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Vairimorpha necatrix infected and replicated in the Heliothis zea cell line HZBCIRLAM1. Spores were purified from caterpillars by Percoll density gradient centrifugation. After priming with 0.01 n KOH in 0.17 m KCl, the spores were mixed with H. zea cells suspended in Tris-HCl ( p H 8) containing 0.17 m KCl, and EDTA as a chelator. Infected cells were cultured at 28°C in L-15B medium with 5% serum. Sporoplasms and early stages were noted in cells stained with Giemsa's solution within the first 24 hr post-infection (pi). The sporoplasms developed into bipolar meronts but did not divide during this time. Using 5 to 10 spores per cell, we initially infected 23.64 ± 4.27% of the cells in three duplicate studies. Three-fourths of the infected cells harbored a single sporoplasm, and the remainder were infected with two to four parasites each. The proportion of infected cells rose to 36.03 ± 7.02% during the first 2 to 4 days, and after 1 week, meronts were rarely seen. Between day 1 and 3 pi V. necatrix multiplied with a doubling time of 8 to 12 hr. Three to 4 days pi, V. necatrix sporonts were seen and by 6 days pi mature spores developed. Within the first 4 days of culture, large multinucleated syncytia formed by cell fusion. The number of host cell nuclei in syncytia correlated with the number of V. necatrix; those with 3 to 5 H. zea nuclei and 20 to 40 V. necatrix were most common. There was considerable asynchrony in parasite development and variability in the number of V. necatrix per host cell.

Until the recent discovery of pRF inRickettsia felis, the obligate intracellular bacteria of the genusRickettsia(Rickettsiales:Rickettsiaceae) were thought not to possess plasmids. We describe pRM, a plasmid fromRickettsia monacensis, which was detected by pulsed-field gel electrophoresis and Southern blot analyses of DNA from two independentR. monacensispopulations transformed by transposon-mediated insertion of coupled green fluorescent protein and chloramphenicol acetyltransferase marker genes into pRM. Two-dimensional electrophoresis showed that pRM was present in rickettsial cells as circular and linear isomers. The 23,486-nucleotide (31.8% G/C) pRM plasmid was cloned from the transformant populations by chloramphenicol marker rescue of restriction enzyme-digested transformant DNA fragments and PCR using primers derived from sequences of overlapping restriction fragments. The plasmid was sequenced. Based on BLAST searches of the GenBank database, pRM contained 23 predicted genes or pseudogenes and was remarkably similar to the larger pRF plasmid. Two of the 23 genes were unique to pRM and pRF among sequenced rickettsial genomes, and 4 of the genes shared by pRM and pRF were otherwise found only on chromosomes ofR. felisor the ancestral group rickettsiaeR. belliiandR. canadensis. We obtained pulsed-field gel electrophoresis and Southern blot evidence for a plasmid inR. amblyommiiisolate WB-8-2 that contained genes conserved between pRM and pRF. The pRM plasmid may provide a basis for the development of a rickettsial transformation vector.

By using degenerate primers deduced from conserved patterns in the flavivirus polymerase gene, a novel RNA virus was discovered in Rhipicephalus ticks sampled from members of the family Bovidae in Senegal. It was named Ngoye virus (NGOV) after the location from which it was isolated. Viral particles could be observed by electron microscopy, but isolation in vertebrate or invertebrate cell lines or by intracerebral infection of newborn mice remained unsuccessful. This is atypical of recognized arboviruses. The characterization of 4176 nt of the non-structural genes revealed that NGOV is a novel flavivirus species. It forms a distinct phylogenetic lineage related distantly to previously identified members of the genus Flavivirus. Analysis of genetic data suggested that the processing of the NGOV polyprotein and the organization of its replication complex are similar to those of flaviviruses. Together with other recent data, these findings suggest that a large number of viruses related distantly to ‘classical’ arthropod-borne flaviviruses remain to be discovered.

Ixodes scapularis and Dermacentor andersoni cell lines were stimulated with heat-killed Escherichia coli and Micrococcus luteus to investigate whether infection by Rickettsia peacockii, an endosymbiont of D. andersoni, modifies humoral immune responses. Radial diffusion assays, western blotting, flow cytometry, and quantitative reverse-transcription PCR were used to determine if expression of bacteriolytic peptides, including lysozyme and defensin, was upregulated by bacterial stimulation or infection with R. peacockii. The I. scapularis line IDE12 upregulated expression of lysozyme and defensin following stimulation. The D. andersoni cell line DAE15 also expressed defensin and lysozyme, but only lysozyme was upregulated by bacterial stimulation. R. peacockii infection alone, or in cells stimulated with bacteria, did not modify defensin or lysozyme expression in either cell line. These results suggest tick endosymbionts may avoid recognition by the tick immune system, and infection may not affect humoral immune responses to bacteria not normally associated with ticks.

Ehrlichia chaffeensis and Ehrlichia canis are tick-transmitted rickettsial pathogens that cause human and canine monocytic ehrlichiosis respectively. We tested the hypothesis that these pathogens express unique proteins in response to their growth in vertebrate and tick host cells and that this differential expression is similar in closely related Ehrlichia species. Evaluation of nine E. chaffeensis isolates and one E. canis isolate demonstrated that protein expression was host cell-dependent. The differentially expressed proteins included those from the p28/30-Omp multigene locus. E. chaffeensis and E. canis proteins expressed in infected macrophages were primarily the products of the p28-Omp 19 and 20 genes or their orthologues. In cultured tick cells, E. canis expressed only the p30-10 protein, an orthologue of the E. chaffeensis p28-Omp 14 protein which is the only protein expressed by E. chaffeensis propagated in cultured tick cells. The expressed Omp proteins were post-translationally modified to generate multiple molecular forms. E. chaffeensis gene expression from the p28/30-Omp locus was similar in tick cell lines derived from both vector (Amblyomma americanum) and non-vector (Ixodes scapularis) ticks. Differential expression of proteins within the p28/p30-Omp locus may therefore be vital for adaptation of Ehrlichia species to their dual host life cycle.

Anaplasma phagocytophilum, the causative agent of human granulocytotropic anaplasmosis, can infect white-tailed deer (WTD; Odocoileus virginianus), and this species is a crucial host for adult Ixodes scapularis, the primary vector of A. phagocytophilum. The goal of this study was to determine the geographic distribution of A. phagocytophilum among WTD across a 19 state region and to evaluate the utility of WTD as natural sentinels. Serologic testing using the indirect fluorescent antibody (IFA) assay was conducted on WTD serum samples and molecular and xenodiagnostic tests were performed to confirm serologic results. The surveillance system was assessed through examination of vital attributes including WTD age and gender associations with serologic status, sample size adequacy for accurate infection status classification, and presence of the vector, I. scapularis. Six hundred thirty-three of 2,666 (24%) WTD in 17 states tested positive for antibodies (>or=128) when tested by IFA assay. Testing for p44 and/or 16S rRNA gene targets identified 73 (16%) PCR positive WTD among 458 animals tested, all of which originated from seropositive populations. Attempts to culture A. phagocytophilum from WTD were unsuccessful; however, xenodiagnostic mice inoculated with blood from 3 WTD became infected. Seroprevalence did not differ by deer age or gender; however, WTD

Ixodes scapularis ticks transmit Anaplasma phagocytophilum (Ap), agent of human granulocytic anaplasmosis (HGA). Invasion of neutrophil granulocytes (PMN) by Ap is the hallmark of the disease, but these short-lived phagocytes are not likely the sole cell type required for productive infection. We analyzed infection of microvascular endothelial cells during pathogenesis of anaplasmosis in vivo and in vitro. Organs from Ap-infected mice were processed for confocal microscopy 41 days p.i. Fluorescent labeling of heart and liver sections using anti-factor VIII and anti-MSP2 antibodies allowed colocalization of Ap and vascular endothelium, indicating infection. Ap rapidly invaded and grew within HMEC-1 human microvascular endothelial cells and readily transferred to PMN. Over 50% of PMN became infected within two hours of coincubation with HMEC-1. PMN adhered to, polarized, and migrated upon infected endothelial monolayers. The Ap receptor on human PMN is PSGL-1, and infected endothelial cells upregulate ICAM-1 (CD54), but the mechanisms of transfer of Ap remain unknown. To elucidate the cellular determinants involved, we tested relevant antibodies and lectins. Anti-PSGL-1 reduced infection of PMN, but did not inhibit adherence of PMN to Ap infected HMEC-1 cells while anti-CD18 did. Sialidase pretreatment increased, and EDTA and fucoidan decreased binding of Ap to HMEC-1, whereas several other lectins had no effect. An endothelial reservoir of Ap offers opportunities for ongoing, direct cell-to-cell infection of PMN, avoidance of host immune effectors, and completion of the Ap life cycle by infection of circulating leukocytes available for transfer to blood-feeding ticks.

We used tick cell culture to isolate a bacterium previously referred to as the “white-tailed deer (WTD) agent” from two captive fawns inoculated with blood from wild WTD ( Odocoileus virginianus ). Buffy coat cells were added to ISE6 tick cell cultures and incubated at 34°C, and 8 days later, Anaplasma -like inclusions were demonstrated in Giemsa-stained culture samples. The microbes became established and could be continuously passaged in tick cells. The identity of a culture isolate designated WTD76 was verified as the WTD agent by using specific PCR primers and by DNA sequencing. Comparison with sequences available in GenBank indicated that the isolate was most closely related first to Anaplasma platys and second to Anaplasma phagocytophilum , supporting its placement in the genus Anaplasma . Transmission electron microscopy of this Anaplasma sp. organism in tick cell cultures revealed large inclusions filled with pleomorphic and rod-shaped bacteria. Tick cells infected with the Anaplasma sp. organism were used to successfully infect a naive deer, thereby proving the infectivity of the isolate for deer.

Anaplasma phagocytophilum is the causative agent of an emerging tick-borne zoonosis in the United States and Europe. The organism causes a febrile illness accompanied by other nonspecific symptoms and can be fatal, especially if treatment is delayed. Persistence of A. phagocytophilum within mammalian reservoir hosts is important for ensuring continued disease transmission. In the related organism Anaplasma marginale , persistence is associated with antigenic variation of the immunoprotective outer membrane protein MSP2. Extensive diversity of MSP2 is achieved by combinatorial gene conversion of a genomic expression site by truncated pseudogenes. The major outer membrane protein of A. phagocytophilum , MSP2(P44), is homologous to MSP2 of A. marginale , has a similar organization of conserved and variable regions, and is also encoded by a multigene family containing some truncated gene copies. This suggests that the two organisms could use similar mechanisms to generate diversity in outer membrane proteins from their small genomes. We define here a genomic expression site for MSP2(P44) in A. phagocytophilum . As in A. marginale , the msp2 ( p44 ) gene in this expression site is polymorphic in all populations of organisms we have examined, whether organisms are obtained from in vitro culture in human HL-60 cells, from culture in the tick cell line ISE6, or from infected human blood. Changes in culture conditions were found to favor the growth and predominance of certain msp2 ( p44 ) variants. Insertions, deletions, and substitutions in the region of the genomic expression site encoding the central hypervariable region matched sequence polymorphisms in msp2 ( p44 ) mRNA. These data suggest that, similarly to A. marginale , A. phagocytophilum uses combinatorial mechanisms to generate a large array of outer membrane protein variants. Such gene polymorphism has profound implications for the design of vaccines, diagnostic tests, and therapy.

We describe the isolation and characterization of Rickettsia monacensis sp. nov. (type strain, IrR/Munich T ) from an Ixodes ricinus tick collected in a city park, the English Garden in Munich, Germany. Rickettsiae were propagated in vitro with Ixodes scapularis cell line ISE6. BLAST analysis of the 16S rRNA, the citrate synthase, and the partial 190-kDa rickettsial outer membrane protein A (rOmpA) gene sequences demonstrated that the isolate was a spotted fever group (SFG) rickettsia closely related to several yet-to-be-cultivated rickettsiae associated with I. ricinus . Phylogenetic analysis of partial rompA sequences demonstrated that the isolate was genotypically different from other validated species of SFG rickettsiae. R. monacensis also replicated in cell lines derived from the ticks I. ricinus (IRE11) and Dermacentor andersoni (DAE100) and in the mammalian cell lines L-929 and Vero, causing cell lysis. Transmission electron microscopy of infected ISE6 and Vero cells showed rickettsiae within the cytoplasm, pseudopodia, nuclei, and vacuoles. Hamsters inoculated with R. monacensis had immunoglobulin G antibody titers as high as 1:16,384, as determined by indirect immunofluorescence assay. Western blot analyses demonstrated that the hamster sera cross-reacted with peptides from other phylogenetically distinct rickettsiae, including rOmpA. R. monacensis induced actin tails in both tick and mammalian cells similar to those reported for R. rickettsii. R. monacensis joins a growing list of SFG rickettsiae that colonize ticks but whose infectivity and pathogenicity for vertebrates are unknown.

Borrelia burgdorferi sensu stricto (s.s.), the causative agent of Lyme disease in North America is transmitted to the mammalian host by ticks belonging to the genus, Ixodes. Antibodies to several spirochetal proteins, most notably outer surface protein C (OspC), have been observed in early infection in both humans and laboratory animals. Thus, the expression of these proteins have been postulated to play a role in tick transmission and spirochetal infectivity for the mammalian host. B. burgdorferi strain JMNT was induced to produce increased levels of OspC by cultivation in BSK medium at 37 degrees C. To diminish expression of OspC, spirochetes were cultivated at 31 degrees C. Spirochetes shifted down from 37 degrees C to 31 degrees C or up from 31 degrees C to 37 degrees C for 1 week contained equivalent amounts of OspC. To evaluate spirochetal infectivity, hamsters were inoculated subcutaneously with 1 x 10(4) or 1 x 10(6) spirochetes grown at the above-mentioned temperatures. Hamsters inoculated with spirochetes expressing high amounts of OspC all became infected, irrespective of the inoculum size. None of the hamsters inoculated with 1 x 10(4) spirochetes grown at 31 degrees C or in cultures shifted down from 37 degrees C to 31 degrees C were infected. All infected hamsters, confirmed by isolation of spirochetes in ear and/or bladder cultures, had an antibody response to OspC. In contrast, all non-infected hamsters lacked antibodies to OspC. We conclude that cultivation of spirochetes at 37 degrees C enhances their infectivity for hamsters. This study also suggests there is a correlation between enhancement of OspC expression and spirochetal infectivity for hamsters.

Background The large amounts of data generated by genomics, transcriptomics and proteomics have increased our understanding of the biology of Anaplasma marginale. However, these data have also led to new assumptions that require testing, ideally through classical genetic mutation. One example is the definition of genes associated with virulence. Here we describe the molecular characterization of a red fluorescent and spectinomycin and streptomycin resistant A. marginale mutant generated by Himar1 transposon mutagenesis. Results High throughput genome sequencing to determine the Himar1-A. marginale genome junctions established that the transposon sequences were integrated within the coding region of the omp10 gene. This gene is arranged within an operon with AM1225 at the 5’ end and with omp9, omp8, omp7 and omp6 arranged in tandem at the 3’ end. RNA analysis to determine the effects of the transposon insertion on the expression of omp10 and downstream genes revealed that the Himar1 insertion not only reduced the expression of omp10 but also that of downstream genes. Transcript expression from omp9, and omp8 dropped by more than 90% in comparison with their counterparts in wild-type A. marginale. Immunoblot analysis showed a reduction in the production of Omp9 protein in these mutants compared to wild-type A. marginale. Conclusions These results demonstrate that transposon mutagenesis in A. marginale is possible and that this technology can be used for the creation of insertional gene knockouts that can be evaluated in natural host-vector systems.

In the United States, human ehrlichiosis is a complex of emerging tick-borne diseases caused by 3 distinct Ehrlichia species: Ehrlichia chaffeensis, Ehrlichia ewingii, and the human granulocytotropic ehrlichiosis agent. Ehrlichioses are characterized by a mild to severe illness, and approximately 4% of cases are fatal. Because these obligate intracellular bacteria are difficult to resolve with routine histologic techniques, their distribution in tissues has not been well described. To facilitate the visualization and detection of ehrlichiae, immunohistochemistry (IHC), in situ hybridization (ISH), and polymerase chain reaction (PCR) assays were developed by use of tissues from 4 fatal cases of E. chaffeensis infection. Evidence of E. chaffeensis via IHC, ISH, and PCR was documented in all 4 cases. Abundant immunostaining and in situ nucleic acid hybridization were observed in spleen and lymph node from all 4 patients. Significantly, in 2 of these patients, serologic evidence of infection was absent. Use of IHC, ISH, and PCR to visualize and detect Ehrlichia in tissues can facilitate diagnosis of ehrlichial infections.

The human granulocytic ehrlichiosis agent (HGEa) survives extreme differences between ticks and humans, possibly by use of differential expression of specific antigens for survival in different hosts. The role of the immunodominant p44 antigens is unknown. In this study, HGEa cultured in human or tick cells was probed with human, mouse, and hamster serum and with monoclonal antibodies (MAbs). p44 antigens were strongly expressed in human HL-60 cells but were strikingly reduced in tick cells. In HGEa alternately grown in HL-60 or tick cells, a p44 epitope recognized by MAb R5E4 was expressed in human but not tick cells. This was not a temperature effect, because incubation of infected tick cells at 37 degrees C did not induce expression of the p44 epitope. The p44 antigen predominates in human but not tick cells and may be involved in regulatory changes that mediate survival of the HGEa by immune modulation after tick transmission.

An embryonic cell line (DAE100) of the Rocky Mountain wood tick, Dermacentor andersoni , was observed by microscopy to be chronically infected with a rickettsialike organism. The organism was identified as a spotted fever group (SFG) rickettsia by PCR amplification and sequencing of portions of the 16S rRNA, citrate synthase, Rickettsia genus-specific 17-kDa antigen, and SFG-specific 190-kDa outer membrane protein A (rOmpA) genes. Sequence analysis of a partial rompA gene PCR fragment and indirect fluorescent antibody data for rOmpA and rOmpB indicated that this rickettsia was a strain (DaE100R) of Rickettsia peacockii , an SFG species presumed to be avirulent for both ticks and mammals. R. peacockii was successfully maintained in a continuous culture of DAE100 cells without apparent adverse effects on the host cells. Establishing cell lines from embryonic tissues of ticks offers an alternative technique for isolation of rickettsiae that are transovarially transmitted.

Previous studies with different Borrelia burgdorferi sensu stricto (s.s.) strains revealed that temperature as well as cocultivation with tick cells modulates the expression of outer surface proteins (Osp) A and C. We investigated the effects of temperature and of interaction with tick cells in culture on the expression of OspA and OspC of the B. afzelii clones cPKo97 and cPKo345 in comparison to the B. burgdorferi s.s. strain N40. To follow the dynamics of Osp expression of single borreliae we used indirect immunofluorescence microscopy with double staining of OspA and OspC. Clone PKo345 always showed expression of only OspA, regardless the conditions it was subjected to. Sequencing of the ospC gene disclosed a insertion leading to a stop codon after base 222 and inability to produce OspC. In cPKo97 and N40 OspC is down-regulated at lower temperatures and up-regulated at higher temperatures, which was especially pronounced on cocultivation with tick cells. Borreliae adherent to tick cells showed greater OspA expression compared to the nonadherent ones, an indication that OspA might play a role as adhesin for tick cells. Interestingly, cPKo97 and N40 displayed different patterns of Osp expression: cPKo97 simultaneously presents OspA and OspC on single borreliae, while N40 has either OspA or OspC on single cells. Adaptation of OspC expression in cPKo97 seems to occur by up- or down-regulation of this protein on single borreliae, as shown by alternating intensities of OspC expression at different temperatures. In contrast, N40 seem to consist of two subsets of borreliae one expressing only OspA and the other only OspC, and change in temperature results in growth benefit for one of these subtypes. Our findings indicate that, regarding OspA and OspC expression, response to temperature and cocultivation with tick cells of B. afzelii is comparable to B. burgdorferi s.s., but the mode of regulation seems phenotypically different. Further European isolates should be investigated for OspA and OspC regulation, especially in the face of vaccine development for the European situation.

The tick-borne rickettsia Cowdria ruminantium has been propagated continuously for over 500 days in the Ixodes scapularis tick cell line IDE8 by using the Gardel isolate from bovine endothelial cells as an inoculum. Infection of the tick cells was confirmed by PCR, karyotyping, electron microscopy, and reinfection of bovine cells.

Human granulocytotropic ehrlichias are tick-borne bacterial pathogens that cause an acute, life-threatening illness, human granulocytic ehrlichiosis (HGE). Ehrlichias within neutrophil granulocytes that invade tick bite sites are likely ingested by the vector, to be transmitted to another mammalian host during the tick’s next blood meal. Thus, the cycle of replication and development in the vector is prerequisite to mammalian infection, and yet these events have not been described. We report tick cell culture isolation of two strains of the HGE agent directly from an infected horse and a dog and have also established a human isolate from HL60 culture in tick cells, proving that the blood stages of the HGE agent are infectious for tick cells, as are those replicating in the human cell line HL60. This required changes to the culture system, including a new tick cell line. In tick cell layers, the HGE agent induced foci of infection that caused necrotic plaques and eventual destruction of the culture. Using the human isolate and electron microscopy, we monitored adhesion, internalization, and replication in vector tick cells. Both electron-lucent and -dense forms adhered to and entered cells by a mechanism reminiscent of phagocytosis. Ehrlichial cell division was initiated soon after, resulting in endosomes filled with numerous ehrlichias. During early development, pale ehrlichias with a tight cell wall dominated, but by day 2, individual bacteria condensed into dark forms with a rippled membrane. These may become compacted into clumps where individual organisms are barely discernible. Whether these are part of an ehrlichia life cycle or are degenerating is unknown.

A rickettsial isolate (isolate MOAa) belonging to the spotted fever group (SFG) was obtained from the lone star tick Amblyomma americanum . We used PCR to characterize the genes for the rickettsial outer membrane proteins rOmpA and rOmpB. We sequenced the PCR products (domains I of both the rompA gene and the rompB gene) of MOAa and WB-8-2, another rickettsial isolate from A. americanum . To place MOAa and WB-8-2 and two other nonpathogenic isolates ( Rickettsia rickettsii Hlp2 and Rickettsia montana M5/6) with respect to their putative sister species, we included them in a phylogenetic analysis of 9 Rickettsia species and 10 Rickettsia strains. Our phylogenetic results implied three evolutionary lineages of SFG rickettsiae and that WB-8-2 and MOAa were most closely related to R. montana . New World isolates were not the most closely related to each other (they did not form a clade). Rather, our results supported four independent origins (introductions) of rickettsiae into North America from different Old World regions. The results of our phylogenetic analysis did not support the hypothesis of a stable coevolution of rickettsiae and their tick hosts. Finally, we examined the rompA gene of a nonpathogenic rickettsial symbiont isolated from the tick Ixodes scapularis . In a phylogenetic analysis, the symbiont was placed as the sister to R. montana and its isolates. The relationship of this symbiont to R. montana raised questions as to the potential origin of pathogenic SFG rickettsiae from nonpathogenic tick symbionts, or vice versa.

Hamsters were immunized with thimerosal-killed Borrelia burgdorferi 297 or a mutant of 297 (M297) that lacks the 49-kb linear plasmid and expression of outer surface proteins A and B (OspA and OspB). Ixodes scapularis nymphs infected with either the B. burgdorferi sensu stricto strain 297 or JMNT, similar in OspA and OspB but differing in OspC expression, were used to evaluate protection. In a homologous challenge, 24 hamsters were vaccinated, 8 each with 297 or M297 and 8 sham (adjuvant)-vaccinated controls. Hamsters vaccinated with either bacterin were completely protected against a natural tick bite or subcutaneous (s.c.) inoculation of 297. Borreliae were effectively eliminated from 80 to 90% of the 297-infected ticks that fed on four hamsters immunized with the 297 bacterin. Cultures of spirochetes isolated from the ticks that remained infected were infectious and induced joint inflammation in naive hamsters. There was no reduction of strain 297 spirochetes in ticks that fed on four hamsters immunized with M297, but the hamsters were protected. Results with the M297 bacterin indicate that proteins other than OspA or OspB can protect hamsters against a tick challenge without eliminating B. burgdorferi in the tick. In a heterologous challenge, 36 hamsters were vaccinated, 12 with each bacterin and 12 controls. None of the hamsters immunized with either bacterin were protected from a challenge involving JMNT-infected ticks, while two of four were protected against an s.c. challenge. Hamsters challenged s.c. with strain 297 spirochetes were protected. There was partial elimination of JMNT spirochetes in ticks that fed on the group of four hamsters immunized with the 297 bacterin, and infection rates were reduced by 50 to 60%. JMNT spirochetes reisolated from the ticks that fed on 297-vaccinated hamsters also remained infectious for hamsters. In the JMNT-infected ticks that fed on four M297-immunized hamsters, there was no decline in the proportion of infected ticks. Destruction of spirochetes in ticks that fed on the hamsters vaccinated with the 297 bacterin suggests that antibodies to OspA and OspB may have been responsible, since the mutant did not induce this activity.

The equine granulocytic ehrlichiosis agent, Ehrlichia equi, is closely related or identical to the human granulocytic ehrlichiosis (HGE) agent. Both are suspected of being transmitted by ticks. We have successfully isolated E. equi in a cell line, IDE8, derived from a putative vector, the tick Ixodes scapularis. Peripheral blood leukocytes from an experimentally infected horse were inoculated onto IDE8 monolayers. Cultures were incubated in a candle jar at 34 degrees C in tick cell culture medium with NaHCO3 and an organic buffer [3-(N-morpholino)-propanesulfonic acid] (MOPS). Within 2 weeks, infected cells were detected in Giemsa-stained culture samples, and the organisms subsequently spread to uninfected cells in the cultures. E. equi was passaged serially by transferring a portion of an infected culture to new cell layers every 2 to 3 weeks. The identity of the organisms was confirmed by PCR using oligonucleotide primers specific for E. equi and the HGE agent and by immunocytology. Homologous equine antibodies and human anti-HGE convalescent serum recognized E. equi grown in tick cell culture. Electron microscopy revealed electron-lucent and -dense ehrlichia-like forms developing within host cell endosomes. E. equi passaged twice in tick cell culture retained infectivity and pathogenicity for the equine host, as demonstrated by intravenous inoculation of a suspension of infected tick cells and subsequent reisolation from peripheral blood, in fulfillment of Koch's postulates. The horse developed severe clinical signs, i.e., fever, inappetence, thrombocytopenia, icterus, and limb edema, typical of granulocytic equine ehrlichiosis, within 1 week.

Background Anaplasma phagocytophilum is an intracellular organism in the Order Rickettsiales that infects diverse animal species and is causing an emerging disease in humans, dogs and horses. Different strains have very different cell tropisms and virulence. For example, in the U.S., strains have been described that infect ruminants but not dogs or rodents. An intriguing question is how the strains of A. phagocytophilum differ and what different genome loci are involved in cell tropisms and/or virulence. Type IV secretion systems (T4SS) are responsible for translocation of substrates across the cell membrane by mechanisms that require contact with the recipient cell. They are especially important in organisms such as the Rickettsiales which require T4SS to aid colonization and survival within both mammalian and tick vector cells. We determined the structure of the T4SS in 7 strains from the U.S. and Europe and revised the sequence of the repetitive virB6 locus of the human HZ strain. Results Although in all strains the T4SS conforms to the previously described split loci for vir genes, there is great diversity within these loci among strains. This is particularly evident in the virB2 and virB6 which are postulated to encode the secretion channel and proteins exposed on the bacterial surface. VirB6-4 has an unusual highly repetitive structure and can have a molecular weight greater than 500,000. For many of the virs, phylogenetic trees position A. phagocytophilum strains infecting ruminants in the U.S. and Europe distant from strains infecting humans and dogs in the U.S. Conclusions Our study reveals evidence of gene duplication and considerable diversity of T4SS components in strains infecting different animals. The diversity in virB2 is in both the total number of copies, which varied from 8 to 15 in the herein characterized strains, and in the sequence of each copy. The diversity in virB6 is in the sequence of each of the 4 copies in the single locus and the presence of varying numbers of repetitive units in virB6-3 and virB6-4. These data suggest that the T4SS should be investigated further for a potential role in strain virulence of A. phagocytophilum.

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.

Antigenic variation of major surface proteins is considered an immune-evasive maneuver used by pathogens as divergent as bacteria and protozoa. Likewise, major surface protein 2 (Msp2) of the tick-borne pathogen, Anaplasma marginale, is thought to be involved in antigenic variation to evade the mammalian host immune response. However, this dynamic process also works in the tick vector in the absence of immune selection pressure. We examined Msp2 variants expressed during infection of four tick and two mammalian cell-lines to determine if the presence of certain variants correlated with specific host cell types. Anaplasma marginale colonies differed in their development and appearance in each of the cell lines (P

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.

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.

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 × 107 spirochetes/ml. After 6 h, >90% of the cells bound an average of 3*.xml5 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*.xml60%, whereas preheatment 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.

We describe an in vitro system that will facilitate molecular analysis of the association between Lyme disease spirochetes and vector cells. We cocultured Borrelia burgdorferi continuously with two tick cell lines, RAE25 (from Rhipicephalus appendiculatus) and IDE8 (from Ixodes scapularis). A clone isolated after twenty-two passages with RAE25 cells had lost the largest (49 kb) plasmid, and probes containing information normally encoded on it, including genes for two surface proteins, hybridized to smaller plasmids. Spirochetes maintained with IDE 8 cells showed a new 43 kb plasmid that hybridized to a probe made from the 49 kb plasmid. After reisolation from hamsters, these spirochetes carried a large plasmid (100 kb) that hybridized with the 49 kb plasmid. These changes may illustrate a plasticity that enables B. burgdorferi to adapt to different environments.

Background Rickettsia spp. (Rickettsiales: Rickettsiaceae) are Gram-negative, obligate intracellular, α-proteobacteria that have historically been associated with blood-feeding arthropods. Certain species cause typhus and spotted fevers in humans, but others are of uncertain pathogenicity or may be strict arthropod endosymbionts. Genetic manipulation of rickettsiae should facilitate a better understanding of their interactions with hosts. Methodology/Principal Findings We transformed a species never associated with human disease, Rickettsia montanensis, by electroporation with a TN5 transposon (pMOD700) containing green fluorescent protein (GFPuv) and chloramphenicol acetyltransferase (CAT) genes under regulation of promoters cloned from the Rickettsia rickettsii ompB gene, and isolated a Chloramphenicol-resistant GFP-fluorescent rickettsiae population (Rmontanensis700). The Rmontanensis700 rickettsiae contained a single transposon integrated near an acetyl-CoA acetyltransferase gene in the rickettsial chromosome. Northern blots showed that GFPuv and CAT mRNAs were both expressed as two transcripts of larger and smaller than predicted length. Western immunoblots showed that Rmontanensis700 and E. coli transformed with a plasmid containing the pMOD700 transposon both expressed GFPuv proteins of the predicted molecular weight. Conclusions/Significance Long-standing barriers to transformation of rickettsiae have been overcome by development of transposon-based rickettsial transformation vectors. The ompB promoter may be the most problematic of the four promoters so far employed in those vectors.

Rickettsia peacockii, also known as the East Side Agent, is a non-pathogenic obligate intracellular bacterium found as an endosymbiont in Dermacentor andersoni ticks in the western USA and Canada. Its presence in ticks is correlated with reduced prevalence of Rickettsia rickettsii, the agent of Rocky Mountain Spotted Fever. It has been proposed that a virulent SFG rickettsia underwent changes to become the East Side Agent. We determined the genome sequence of R. peacockii and provide a comparison to a closely related virulent R. rickettsii. The presence of 42 chromosomal copies of the ISRpe1 transposon in the genome of R. peacockii is associated with a lack of synteny with the genome of R. rickettsii and numerous deletions via recombination between transposon copies. The plasmid contains a number of genes from distantly related organisms, such as part of the glycosylation island of Pseudomonas aeruginosa. Genes deleted or mutated in R. peacockii which may relate to loss of virulence include those coding for an ankyrin repeat containing protein, DsbA, RickA, protease II, OmpA, ScaI, and a putative phosphoethanolamine transferase. The gene coding for the ankyrin repeat containing protein is especially implicated as it is mutated in R. rickettsii strain Iowa, which has attenuated virulence. Presence of numerous copies of the ISRpe1 transposon, likely acquired by lateral transfer from a Cardinium species, are associated with extensive genomic reorganization and deletions. The deletion and mutation of genes possibly involved in loss of virulence have been identified by this genomic comparison. It also illustrates that the introduction of a transposon into the genome can have varied effects; either correlating with an increase in pathogenicity as in Francisella tularensis or a loss of pathogenicity as in R. peacockii and the recombination enabled by multiple transposon copies can cause significant deletions in some genomes while not in others.

Ticks harbor several different genera of endosymbiotic bacteria. These obligate intracellular bacteria, most of which appear to be nonpathogenic for the tick, persist in nature by being transmitted transovarially from generation to generation. Those in the genus Rickettsiabelong to the spotted fever group (SFG) of rickettsia which includes both mammalian pathogenic and nonpathogenic species. The species that cause spotted fevers in humans, e.g. Rickettsia rickettsii, are well characterized but those not known to cause disease remain poorly understood. Transmission electron microscopy on SFG rickettsia cultured in tick cell cultures offers a powerful way to examine dynamic host parasite interactions in these apparently nonpathogenic SFG species.We have successfully isolated two unknown, nonpathogenic rickettsia strains in tick cell lines. T2 was isolated from an Ixodes ricinus tick collected in the English Garden in Munich, Germany and is propagated in an Ixodes scapularis cell line, ISE6.

Tick-borne pathogens cause emerging zoonoses, and include fastidious organisms such as Anaplasma phagocytophilum. Because of their obligate intracellular nature, methods for mutagenesis and transformation have not been available. To facilitate genetic manipulation, we transformed A. phagocytophilum (Ap) to express a green fluorescent protein (GFP) with the Himar1 transposase system and selection with the clinically irrelevant antibiotic spectinomycin. These transformed bacteria (GFP/Ap) grow at normal rates and are brightly fluorescent in human, monkey, and tick cell culture. Molecular characterization of the GFP/Ap genomic DNA confirmed transposition and the flanking genomic insertion locations were sequenced. Three mice inoculated with GFP/Ap by intraperitoneal injection became infected as demonstrated by the appearance of morulae in a peripheral blood neutrophil and re-isolation of the bacteria in culture.

We examined the effectiveness of bovine cholesterol concentrate in reducing the high level (10-20%) of fetal bovine serum (FBS) necessary to promote tick cell growth in vitro. Tick cell lines isolated from embryos of Anocentor nitens (ANE 58), Boophilus microplus (BME 26), and Rhipicephalus appendiculatus (RAE 25) were used. They were incubated in L-15 (BME 26) or L-15B (ANE 58 and RAE 25) supplemented with 10% tryptose phosphate broth (TPB), 5% (ANE 58 and BME 26) or 3% FBS, 10-90 microns/ml cholesterol. A concentration of 10 micrograms/ml cholesterol stimulated the growth rate of all three lines but more than 30 micrograms/ml depressed growth in ANE 58 and RAE 25 cells, while multiplication of BME 26 cells was enhanced by all cholesterol concentrations tested. All three lines could be continuously grown in 5% FBS, provided that 10 micrograms/ml cholesterol was included. Nutrients added to L-15 in the formulation of L-15B were tested singly or in combination for their ability to support tick cell growth in medium supplemented only with 5% FBS and 10 microns/ml cholesterol. In L-15 alone, RAE 25 cells did not multiply. Adding glucose (Glc), glutamic acid (Glu), or alpha-ketoglutaric acid (alpha K) had little or no effect, and the same was true for combinations of Glc plus alpha K, aspartic acid (Asp) plus proline (Pro) and glutamine (Gln), and minerals plus vitamins (MV). When Asp, Gln, Pro, and alpha K were combined with Glc and/or MV and added to L-15, there was appreciable growth stimulation, but best results were obtained when Glu was also included. In this medium, i.e., L-15B with 5% FBS and 10 mu/ml cholesterol, lines BME 26 and RAE 25 could be continuously subcultured.

The cellular origin and development of bovine lymphoblastoid cell lines persistently infected with macroschizonts of Theileria parva was studied. Cultures of lymphoblastoid cells isolated from cattle with patent East Coast fever were compared with those obtained by infecting normal lymphocytes in vitro with sporozoites. The young lines were contrasted with a continuous line which had been isolated earlier. The mononuclear cells were separated from the blood and the inoculum enriched for lymphoblastoid cells and/or lymphocytes by removing the monocytes. The lines arose directly from lymphoblastoid cells transplanted into culture or from lymphocytes infected by sporozoites. In primary cultures of lymphoblastoid cells from the peripheral blood, there was an increase in the proportion of infected cells without the eclipse of the parasite, the macroschizonts were larger than those observed in the inoculum or the continuous line, and there was concurrent microschizont differentiation. In lymphocyte cultures challenged with sporozoites, small mononucleated trophozoites were observed after 2 days which differentiated into typical macroschizonts but microschizonts were rare. In all cultures, the infected cells had mitotic indices of 4 to 5%. As the young lines were passaged, the parasites came to resemble those of the continuous line. The macroschizont size in the continuous line was stable and most had six to eight nuclei but when cultured at high cell concentrations the number of parasite nuclei increased. Minicultures of lymphocytes were used to quantitate the infectivity of sporozoites obtained from organ cultures of Rhipicephalus appendiculatus savliary glands. Sporozoites from ticks fed on rabbits for 5 days were approximately six times more infective than those from glands of ticks fed for 2 days and then cultured at 32 °C for 3 days. Glands from unfed ticks cultured for 5 days failed to yield infective sporozoites.

The tick-borne pathogen, Anaplasma marginale, has a complex life cycle involving ruminants and ixodid ticks. It causes bovine anaplasmosis, a disease with significant economic impact on cattle farming worldwide. The obligate intracellular growth requirement of the bacteria poses a challenging obstacle to their genetic manipulation, a problem shared with other prokaryotes in the genera Anaplasma, Ehrlichia, and Rickettsia. Following our successful transformation of the human anaplasmosis agent, A. phagocytophilum, we produced plasmid constructs (a transposon bearing plasmid, pHimarAm-trTurboGFP-SS, and a transposase expression plasmid, pET28Am-trA7) designed to mediate random insertion of the TurboGFP and spectinomycin/streptomycin resistance genes by the Himar1 allele A7 into the A. marginale chromosome. In these trans constructs, expression of the fluorescent and the selectable markers on the transposon, and expression of the transposase are under control of the A. marginale tr promoter. Constructs were co-electroporated into A. marginale St. Maries purified from tick cell culture, and bacteria incubated for 2 months under selection with a combination of spectinomycin and streptomycin. At that time, ≤1% of tick cells contained colonies of brightly fluorescent Anaplasma, which eventually increased to infect about 80–90% of the cells. Cloning of the insertion site in E. coli and DNA sequence analyses demonstrated insertion of the entire plasmid pHimarAm-trTurboGFP-SS encoding the transposon in frame into the native tr region of A. marginale in an apparent single homologous crossover event not mediated by the transposase. Transformants are fastidious and require longer subculture intervals than wild type A. marginale. This result suggests that A. marginale, as well as possibly other species of Anaplasma and Ehrlichia, can be transformed using a strategy of homologous recombination.

Borrelia burgdorferi, strain 297, was incubated with tick cell lines isolated from Rhipicephalus appendiculatus (Neumann), R. sanguineus (Latreille), Anocentor nitens (Neumann), Dermacentor variabilis (Say), and Boophilus microplus (Canestrini). A tick cell culture medium, L-15B, was modified by the addition of gelatin and N'-acetylglucosamine (L-15B/S) to permit the cocultivation of B. burgdorferi and tick cells. Spirochetes continuously passaged axenically in L-15B/S had longer population doubling times (27.1 ± 4.5 h) than those grown in BSK medium (11.7 ± 2.2 h), which was unsuitable for tick cells. Growth of spirochetes cocultured with five of six lines did not change, but when B. burgdorferi was incubated with R. sanguineus cells, the spirochetes disappeared and could not be detected after 1 wk. Spirochetes bound themselves to tick cells by one end within 30 min after being added to cultures. Not all spirochetes attached to tick cells nor did all cells of a given line bind spirochetes. B. burgdorferi had the greatest affinity for embryonic R. appendiculatus cells and the least for D. variabilis cells. Scanning electron microscopy revealed numerous intertwined, coiled spirochetes attached to foci at the surface of R. appendiculatus cells, apparently causing the formation of round bodies. Fewer spirochetes attached to A. nitens cells. High spirochete concentrations (>107 per ml) elicited cytopathic effects—loss of surface membrane extensions, rounding up, detachment, and lysis. This system should be applicable in the study of tick cell-spirochete interactions and the analysis of spirochete tropism and binding to tick cells.

Two strains of Borrelia burgdorferi, B31 and 297, formed colonies when plated onto Barbour-Stoenner-Kelly medium solidified with agarose (1.3%) and incubated in a candle jar at 34 degrees C. Colonies differing in morphology were observed in both strains after 2 to 3 weeks of incubation. Strain B31 colonies were either compact, round (mean diameter, 0.43 mm), and restricted to the surface of the agarose medium or diffuse (mean diameter, 1.80 mm) and penetrating into the solid medium. Strain 297 colonies (mean diameter, 1.43 mm) either showed a raised center surrounded by a diffuse ring of spirochetes or consisted of numerous small spirochetal aggregates. Both colony types expanded into the agarose medium. Scanning electron and light microscopy confirmed that the colonies were formed by spirochetes. Twisted tangles of intertwined spirochetes were visible on the surface, with numerous spherical bodies among them, especially in the central regions. At the periphery, the borreliae were more loosely packed, and individual coils were discernible.

Publisher Summary This chapter presents an overview of the mosquito cell culture. Mosquito cell culture has matured to a technique that enables the use of continuous cell lines as substrates for cultivation of mosquito-borne pathogens of man and animals or as prototype insect cells for studies on the physiology, molecular biology, and genetics of eukaryotic cells. Today, the isolation and maintenance of continuous cell lines has become a routine based on standardized techniques. A number of media have been formulated, including those that support the growth of the cells under reduced serum or serum-free conditions. Although it is not yet possible to isolate lines of specific cell types from a given tissue or organ, suitable sources of seed cells for the general isolation and establishment of cell lines have been identified. Cells from several species of mosquitoes are now in continuous culture and some of the lines are well characterized. The chapter presents the current knowledge of mosquito cell culture and describes the characteristics of the more commonly used lines.

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.

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.

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 106 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.

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 < 0.02), but 200 nMolar or more inhibited growth (P < 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 ,uMolar) 20-HE was needed to achieve significant growth suppression (P < 0.025 and < 0.005). The growth of both lines declined (P < 0.01) by 20% (RAE 25) or 30% (ANE 58) when the medium contained 38 AMolar of JH III. The bimodal growth response of line RAE 25 to 20-HE also occurred in the presence of 3.8 and 38 ,Molar JH III, and 2 nMolar 20-HE counteracted the suppressive effect of 38 AMolar JH III. Under the influence of 20-HE, the epithelial-like RAE 25 cells aggregated, detached and formed vesicles after certain times, depending on hormone concentration: 5 to 6 days with 2 nMolar, 2 days with 20 nMolar, and 1 day with 200 nMolar or more. The hemocytelike cells of ANE 58 were less sensitive to 20-HE. In the presence of 2 to 20 4Molar 20-HE, the pseudopodia retracted and 70% of the cell population detached. The responses of acarine cells to the hormones 20-HE and JH III are contrasted with those reported for insect cells in culture.

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.

Background The Arthropods are a diverse group of organisms including Chelicerata (ticks, mites, spiders), Crustacea (crabs, shrimps), and Insecta (flies, mosquitoes, beetles, silkworm). The cattle tick, Rhipicephalus (Boophilus) microplus, is an economically significant ectoparasite of cattle affecting cattle industries world wide. With the availability of sequence reads from the first Chelicerate genome project (the Ixodes scapularis tick) and extensive R. microplus ESTs, we investigated evidence for putative RNAi proteins and studied RNA interference in tick cell cultures and adult female ticks targeting Drosophila homologues with known cell viability phenotype. Results We screened 13,643 R. microplus ESTs and I. scapularis genome reads to identify RNAi related proteins in ticks. Our analysis identified 31 RNAi proteins including a putative tick Dicer, RISC associated (Ago-2 and FMRp), RNA dependent RNA polymerase (EGO-1) and 23 homologues implicated in dsRNA uptake and processing. We selected 10 R. microplus ESTs with >80% similarity to D. melanogaster proteins associated with cell viability for RNAi functional screens in both BME26 R. microplus embryonic cells and female ticks in vivo. Only genes associated with proteasomes had an effect on cell viability in vitro. In vivo RNAi showed that 9 genes had significant effects either causing lethality or impairing egg laying. Conclusion We have identified key RNAi-related proteins in ticks and along with our loss-of-function studies support a functional RNAi pathway in R. microplus. Our preliminary studies indicate that tick RNAi pathways may differ from that of other Arthropods such as insects.