In vitro culture of a novel genotype of Ehrlichia sp. from Brazil.

Ehrlichiae are obligate intracytoplasmic Gram-negative, tick-borne bacteria belonging to the Anaplasmataceae family. Ehrlichioses are considered emerging diseases in both humans and animals. Several members of the genus Ehrlichia have been isolated and propagated in vitro. This study describes the continuous propagation of a Brazilian Ehrlichia sp. isolate in IDE8 tick cells, canine DH82 cells and bovine aorta cells. Initially, the organisms were isolated from the haemolymph of a Rhipicephalus (Boophilus) microplus tick into IDE8 cells. Infected IDE8 cells were brought from Brazil to Germany, where the organisms were continuously propagated in IDE8, DH82 and bovine aorta cells. Bovine aorta cells were infected and propagated for 3 months, corresponding to six subcultures, whereas the other two infected cell lines were kept for more than 1 year. During the cultivation period, 36 and 14 subcultures were carried out in IDE8 and DH82 cell cultures, respectively. Reinfection of IDE8 cells with organisms grown in DH82 cells was achieved. Sequence analysis made with a fragment of the 16S rRNA gene showed that this Ehrlicha sp. is closely related to Ehrlichia canis. However, the maximum likelihood phylogenetic tree shows that it falls in a separate phylogenetic clade from E. canis.

First record of Ixodes frontalis collected by flagging in Germany.

For the first time, a female specimen of Ixodes frontalis was collected using the flagging method in a public park in the Bavarian city of Ingolstadt, Germany. Previous reports were from migratory birds only. Therefore, this is the first evidence that I. frontalis may be part of the tick fauna of Germany.

Embryogenesis and the first-stage larva of Thelazia lacrymalis.

The female reproductive system of Thelazia lacrymalis (Nematoda: Thelaziidae) was investigated by light and scanning electron microscopy (SEM) with regard to the developmental stages and the stage deposited by the gravid nematode. Female T. lacrymalis have a didelphic and opisthodelphic type of reproductive system with paired ovaries, oviducts and uteri and a single vagina and vulva. Round and spindle-shaped primary oocytes are documented within the ovaries and oviducts, respectively. The distal part of each uterus provides a fertilization chamber filled with spermatozoa, followed by a sphincter-like part. Further anterior, the uteri broaden gradually containing dividing zygotes, small and large morulae, tadpole-stage embryos and horseshoe-shaped embryos which increase in length and become slimmer forming pretzel-stage embryos and larvae rolled up. The larvae stretch gradually and finally lie straight but still covered with their egg membrane in the vagina. The egg membrane encloses the whole larva and is enlarged at the pointed tail of the larva forming a bulb. At the SEM level, the first-stage larva is shown to have a terminal mouth and three hooks directed posteriorly and a striated cuticle. As morphologically identical larvae were also found in lavages of the conjunctival sac of horses infected with T. lacrymalis, this nematode species can be described as ovoviviparous.

Morphology and structural organization of gené's organ in Dermacentor reticulatus (Acari: Ixodidae).

Scanning and transmission electron microscopical investigations revealed that Genés organ in unfed and ovipositing females of Dermacentor reticulatus is formed as a double-sac-structure consisting of an outer epithelial and an inner cuticular sac. In ovipositing ticks the latter emerges through the camerostomal aperture to the exterior. Genés organ in unfed ticks consists of a corpus, two posterior horns and a pair of undeveloped glands at each side, which differentiate in ovipositing ticks to compound, branched tubular glands with a main efferent duct for each gland opening into the lumen between the epithelial and the cuticular sac. Genés organ of egg-laying females corresponds basically in morphology and structural organization to that of unfed ticks. Compared with unfed ticks, however, in ovipositing ticks the corpus and horns are longer and broader, the glands are fully developed and the cuticular sac is evertable. The epithelial sac as the outermost part of Genés organ is continuous with the hypodermis of the basis capituli and the scutum, arises at the camerostomal aperture, forms the corpus and the two blind-ending horns, passes into the epithelium of the main excretory ducts of the glands and envelops the cuticular sac. The cuticular sac passes into the cuticle of the basis capituli and the scutum, arises at the camerostomal aperture, is folded, expands into the horn tips and consists inwards of a smooth epicuticula and outwards of a fibrous endocuticula. Muscles originating from the scutum pass caudomedially through the epithelial sac and are inserted into the cuticular sac. The entire surface of the maximally everted cuticular sac is covered with an amorphous mass. In cleaned samples, ledge-like structures appear on the lateral surface. These ledges turn into balloon-like structures which extend over the medial and dorsal surface. The entire surface including the balloon-like structures and the ledges are provided with numerous cribrate pits.

Morphology and structural organization of Gene's organ in Argas walkerae.

Electron microscopy revealed that Gene's organ in females of Argas walkerae Kaiser & Hoogstraal (Ixodida: Argasidae) is formed as a double-sac structure consisting of an outer epithelial and an inner cuticular sac. The latter emerges through the camerostomal aperture to the exterior in ovipositing ticks. The epithelial sac forms the corpus and the two blind-ending horns, which pass into the epithelium of the excretory duct of a gland at each side of Gene's organ and envelop the cuticular sac. Both excretory ducts open into the lumen between the epithelial and the cuticular sac. The cuticular sac is folded and consists of a fibrous endocuticula outwards towards the lumen between the epithelial and the cuticular sac and of a smooth epicuticula inwards. Parallel running grooves occur over the lateral epicuticular surface turning medially into cobble-stone pavement-like rises. Tubuli pass through the cuticular sac ending in pores on the epicuticular surface and open into the lumen between the epithelial and the cuticular sac. Muscle fibres pass through the epithelial sac at the horn tips and are inserted to the cuticular sac. In ovipositing females, the glands are fully developed and the lumen between the epithelial and the cuticular sac is filled with an amorphous mass.

Factors affecting proliferation and dedifferentiation of primary bovine oviduct epithelial cells in vitro.

The oviduct is the physiological site for key events in reproduction, such as capacitation of spermatozoa, fertilization and early embryonic development. Interactions between oviduct epithelial cells and gametes or embryos cannot sufficiently be studied in vivo. Therefore, model systems are needed which mimic in vivo conditions most closely. In this study we optimised the method for isolating bovine oviduct cells and compared different cell support materials as well as two culture systems (perfusion vs static culture) for their ability to maintain characteristic morphological and functional features of oviduct cells. Out of nine different cell support materials tested, cellulose nitrate (0.45 micron pore size) was the most suitable to maintain cells in a manner similar to freshly isolated oviduct epithelial cells. Comparing static vs perfusion culture by electron microscopy, morphological differences of the cells were insignificant in the first days of culture, while they became more evident after 8 days. The cells in the static system lost typical characteristics such as columnar shape, cilia and secretory protrusions, while these features were still present in perfusion culture. In addition, intense ciliogenesis and cytoplasmic organelles for protein synthesis were found under perfusion conditions. These findings were underlined by differences in expression of the oviduct-specific oestrus-associated glycoprotein 85-97 kDa (GP 85-97) gene as revealed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The RNA levels of this specific gene were significantly higher in perfusion compared to the static culture system. Our data show clear advantages of perfusion vs static culture for primary bovine oviduct epithelial cells.

Morphology of the eyes in adult Hyalomma truncatum ticks (Acari: Ixodidae).

The eyes of Hyalomma truncatum ticks are morphologically similar in both sexes. They appear as paired hemispherical structures situated dorsally between the first and second pair of legs on the lateral scutal margin. Each eye consists of a lens and photoreceptor cells, which are separated by a fine fibrillar layer and the hypodermis. The lens contains numerous channels which open beneath the epicuticle, converge uninterruptedly to the interior of the lens and end below the fine fibrillar layer. The inner closure of the lens is formed as an oval plateau with a cone-like projection situated caudolaterally and eccentrically to the longitudinal axis of the lens. The hypodermis is a single layer of cells, situated immediately below the fine fibrillar layer. Beneath the hypodermis, directly below the cone-like projection of the inner lens are the photoreceptor cells localized in a rosette-like arrangement. Facing the hypodermis, each photoreceptor cell is provided with numerous microvilli. The microvilli consistently border the microvilli regions of other cells at different angles but are always oriented at a right angle to the lenticular channels. The photoreceptor cells are unipolar neurons, whose axons arise from the basal portion of the cell and join to nerve fibre bundles forming the optic nerve. It is concluded that the eyes in adult H. truncatum ticks possess all the structures necessary to perceive and conduct light stimuli and visual signals.

[Stone age deerfly (Lipoptena cervi) found with a mummy in a glacier]. / Steinzeitliche Hirschlausfliegen (Lipoptena cervi) in Beifunden einer Gletschermumie.

In hair samples from the accompanying equipment of a human mummy preserved in glacial ice in South Tyrol for more than 5000 years, numerous remains of insects were found. These fragments corresponded in form, size and structural organization to the features of the deer ked, Lipoptena cervi. Considering the biology and ecology of L. cervi with regard to the finding place of the mummy at an altitude of 3210 m above sea level in a glacier it is concluded that the deer keds invaded the equipment before and not after the death of the Stone Age man.

Comparative studies of synthesis, phosphorylation, DNA binding and proteolytic characteristics of a novel protein during phases of the mouse spleen cell cycle.

1. Cultured mouse spleen cells were exposed to the mitogen Concanavalin A followed by isoproterenol, and nuclei were electronically sorted from seven partitions of the cell cycle. 2. Several nuclear proteins, including stress proteins, which were cell-cycle-stage specific, were elicited by isoproterenol as determined by micro-electrophoresis and fluorography. 3. Two novel S-phase proteins (X0 and X') demonstrated differing synthesis and phosphorylation patterns during the cell-cycle phases. 4. X' showed DNA binding characteristics and proteolytic properties (hydrolyzing X0 or beta-galactosidase); both proteins were cell-cycle regulated.

[Trichomonad infections of the oral cavity in horses in southern Germany]. / Trichomonaden-Infektionen der Mundhöhle bei Pferden in Süddeutschland.

Trichomonads of the oral cavity were found in 9 of 60 investigated horses. Apart from dental tartar, the oral cavity showed no clinical signs in all positive horses. The clinical investigation of these horses additionally revealed colic in 4 and coughing in 4 horses as well as lumbago in 1 animal. By means of scanning electron microscopy the trichomonads were shown to be round or piriform with an average length of 7.6 microns and greatest width of 6.3 microns. They had 4 anterior flagella with an average length of 8.3 microns, an undulating membrane measuring 8.7 microns with no trailing flagellum as well as an axostyle extending on average 7.8 microns beyond the body, and therefore have to be placed into the genus Trichomonas.

Morphology and structural organization of spiracles in female Argas (Persicargas) walkerae (Acari: Argasidae).

Scanning electron microscopical investigations of fractures and corrosion casts of the spiracles from female A. walkerae ticks revealed a four-part structure, consisting of spiracular plate, ostium and macula forming the external closure, followed by the subostial space and the vestibulum of regulable volume, as well as the atrial chamber as the innermost part from which the main tracheal trunks originate. On the average, the spiracular plate was 158 microns long and 188 microns at the broadest width. It consisted of a thin, highly perforated external and a thick internal layer, which enclosed the interpedicellar space with numerous stout pedicels. In its posterior region, the spiracular plate was covered by the macula, which was up to 80 microns in length and 110 microns in width. The interpedicellar cavity opened into the subostial space measuring 95.5 microns in length and 159.6 microns in width, which proceeded into the 112-microns long vestibulum. The roof of the vestibulum was flexible and could be everted and inverted. Inverted, the roof formed a quadratic bulge with numerous deep cuticular folds, which confined the lumen of the vestibulum either partially or completely. In corrosion casts, the roof was everted to a length of up to 89.3 microns. In the posterior part of the vestibulum, as well as in the initial fourth of the atrial chamber, numerous anvil-, cone- or drop-like cuticular projections were arranged in wedge-like fashion. The atrial chamber was almost spherical with a diameter of 138.4 microns. Five main tracheal trunks of different luminal diameter as well as numerous channels opened into the atrial chamber.

[Trichomonad infections of the oral cavity in cats in south Germany]. / Trichomonaden-Infektionen der Mundhöhle bei Katzen in Süddeutschland.

In this investigation trichomonads were isolated from the oral cavity in 21 of 110 examined cats, and only from those which were simultaneously FeLV, FIV or FIP positive. By means of scanning electron microscopy the trichomonads were shown to be round or piriform parasites which were on average 7.9 microns long and 6.2 microns wide at maximum width. They had 4 anterior flagella, which were on average 9.4 microns long, an undulating membrane measuring 6.7 microns with no trailing flagellum as well as an axostyle extending on average 5.0 microns beyond the body, and therefore should be attributed to the genus Trichomonas.

Nitrobenzo[a]pyrene-induced DNA amplification in SV40-transformed Chinese hamster embryo cells.

Nitrobenzo[a]pyrenes (NBaPs) are ubiquitous environmental pollutants that produce mutations in Salmonella typhimurium and Chinese hamster ovary cells. In this study, 1-, 3-, and 6-NBaP induced amplification of SV40 DNA sequences in an SV40-transformed Chinese hamster embryo cell line which is sensitive to DNA amplification by various known carcinogens. Of the three isomers, 3-NBaP produced the highest level of gene amplification, which was 4.8 relative to untreated controls at a dose of 5 micrograms/ml. Considering the relationship between gene amplification and tumorigenesis, it seems prudent to carry out a more exhaustive analysis of the carcinogenic potential of these agents.

Morphology and structural organization of Haller's organ during postembryonic development of Argas (Persicargas) walkerae (Ixodoidea: Argasidae).

Scanning-electron-microscopic investigations of Haller's organ in larvae, nymphs I, II, III and IV, and male and female adult Argas (Persicargas) walkerae ticks showed that morphology and structural organization change during postembryonic development. Stage-dependent differences existed regarding setal numbers of the anterior pit as well as formation and reticulation of cuticular projections in the capsule cavity. The anterior pit increased in size in the course of postembryonic development. It contained only seven setae in larvae, one conical, setiform and grooved seta each as well as two porose and fine setae. Nymphs I, II, III and IV adult ticks had equal numbers of setae; however, one additional unilaterally serrate and grooved seta each were present. Setal length increased continuously during postembryonic development and attained maximum values in adult ticks. The capsule consisted of roof and cavity and was located distinctly lateral in larvae, slightly lateral in nymphs I and II, and in all other stages directly on the longitudinal axis of tarsus. The capsule roof showed a reticular structure. The slit-like main aperture was located peripherally and arranged transversally to the longitudinal axis of tarsus I in larvae. Nymphs and adult ticks had a central, circular main aperture. Stage-dependent cuticular projections of varying form protruded into the capsule cavity. Larvae had only single, free-standing projections which ramified slightly and communicated with each other. Projections were more heavily reticulated in nymphs I and II. In nymphs III and IV as well as male and female adult ticks, a long centrally situated tube of reticular appearance was seen, which was supported by a large number of radially organized and interlocking pillars and communicated with the capsule roof. In all tick stages there were always four porose setae present, arranged on the capsule floor.

[Imported infestations of nasopharyngeal parasites in dogs]. / Importierte Infestationen nasopharyngealer Parasiten beim Hund.

In this paper infestations of the nasal cavity with Myxobdella annandalei in a dog after a sojourn in Nepal and with Linguatula serrata in 2 stray dogs from Turkey, which were imported to Germany, are described.

Synthesis and biochemical characteristics of nucleoproteins following a toxic dose of retinoic acid in cycling and differentiating HL-60 cells.

Exposure of HL-60 cells to 2 microM retinoic acid (RA), twice the dose necessary for differentiation, initiated rapid synthesis (2 h) of the nuclear stress proteins (SPs) e.g., 90, 70c, 70x, 22 (Mr x 10(-3)) during the G0 + G1 phase of the cell cycle as observed by polyacrylamide gel electrophoresis (PAGE). Synthesis of SPs was cell cycle correlated and not dependent on time-after-dosing, and all labeling disappeared from these proteins within 48 h of RA exposure. Stress proteins were not elicited with a 1 microM dose or less of retinoic acid. Non-stress nuclear proteins revealed changes in synthesis levels (e.g., actin, lamins, tubulins) which were cell cycle related and temporally associated with dosing. A major non-stress nuclear protein (Mr 120,000) which possessed an affinity for actin in binding assays, was cell cycle related in control cells, and was suppressed in synthesis in cells exposed to 2 microM retinoic acid. Two additional nuclear non-SPs 51 and 55 (Mr x 10(-3)) covalently bound the isotope [3H]retinoic acid, and their incorporation was cell cycle correlated during early periods of RA exposure. Except for the induction of SPs, the autoradiographs of nuclear proteins of RA dosed HL-60 cells, showed more quantitative than qualitative changes.

The induction of aneuploidy by 3-nitrobenzo[a]pyrene in Chinese hamster ovary cells.

Nitrobenzo[a]pyrenes are found in urban air particulates and particulates from diesel exhaust, gasoline engines and wood burning stoves. Following exposure of Chinese hamster ovary cells (CHO-K1-BH4) to 3-nitrobenzo[a]pyrene (3-NB[a]P), cells with multiple nuclei and/or nuclei with multiple lobes were observed. When CHO cells were treated with 5 micrograms/ml 3-NB[a]P for 5 or 20 h, aneuploidy was noted in these cells at 24-96 h post-exposure. The addition of N6, O2'-dibutyryl adenosine 3':5'-cyclic monophosphate to 3-NB[a]P-exposed CHO cell cultures appeared to reduce the amount of aneuploidy in treated cultures. Structure--activity studies showed that 1-NB[a]P was a much less effective inducer of aneuploidy than 3-NB[a]P and 6-NB[a]P was ineffective. 1-, 3- and 6-nitrosobenzo[a]pyrenes were not effective inducers of aneuploidy in CHO cells, and aneuploidy was not observed in cultures treated with 3-NB[a]P in the presence of S9 activation. It appears that the parent 3-NB[a]P is responsible for producing aneuploidy in CHO cells.

Effect of interleukin-2 on cell proliferation, sister-chromatid exchange induction, and nuclear stress protein phosphorylation in PHA-stimulated Fischer 344 rat spleen lymphocytes: modulation by 2-mercaptoethanol.

The effect of interleukin-2 (IL-2) on cell proliferation, sister-chromatid exchange (SCE) frequency, and the phosphorylation of nuclear stress proteins was evaluated in phytohemagglutinin (PHA)-stimulated spleen lymphocytes isolated from Fischer 344 rats. In addition, the ability of 2-mercaptoethanol (2-ME) to modulate the induction of these biological responses was characterized. Cell proliferation, as measured by the mitotic index, increased significantly (P less than .003) from a range of 3-4% in PHA-stimulated cultures to a range of 8-11% in PHA-stimulated cultures exposed to IL-2. The average generation time (AGT) did not respond to IL-2 in a concentration-dependent manner and decreased significantly (P less than .05) when 20 microM 2-ME was included with IL-2 in the culture medium. The number of SCE increased significantly (P less than .004) from control frequencies, which ranged from 13.1 to 15.6 SCE per cell, to frequencies of 18.5 to 21.5 SCE per cell as the concentration of IL-2 in the culture medium increased to 50 half-maximal units per ml. A reduction in SCE frequency was observed when cells were cultured with 20 microM 2-ME and IL-2 compared to IL-2 alone. Three nuclear proteins, with relative molecular masses of approximately 13,000-18,000, 20,000, and 80,000, were phosphorylated in IL-2-exposed G1-phase nuclei. Elicitation of these nuclear proteins in IL-2-exposed cells was not affected by exposure to 2-ME.