally, thin extensions of the basement membrane separate the cell types. We have found no morphological evidence for the existence of electrical coupling (gap junctions) between the saliva-producing cells. Our ultrastructural data support the view that the peripheral cells are responsible for water and electrolyte transport, whereas only central cells secrete the proteinaceous components of the saliva. We have found that the duct cells distal to the acini are also specialized for ion and water transport. They have a prominent basal labyrinth containing numerous mitochondria and a highly folded apical surface. The folded apical membrane surface is coated with electron-dense particles on its cytoplasmic side; these particles are probably portasomes. Our investigation provides morphological evidence that the duct cells distal to the secreting acini are able to modify primary saliva.

Among 310 fl eas collected from dogs and cats in Germany, Rickettsia felis was detected in all specimens (34) of Archaeopsylla erinacei (hedgehog fl ea) and in 9% (24/226) of Ctenocephalides felis felis (cat fl ea). R. helvetica was detected in 1 Ceratophyllus gallinae (hen fl ea). R ickettsia felis, the causative agent of the fl ea-borne spotted fever rickettsiosis, is pathogenic for humans (1-4). Since the fi rst detection of R. felis from midgut epithelial cells of the cat fl ea, Ctenocephalides felis felis, in 1990 (5), interest in the role of this fl ea species as its main vector has increased. R. felis has been found in cat fl eas on all continents (6). Because R. felis is not lethal for cat fl eas and is transmitted transovarially by these fl eas (4), C. felis could be a vector and a reservoir of this pathogen. For these reasons, the cat fl ea was considered the only fl ea species with a major role in the epidemiology of fl ea-borne spotted fever rickettsiosis. However, R. felis has been reported in other fl ea species (4,6-8), and fl ea-borne spotted fever rickettsiosis is now considered an emerging human infectious disease. We analyzed the presence of R. felis in different fl ea species collected from naturally infested cats and dogs in different locations in Germany. The StudyA total of 310 fl eas were collected from 49 dogs and 54 cats in 11 widely distributed locations in Germany (Berlin, Munich, Brandenburg, Leipzig, Chemnitz, Rostock/Laage, Bremen, Osnabrück, Münster, Freising, and Schongau) (Figure) in 2007. Specimens collected were recorded and kept at -20°C. Samples were shipped on dry ice to our laboratory, and species identifi cation was performed by using light microscopy and following the determination key of Hopkins and Rothschild (9). Because of infestation variations (1-150 fl eas per animal), 3 fl eas per animal host were chosen randomly for species differentiation.Fleas were homogenized individually in 80 μL of phosphate-buffered saline with a RETSCH Tissue Lyser Mixer Mill 300 (QIAGEN, Hilden, Germany) by using 5-mm steel beads. A 100-μL volume of ATL buffer and 20 μL of proteinase K (QIAGEN) were added, and homogenates were incubated at 56°C in an Eppendorf Thermomixer (Eppendorf, Hamburg, Germany) until tissues were completely lysed. DNA was extracted from each fl ea by using a QIAamp DNA Mini Kit (QIAGEN) according to the manufacturer's instructions (tissue protocol) and stored at -20°C until used.PCR amplifi cation of rickettsial DNA was performed by using oligonucleotide primer pairs Rp CS.877p/Rp CS.1258n (10) generated from the rickettsial citrate synthase (gltA) gene. Positive samples were analyzed for a 530-bp portion of the outer membrane protein A (ompA) gene with primer pair Rr 190.70p/Rr 190.602n (10) and for a 765-bp portion of the ompB gene with primer pair 120-1278/120-3599 (11). PCR amplifi cation was accomplished in 50-μL volumes containing 5 μL DNA, 30 μL distilled water, 10 μL 5× Taq buffer (Roche, Mannheim, Germany), 3 μL 25 mmol/L MgCl 2 (Roche), 1 μL 10 mmol/L dN...

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