The Region of Difference Four is a Robust Genetic Marker for Subtyping Mycobacterium caprae Isolates and is Linked to Spatial Distribution of Three Subtypes.

Alpine Mycobacterium caprae isolates found in cattle and red deer display at least three genetic variations in the region of difference four (RD4) that can be used for further differentiation of the isolates into the subtypes 'Allgäu', 'Karwendel' and 'Lechtal'. Each genomic subtype is thereby characterized by a specific nucleotide deletion pattern in the 12.7-kb RD4 region. Even though M. caprae infections are frequently documented in cattle and red deer, little is known about the transmission routes. Hence, robust markers for M. caprae subtyping are needed to gain insight into the molecular epidemiology. For this reason, a rapid and robust multiplex PCR was developed for the simultaneous detection of three M. caprae RD4 subtypes and was used to subtype a total number of 241 M. caprae isolates from animals (145 cattle, 95 red deer and one fox) from Bavaria and Austria. All three subtypes occur spatially distributed and are found in cattle and in red deer suggesting transmission between the two species. As subtypes are genetically stable in both species it is hypothesized that the described genetic variations developed within the host due to 'within-host replication'. The results of this study recommend the genomic RD4 region as a reliable diagnostic marker for M. caprae subtype differentiation.

A miniature temperature high germanium doped PCF interferometer sensor.

We report in this paper a high thermal sensitivity (78 pm/°C) modal interferometer using a very short Photonic Crystal Fiber stub with a shaped Germanium doped core. The Photonic Crystal Fiber is spliced between two standard fibers. The splice regions allow the excitation of the core and cladding modes in the PCF and perform an interferometric interaction of such modes. The device is proposed for sensitive temperature measurements in transmission, as well as in reflection operation mode with the same high temperature sensitivity.

Polymerase chain reaction detection of Rickettsia felis-like organism in Archaeopsylla erinacei (Siphonaptera: Pulicidae) from Bavaria, Germany.

Fleas occur as ectoparasites of vertebrates around the world. The obligate intracellular bacterium Rickettsia felis has been detected globally in several flea species, causing a murine-typhus like disease in humans. In this study, a total of 150 hedgehog fleas (Archaeopsylla erinacei Bouché) were collected from 18 hedgehogs coming from four locations in southern Germany for the detection of R. felis. Individual DNA extracts were tested with polymerase chain reaction (PCR) for the amplification of the rickettsial ompB, gltA, ompA, and 16S rRNA genes. A total of 144 samples (96%) were positive using ompB PCR. Sequencing and phylogenetic analysis showed an organism very closely related to R. felis with 96% similarity. These results provided evidence that hedgehog fleas in Germany may be nearly 100% infected with a rickettsial species closely related to R. felis. Further studies are needed for its molecular and pathogenetic characterization. The hedgehog as a potential reservoir for the emergent pathogen R. felis or closely related strains also needs further study.

Disseminated angiostrongylosis with fatal cerebral haemorrhages in two dogs in Germany: a clinical case study.

Canine angiostrongylosis is a nematode infection in domestic dogs and wild carnivores. Few single case reports describing the occurrence of this disease in Germany exist and until recently angiostrongylosis has not been considered endemic in this country. The present report focuses on clinical, pathological and parasitological findings in two cases of fatal disseminated canine angiostrongylosis associated with multifocal haemorrhages in the central nervous system. Both animals, which lived in Germany, presented with rapidly progressive neurological signs including depression, ataxia, unilateral central blindness and epileptic seizures. Blood work revealed grossly elevated D-dimers and mild thrombocytopenia. Both animals were subsequently euthanised due to progressive clinical aggravation. Necropsy showed cerebral and lung haemorrhages in both animals. Multiple sections of nematode larvae consistent with Angiostrongylus vasorum were identified on histopathological sections of the brain, heart, kidney and lung in both animals and a predominantly granulomatous inflammation with the occurrence of multinucleated giant cells was observed. Adult nematodes were found in the larger lung arteries of one dog and Angiostrongylus infection was subsequently confirmed by PCR-analysis and sequencing in both dogs. A. vasorum larvae were not detected by faecal Baermann examination performed in one of the dogs. It was concluded that canine angiostrongylosis should be considered as differential diagnosis in dogs in Germany, even if faecal examination is negative. There is currently still a lack of studies investigating the occurrence of angiostrongylosis in dogs and intermediate hosts in Germany which would be necessary to survey the endemic realities of this disease.

Molecular evidence for Bartonella spp. in cat and dog fleas from Germany and France.

Nine hundred and fifty-two fleas were collected from 148 cats and 133 dogs at 18 widely distributed geographic locations in Germany and France and examined for the presence of six different Bartonella spp. (Bartonella bacilliformis, Bartonella clarridgeiae, Bartonella elizabethae, Bartonella henselae, Bartonella quintana, Bartonella vinsonii subsp. berkhoffii) by PCR. Thirty-five specimens (3.7%) tested positive for either B. henselae (14 positive fleas) or B. clarridgeiae (21 positive fleas). DNA of other Bartonella spp. were not detected. Bartonella clarridgeiae was the dominating species in samples from France (19 out of 22 positive fleas), whereas B. henselae was more frequent in Germany (11 out of 13 positive fleas). With 3.5% (22 out of 632 fleas) in France and 4.1% (13 out of 320 fleas) in Germany, the overall prevalences of pathogen did not vary significantly between the flea populations of both countries. 5.4% of cats in France versus 16.1% of cats from Germany were infested by fleas carrying Bartonella, whereas 9.5% of dogs in France but none of the examined dogs from Germany were infested by Bartonella positive fleas. The molecular evidence of Bartonella infections reveals that agents of zoonotic potential are established in flea populations in Germany and France and that the spectrum of species can vary significantly from country to country.

Characterization of an iridescent virus isolated from Gryllus bimaculatus (Orthoptera: Gryllidae).

We have isolated an iridescent virus from commercially produced colonies of Gryllus bimaculatus in Germany, which showed apparent mortality. Transmission electron microscopy studies on adult cricket specimens revealed the paracrystalline assembly of icosahedral virus particles in the cytoplasm of hypertrophied abdominal fat body cells. The infecting agent could be cultivated in the lepidopteran cell line sf-9, where it caused cytopathogenic effects such as cell hypertrophy, cytoplasmic vacuolization, and cell death within 8 days postinfection. Infection titers of the first virus passage reached 10(7.5) TCID(50)/ml. Negatively stained virus particles (n = 100) had dimensions of 172 +/- 6 nm (apex to apex) and 148 +/- 5 nm (side to side). SDS-polyacrylamide gel electrophoresis of virus proteins showed more than 20 distinct polypeptides with a major species of approximately 50 kDa. Analysis of the restriction fragment length profiles from digestion of purified viral DNA with the endonucleases EcoRI, BamHI, and HindIII showed marked differences from the profiles of iridoviruses of lower vertebrates (genus Ranavirus), e.g., Rana esculenta Iridovirus and Frog virus 3. Restriction enzyme digests with the endonucleases MspI and HpaII indicated the lack of methylation of viral DNA. Polymerase chain reaction led to the amplification of a 420-bp gene fragment with 97% sequence homology to the major capsid protein gene of Chilo iridescent virus. These data indicate that this new isolate, which we propose to be termed Gryllus bimaculatus iridescent virus, belongs to the genus Iridovirus of the family Iridoviridae.

Occurrence of an invertebrate iridescent-like virus (Iridoviridae) in reptiles.

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.

The effects of serotonin and dopamine on salivary secretion by isolated cockroach salivary glands

We have studied the effects of 3-hydroxytyramine (dopamine) and 5-hydroxytryptamine (serotonin) on (1) the rates of salivation from isolated salivary glands of the cockroach Periplaneta americana, (2) the protein content of the saliva, and (3) the ultrastructure of the salivary gland epithelium. The rates of neurotransmitter-induced salivation varied in a dose-dependent manner within the concentration range 10(-9) to 10(-4) mol l-1. Half-maximal secretory rates were induced by 6x10(-7) mol l-1 serotonin and 1.1x10(-7) mol l-1 dopamine. Stimulation of the glands by serotonin resulted in the production of a protein-rich saliva, whereas saliva was protein-free after stimulation by dopamine. Electron microscopic studies revealed that the central cells, which are believed to produce the proteinaceous components of the saliva, secrete their vesicular content after stimulation by 10(-6) mol l-1 serotonin for 20 min. In contrast, no morphological changes could be detected after stimulation by 10(-6) mol l-1 dopamine. These data indicate that dopamine stimulates only the secretion of the fluid component of the saliva, whereas serotonin is necessary to stimulate secretion of the proteinaceous components.

Localization of carbonic anhydrase in the salivary glands of the cockroach, Periplaneta americana.

Carbonic anhydrase (CA) activity was localized in the salivery glands of the cockroach, Periplaneta americana, by (1) Hansson's histochemical technique, and (2) the use of the fluorescent sulphonamide, 5-dimethyl-amino-naphthalene-1-sulphonamide (DNSA). Both techniques reveal the same distribution pattern of CA in the four morphologically different cell types of the glands: peripheral cells, central cells, inner acinar duct cells, and distal duct cells. Positive reactions with Hansson's cobalt/phosphate technique were found in the apical regions of the peripheral cells and the distal duct cells, and were inhibited by 10(-5) M acetazolamide in control experiments. No staining could be detected in the central cells and the inner acinar duct cells. The fluorescent CA inhibitor DNSA (10(-4)M) specifically stained the peripheral cells and the distal duct cells in methanol-fixed cryostat sections, whereas the central cells and the inner acinar duct cells remained unstained. The role of CA in the peripheral cells is not clear. CA activity in the distal duct cells may provide the protons needed to run the vacuolar-type H(+)-ATPase on the apical infoldings of the cells. This ATPase may be involved in modification of the primary saliva.

Immunocytochemical localization of Na+/K(+)-ATPase and V-H(+)-ATPase in the salivary glands of the cockroach, Periplaneta americana.

The acinous salivary glands of the cockroach (Periplaneta americana) consist of four morphologically different cell types with different functions: the peripheral cells are thought to produce the fluid component of the primary saliva, the central cells secrete the proteinaceous components, the inner acinar duct cells stabilize the acini and secrete a cuticular intima, whereas the distal duct cells modify the primary saliva via the transport of water and electrolytes. Because there is no direct information available on the distribution of ion transporting enzymes in the salivary glands, we have mapped the distribution of two key transport enzymes, the Na+/K(+)-ATPase (sodium pump) and a vacuolar-type H(+)-ATPase, by immunocytochemical techniques. In the peripheral cells, the Na+/K(+)-ATPase is localized to the highly infolded apical membrane surface. The distal duct cells show large numbers of sodium pumps localized to the basolateral part of their plasma membrane, whereas their highly folded apical membranes have a vacuolar-type H(+)-ATPase. Our immunocytochemical data are supported by conventional electron microscopy, which shows electron-dense 10-nm particles (portasomes) on the cytoplasmic surface of the infoldings of the apical membranes of the distal duct cells. The apically localized Na+/K(+)-ATPase in the peripheral cells is probably directly involved in the formation of the Na(+)-rich primary saliva. The latter is modified by the distal duct cells by transport mechanisms energized by the proton motive force of the apically localized V-H(+)-ATPase.

Salivary glands of the cockroach, Periplaneta americana: new data from light and electron microscopy.

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.