Differentially expressed genes in silkworm cell cultures in response to infection by Wolbachia and Cardinium endosymbionts.

Wolbachia and Cardinium are bacterial endosymbionts that are widely distributed amongst arthropods. Both cause reproductive alterations, such as cytoplasmic incompatibility, parthenogenesis and feminization. Here we studied differentially expressed genes in Wolbachia- and Cardinium-infected Bm-aff3 silkworm cells using a silkworm microarray. Wolbachia infection did not alter gene expression or induce or suppress immune responses. In contrast, Cardinium infection induced many immune-related genes, including antimicrobial peptides, pattern recognition receptors and a serine protease. Host immune responses differed, possibly because of the different cell wall structures of Wolbachia and Cardinium because the former lacks genes encoding lipopolysaccharide components and two racemases for peptidoglycan formation. A few possibly non-immune-related genes were differentially expressed, but their involvement in host reproductive alteration was unclear.

Cytogenetic characteristics of cell lines from Ixodes scapularis (Acari: Ixodidae).

Three new cell lines, IDE8 and IDE12 from embryos of northern specimens of Ixodes scapularis Say and ISE18 from southern specimens of I. scapularis, were compared cytogenetically via conventional karyotyping, C- and G-banding, and nucleolar organizing regions (NORs). The karyotypes were very similar. The standard karyotype in the three cell lines consisted of 28 chromosomes with 26 autosomes and XX (female) or XY (male) sex chromosomes. The X chromosome was the largest, and the Y chromosome the smallest chromosome of the karyotype. Constitutive heterochromatin (C-bands) was almost entirely restricted to the centromeric region. An additional interstitial C-band in chromosome 7 was an important notable characteristic of the three cell lines. In sets showing a similar degree of condensation, individual chromosomes of the three lines had identical G-banding patterns. In addition, there was no difference among the cells in number and position of NORs. There were approximately 100 G-bands per haploid set in chromosomes from cells in metaphase, with three to 18 G-bands in each chromosome arm. After staining with silver nitrate, interstitial NORs were identified in chromosomes 7, 10, and the X chromosome. Male cells had five and female cells had six NORs. These findings support the notion that I. scapularis and I. dammini Spielman et al. are conspecific.

Adhesion to and invasion of cultured tick (Acarina: Ixodidae) cells by Borrelia burgdorferi (Spirochaetales: Spirochaetaceae) and maintenance of infectivity.

Lyme disease spirochetes, Borrelia burgdorferi, interact with cultured tick cells in ways similar to those reported to occur in the vector Ixodes dammini Spielman, Clifford, Piesman & Corwin. Spirochete adhesion and penetration were examined using a cell line from embryos of Rhipicephalus appendiculatus Neumann that morphologically resembles tick gut cells, RAE25. Cocultivation of B. burgdorferi with these cells permitted prolonged maintenance of infectivity for hamsters. Borrelial adherence to RAE25 cells was time- and density-dependent and increased by 10-15% per h during the first 5.5 h of cocultivation when we used a concentration of 4 x 10(7) spirochetes/ml. After 6 h, > 90% of the cells bound an average of 3-5 spirochetes per cell. Low passage, hamster-infective strains of B. burgdorferi (JMNT and CD16) showed a 2-3-fold higher rate of adhesion to RAE25 cells than the highly passaged, noninfectious strain B31. Inactivation of CD16 or JMNT by heat, starvation, or treatment with puromycin reduced adherence by 40-60%, whereas pretreatment with monoclonal antibodies to the outer surface proteins had no effect. Spirochetes adhered to young I. dammini cell lines to a similar degree as they did to RAE25, whereas lines from the ticks Dermacentor variabilis (Say) (RML15) and Boophilus microplus (Canestrini) (BME26) bound 30-60% fewer spirochetes. Electron microscopy revealed epicellular borreliae associated with coated pits and vesicles before endocytosis, and intracellular spirochetes were surrounded by a host cell-derived membrane.

Establishment of the tick (Acari:Ixodidae)-borne cattle pathogen Anaplasma marginale (Rickettsiales:Anaplasmataceae) in tick cell culture.

Anaplasma marginale is a tick-borne rickettsia that causes bovine anaplasmosis worldwide. Despite its importance, A. marginale has thus far not been established in a continuous culture system. We have propagated A. marginale continuously for the 1st time in a tick cell line derived from the black-legged tick, Ixodes scapularis Say, using infected bovine blood as the inoculum. Erythrocytic stages invaded the tick cells and multiplied in membrane-lined vacuoles to form colonies typical of those observed in naturally infected ticks as demonstrated by light and electron microscopy. The rickettsiae have been passaged serially for 3 yr and have been cryopreserved in liquid nitrogen. Antigens present in A. marginale from tick cell culture were recognized by bovine immune serum against the blood stages of A. marginale. A. marginale grown in this tick cell line was infective for calves, and male ticks fed on the calves transmitted A. marginale to a susceptible calf. The ability to culture A. marginale removes a major impediment to the study of Anaplasma biology in vitro, and will enhance development of vaccines and diagnostic tests.

Power and heat measurement by direct calorimetry of individual insect response to allelo- and toxic compounds.

A microcalorimeter was constructed for the individual insect in order to measure the insect's power output as a function of time (the thermogram). The instrument has a figure of merit of 7 microW/microV. It includes a Peltier pumped thermoelectric control loop which protects from intruding ambient thermals, and holds baseline drift to less than 2 microV/day. the design is a conventional twin, or differential heat conduction calorimeter, with two insect-holding vessels of glass. The vessels are connected by a stopcock, to give the insect the option of crawling from the sample chamber to the reference chamber. Continuous, metered air flow is provided. A small pulse of compound may be born in, as vapor with the air flow, for the study of attractants, toxic compounds, anesthetics and allelochemicals. The insect's reaction to such compounds, appears in the thermogram. Cabbage looper larvae were examined in their irritative exothermic reaction to microgram amounts of benzene, and in their neutral behavior toward similar amounts of aliphatic hydrocarbons delivered as a single pulse of vapor. The male northern corn rootworm was monitored in his attraction to female rootworm extract ("pheromone') and his tendency to move upwind, or up airflow, toward the pheromone. The instrument enables discrimination of the transient metabolism of muscle use, from that of the resting state power which is probably the true basal metabolic rate power.

Comparative studies on the infectivity of Plasmodium berghei gametocytes and ookinetes for gnotobiotic and xenobiotic Anopheles stephensi.

Previous studies indicated that gnotobiotic Anopheles stephensi mosquitoes were less susceptible to infection with Plasmodium berghei than xenobiotic ones (Munderloh and Kurtti, 1985). Groups of 100 to 200 mosquitoes were fed on infected hamsters, heparinized gametocytemic blood (via a membrane feeder), and in vitro-formed ookinetes suspended in blood (membrane feeder). Xenobiotic A. stephensi were readily infected by all 3 routes. Gnotobiotic mosquitoes consistently acquired infection after engorging on hamsters (average level of infected females in 8 experiments: 54.1%), but the parasite yield was low (average number of oocysts per infected female: 21.6). In 7 experiments where gnotobiotic A. stephensi were membrane-fed infected hamster blood, an average of only 8.8% of the females became infected, harboring a mean of 2.4 oocysts, and in 7 additional cases no infection was achieved. This pattern was reversed when gnotobiotic A. stephensi were fed ookinetes. A larger proportion of them became infected (mean level of infection in 8 experiments: 76.2%) and they acquired a higher mean number of oocysts per female (94.4) than did xenobiotic mosquitoes. Thus, gnotobiotic A. stephensi are as able as xenobiotic ones to support the sporogonic development of P. berghei, but are less able to support ookinete development.

The effects of 20-hydroxyecdysone and juvenile hormone III on tick cells.

Two cell lines isolated from Rhipicephalus appendiculatus ( RAE 25) and Anocentor (= Dermacentor) nitens (ANE 58) responded to the invertebrate hormones 20-hydroxyecdysone (20-HE) and juvenile hormone III (JH III) in vitro. In the presence of 0.2 or 2 nMolar 20-HE, the cells of the continuous line RAE 25 attached to the culture substrate at a rate of 9% per hr for the first 8 hr, as did cells in growth medium. Twenty or 200 nMolar of 20-HE reduced the rate of cell attachment to 6% per hr, and in the higher hormone concentration the cells ceased to attach after 4 hr. Low concentrations (0.2 and 2 nMolar ) of 20-HE stimulated the growth of the RAE 25 line (P less than 0.02), but 200 nMolar or more inhibited growth (P less than 0.001). Twenty-HE suppressed the growth of the young line ANE 58 in a dose-dependent manner, but the decrease in cell growth was less pronounced than in RAE 25. Ten to 100 times more (2 and 20 mu Molar) 20-HE was needed to achieve significant growth suppression (P less than 0.025 and less than 0.005). The growth of both lines declined (P less than 0.01) by 20% ( RAE 25) or 30% (ANE 58) when the medium contained 38 mu Molar of JH III. The bimodal growth response of line RAE 25 to 20-HE also occurred in the presence of 3.8 and 38 mu Molar JH III, and 2 nMolar 20-HE counteracted the suppressive effect of 38 mu Molar JH III.(ABSTRACT TRUNCATED AT 250 WORDS)

The infectivity and purification of cultured Plasmodium berghei ookinetes.

Plasmodium berghei ookinetes were cultured from hamster blood as described previously (Kurtti and Munderloh, 1986). An average of 7.3 X 10(6) ookinetes was harvested from each ml of blood. Ookinetes were purified by centrifugation on first a 40% and then a 36% Percoll gradient. The final preparation comprised 32.8% of the ookinetes initially obtained, and contained 3.3 other parasite stages or blood cells per ookinete. Unpurified and purified ookinetes were resuspended in hamster blood and fed to Anopheles stephensi. There was a strong linear correlation between the concentration of purified or unpurified ookinetes and the number of oocysts formed. With unpurified ookinetes, a maximum was reached when preparations containing 1 X 10(7) ookinetes/ml were fed, and feeding preparations containing a higher concentration did not produce more oocysts. Sporozoites were found in the salivary glands of mosquitoes fed ookinetes by days 14 (unpurified) or 15 (purified) PI. Approximately 5 times as many purified as unpurified ookinetes were required to produce each oocyst.

Anopheles stephensi and Toxorhynchites amboinensis: aseptic rearing of mosquito larvae on cultured cells.

Aseptic larvae of Anopheles stephensi and Toxorhynchites amboinensis were reared on a continuous cell line (RU TAE 12 V) from the mosquito, T. amboinensis, that grew in suspension as multicellular vesicles. Surface-sterilized eggs were hatched in a 24-well plate containing 0.2 ml of Leibovitz's L-15 medium per well and incubated in a humidified atmosphere. Toxorhynchites amboinensis eggs of 36 hr or older were placed singly to assure hatching and avoid cannibalism. Hatching rates were over 80%. All larval instars were maintained in L-15 medium at 28 C with a 12-hr photoperiod. Anopheles stephensi larvae were reared in 25-cm2 tissue culture flasks containing 10 ml of L-15 medium with 30 to 50 first and second instar larvae or 10 third and fourth instar larvae per flask. Toxorhynchites amboinensis larvae remained in the 24-well plate in 1.5 ml of medium through the second instar; third instar larvae were kept in 12-well plates (3 ml of medium per well) and transferred to 25-cm2 flasks (10 ml per flask) when they reached the fourth instar. First and second instar A. stephensi larvae were fed cultured cells once, and third or fourth instar larvae twice a day. Toxorhynchites amboinensis larvae were fed vesicles once during the first 4 days after hatching, and every 1 or 2 days thereafter. Each A. stephensi larva consumed approximately 2 X 10(6) cells, and T. amboinensis larvae 10 times more cells before pupating. Anopheles stephensi pupated after 7 to 8 days and adults emerged during days 9 to 11. Pupation in T. amboinensis began on day 21 after hatching and adults emerged 5 days later. Cell lines isolated from A. stephensi larvae or embryos of the ticks Rhipicephalus sanguineus and Anocentor (Dermacentor) nitens supported only limited growth of A. stephensi larvae. Defibrinated hamster (Mesocricetus auratus) blood, though readily ingested, did not support the growth of A. stephensi whereas larvae reared on blood cells plus T. amboinensis cells showed limited growth.

Effect of medium supplements on tick cells in culture.

The growth of tick cells in Leibovitz's L--15 medium supplemented with various concentrations of fetal bovine serum (FBS), tryptose phosphate broth (TPB), and tick egg extract (TEE) was evaluated using a protein assay. A continuous cell line from Rhipicephalus appendiculatus (RA 243) was compared with young lines of cells isolated from embryos of R. appendiculatus (RAE 25) and Rhipicephalus sanguineus (RSE 8). We found fetal bovine serum and tryptose phosphate broth both to be essential supplements. The addition of tick egg extract further stimulated growth. The yield of cellular protein in both young and continuous lines of tick cells increased as a function of the concentration of tryptose phosphate broth from 0 to 10%, and fetal bovine serum from 2.5 to 20%. The growth of the RA 243 line correlated negatively with the size of the inoculum and positively with the concentration of fetal bovine serum, as the greatest increase in cell protein was obtained when cells were seeded at a low density into a medium containing 20% fetal bovine serum. The addition of an extract prepared from eggs of R. sanguineus or Hyalomma excavatum improved yields of cultures and promoted cell growth at low population densities. The protein yield increased as a function of tick egg extract concentration, but 0.8% inhibited growth of the RA 243 line. The RA 243 line could be propagated in a medium supplemented with 10% tryptose phosphate broth, 5% fetal bovine serum, and 0.5% tick egg extract.

Establishment, maintenance and description of cell lines from the tick Ixodes scapularis.

Interest in tick-borne pathogens has been enhanced by the emergence of Lyme disease and, more recently, human and animal ehrlichioses. In order to facilitate investigations of the vector phase of tick-borne disease agents in vitro, several new cell lines derived from embryonated eggs of northern (IDE lines) and southern (ISE lines) populations of the tick Ixodes scapularis were developed. The establishment and characteristics of 4 IDE (IDE1, 2, 8, and 12) and 2 ISE (ISE5 and 18) lines were described. Primary cultures were initiated in L-15B medium at 31 C from a single egg mass each and established lines developed a morphologically distinct phenotype. Myoblasts were present during the first year after isolation in several lines as isolated clusters or sheets covering the whole flask. Cell line extracts resolved by isoelectric focusing were characterized for 3 isozymes (lactate dehydrogenase, malate dehydrogenase, and malic enzyme). The combined banding patterns allowed discrimination between Ixodes cell lines and a Rhipicephalus appendiculatus cell line. Two lines, i.e., ISE5 and ISE18, had unique isozyme bands. Chromosome numbers and morphology conformed to those described from tissue squashes of I. scapularis.

Epidemiological aspects of human granulocytic Ehrlichiosis in southern Germany.

Human granulocytic Ehrlichiosis (HGE) is a newly emerging acute febrile illness which is likely transmitted by ticks of the Ixodes ricinus/I. persulcatus complex. First seroepidemiological surveys on the prevalence of HGE antibodies, detection of DNA of granulocytotropic Ehrlichiae in I. ricinus and one case of HGE from Slovenia confirmed by serology and PCR (polymerase chain reaction) suggest that HGE might exist all over Europe. The purpose of the present study was a) to determine the prevalence of antibodies against the HGE agent in sera collected from persons at high risk for exposure to I. ricinus with that of a control population and b) to determine the prevalence of granulocytic Ehrlichiae in I. ricinus ticks from Southern Germany. We studied sera from 150 forestry workers and 105 patients with an established diagnosis of Lyme disease as tick-exposed populations. Sera from 103 healthy blood donors without a history of known tick bites served as controls. A significantly higher prevalence of HGE antibodies (P < or = 0.01) was present among patients with Lyme borreliosis (12 of 105 were positive; 11.4%) and forestry workers (21 of 150 were positive; 14%) compared to blood donors (2 of 103 were positive; 1.9%). Furthermore, 510 adult and nymphal I. ricinus were investigated by PCR for the presence of granulocytic Ehrlichiae with primers specific for the E. phagocytophila group. In eight (1.6%) of the investigated ticks the expected amplification product was detectable, indicating a low prevalence of infected ticks especially when compared with B. burgdorferi. The presented data strongly suggests that the HGE agent or a closely related organism exists in Southern Germany and therefore HGE should be considered in the differential diagnosis of febrile illnesses. However, final evidence can be provided only after isolation of the organism from patients.

Characterization of a new densovirus infecting the German cockroach, Blattella germanica.

A new DNA virus (Parvoviridae: Densovirinae, Densovirus) was isolated and purified from descendants of field-collected German cockroaches, Blattella germanica. Viral DNA and cockroach tissues infected with B. germanica densovirus (BgDNV) were examined by electron microscopy. Virus particles, about 20 nm in diameter, were observed both in the nucleus and in the cytoplasm of infected cells. Virus DNA proved to be a linear molecule of about 1.2 microm in length. BgDNV isolated from infected cockroaches infected successfully and could be maintained in BGE-2, a B. germanica cell line. The complete BgDNV genome was sequenced and analysed. Five open reading frames (ORFs) were detected in the 5335 nt sequence: two ORFS that were on one DNA strand encoded structural capsid proteins (69.7 and 24.8 kDa) and three ORFs that were on the other strand encoded non-structural proteins (60.2, 30.3 and 25.9 kDa). Three putative promoters and polyadenylation signals were identified. Structural analysis of the inverted terminal repeats revealed the presence of extended palindromes. The genome structure of BgDNV was compared with that of other members of the family Parvoviridae; the predicted amino acid sequences were aligned and subjected to phylogenetic analyses.

The dissociation of insect embryos for cell culture.

Procedures and solutions were developed for dissociating embryos of Blattella germanica in preparation for primary cell culture. Trypsin solutions were maximally effective at 0.01% for germ bands but higher concentrations, 0.05 to 0.1% were needed for embryos in later stages.

Cellular and molecular interrelationships between ticks and prokaryotic tick-borne pathogens.

Tick-borne prokaryotic pathogens share a very intimate relationship with the vectors. Ingestion during the bloodmeal places the microbe into the gut lumen whence it must travel to the salivary glands at the right time for transmission during a subsequent feeding. This crucial event requires coordination between pathogen development and arthropod host activities that may be mediated by the expression of genes specific for the vector phase of the pathogen. Invertebrate hormones or factors associated with tick tissues may provide the cues that signal changes in tick physiology that induce necessary steps in the pathogen, such as colonization of ovaries during egg development in preparation for transovarial transmission or dispersion to the salivary glands at the time of a bloodmeal. These hypotheses cannot easily be investigated within the complex environment of the tick, but tick cell culture offers a simplified system with which to examine many of these important interrelationships.

Malarial parasites complete sporogony in axenic mosquitoes.

To obtain sporogonic stages of malaria free from microbial contaminants for in vitro studies, Anopheles stephensi were reared under sterile conditions using a mosquito cell line as larval food. The adult females, kept in sterile humidified containers and allowed to engorge on parasitemic hamsters, supported the sporogonic development of the rodent malarial parasite Plasmodium berghei. In 10 experiments, the proportion of infected mosquitoes varied from 0 to 92%, and the geometric mean number of oocysts per female mosquito from 2.5 to 58.6, with a range of 1 to 548. The average number of salivary gland sporozoites per infected mosquito was determined by direct sporozoite counts in the pooled homogenate of the thoraces of all female mosquitoes. In five experiments, it varied from 2.7 X 10(3) to 9.0 X 10(3). The sterile sporozoites, harvested on day 19 or 20 after the infective blood meal, were as infective for rodents as nonsterile ones.