Isolation and Propagation of Laboratory Strains and a Novel Flea-Derived Field Strain of Wolbachia in Tick Cell Lines

Wolbachia are intracellular endosymbionts of several invertebrate taxa, including insects and nematodes. Although Wolbachia DNA has been detected in ticks, its presence is generally associated with parasitism by insects. To determine whether or not Wolbachia can infect and grow in tick cells, cell lines from three tick species, Ixodes scapularis, Ixodes ricinus and Rhipicephalus microplus, were inoculated with Wolbachia strains wStri and wAlbB isolated from mosquito cell lines. Homogenates prepared from fleas collected from cats in Malaysia were inoculated into an I. scapularis cell line. Bacterial growth and identity were monitored by microscopy and PCR amplification and sequencing of fragments of Wolbachia genes. The wStri strain infected Ixodes spp. cells and was maintained through 29 passages. The wAlbB strain successfully infected Ixodes spp. and R. microplus cells and was maintained through 2–5 passages. A novel strain of Wolbachia belonging to the supergroup F, designated wCfeF, was isolated in I. scapularis cells from a pool of Ctenocephalides sp. cat fleas and maintained in vitro through two passages over nine months. This is the first confirmed isolation of a Wolbachia strain from a flea and the first isolation of any Wolbachia strain outside the “pandemic” A and B supergroups. The study demonstrates that tick cells can host multiple Wolbachia strains, and can be added to panels of insect cell lines to improve success rates in isolation of field strains of Wolbachia.

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Isolation and Propagation of Laboratory Strains and a Novel Flea-Derived Field Strain of Wolbachia in Tick Cell Lines

10.20944/preprints202006.0141.v1 ◽

2020 ◽

Author(s):

Jing Jing Khoo ◽

Timothy Kurtti ◽

Nurul Aini Husin ◽

Alexandra Beliavskaia ◽

Fang Shiang Lim ◽

...

Keyword(s):

Cell Lines ◽

Ixodes Scapularis ◽

Pcr Amplification ◽

Wolbachia Strain ◽

Success Rates ◽

Tick Cell ◽

Mosquito Cell Lines ◽

Cat Fleas ◽

Insect Cell Lines

Wolbachia are intracellular endosymbionts of several invertebrate taxa, including insects and nematodes. Although Wolbachia DNA has been detected in ticks, its presence is generally associated with parasitism by insects. To determine whether or not Wolbachia can infect and grow in tick cells, cell lines from three tick species, Ixodes scapularis, Ixodes ricinus and Rhipicephalus microplus, were inoculated with Wolbachia strains wStri and wAlbB isolated from mosquito cell lines. Homogenates prepared from fleas collected from cats in Malaysia were inoculated into an I. scapularis cell line. Bacterial growth and identity were monitored by microscopy and PCR amplification and sequencing of fragments of Wolbachia genes. The wStri strain infected Ixodes spp. cells and was maintained through 29 passages. The wAlbB strain successfully infected Ixodes spp. and R. microplus cells and was maintained through 2-5 passages. A novel strain of Wolbachia belonging to the supergroup F, designated wCfeF, was isolated in I. scapularis cells from a pool of Ctenocephalides sp. cat fleas and maintained in vitro through two passages over nine months. This is the first confirmed isolation of a Wolbachia strain from a flea and the first isolation of any Wolbachia strain outside the “pandemic” A and B supergroups. The study demonstrates that tick cells can host multiple Wolbachia strains, and can be added to panels of insect cell lines to improve success rates in isolation of field strains of Wolbachia.

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Modulation of Borrelia burgdorferi Stringent Response and Gene Expression during Extracellular Growth with Tick Cells

Infection and Immunity ◽

10.1128/iai.70.6.3061-3067.2002 ◽

2002 ◽

Vol 70 (6) ◽

pp. 3061-3067 ◽

Cited By ~ 27

Author(s):

Julia Bugrysheva ◽

Elena Y. Dobrikova ◽

Henry P. Godfrey ◽

Marina L. Sartakova ◽

Felipe C. Cabello

Keyword(s):

Gene Expression ◽

Borrelia Burgdorferi ◽

Cell Lines ◽

Mrna Level ◽

Stringent Response ◽

Global Regulator ◽

Tick Cell ◽

Fourfold Increase ◽

Reciprocal Interactions

ABSTRACT Borrelia burgdorferi N40 multiplied extracellularly when it was cocultured with tick cells in L15BS medium, a medium which by itself did not support B. burgdorferi N40 growth. Growth of B. burgdorferi N40 in the presence of tick cells was associated with decreased production of (p)ppGpp, the stringent response global regulator, a fourfold decrease in relA/spoT mRNA, an eightfold net decrease in bmpD mRNA, and a fourfold increase in rpsL-bmpD mRNA compared to growth of B. burgdorferi in BSK-H medium. As a result, the polycistronic rpsL-bmpD mRNA level increased from 3 to 100% of the total bmpD message. These observations demonstrate that there are reciprocal interactions between B. burgdorferi and tick cells in vitro and indicate that the starvation-associated stringent response mediated by (p)ppGpp present in B. burgdorferi growing in BSK-H medium is ameliorated in B. burgdorferi growing in coculture with tick cell lines. These results suggest that this system can provide a useful model for identifying genes controlling interactions of B. burgdorferi with tick cells in vitro when it is coupled with genetic methods to isolate and complement B. burgdorferi mutants.

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In Vitro Infection of Ovine Cell Lines by Jaagsiekte Sheep Retrovirus

Journal of Virology ◽

10.1128/jvi.73.12.10070-10078.1999 ◽

1999 ◽

Vol 73 (12) ◽

pp. 10070-10078 ◽

Cited By ~ 44

Author(s):

Massimo Palmarini ◽

J. Michael Sharp ◽

Christine Lee ◽

Hung Fan

Keyword(s):

Cell Lines ◽

Pcr Amplification ◽

Heat Inactivation ◽

Clara Cells ◽

Viral Dna ◽

Jaagsiekte Sheep Retrovirus ◽

Viral Receptor ◽

Infected Cells

ABSTRACT Sheep pulmonary adenomatosis (SPA), also known as jaagsiekte or ovine pulmonary carcinoma, is a contagious lung cancer of sheep, originating from type II pneumocytes and Clara cells. Previous studies have implicated a type D retrovirus (jaagsiekte sheep retrovirus [JSRV]) as the causative agent of SPA. We recently isolated a proviral clone of JSRV from an animal with a spontaneous case of SPA (JSRV21) and showed that it harbors an infectious and oncogenic virus. This demonstrated that JSRV is necessary and sufficient to induce SPA. A major impediment in research on JSRV has been the lack of an in vitro tissue culture system for the virus. The experiments reported here show the first successful in vitro infection with this virus, using the JSRV21 clone. JSRV21virus was obtained by transiently transfecting human 293T cells with a plasmid containing the JSRV21 provirus driven by the human cytomegalovirus immediate-early promoter. Virus produced in this manner exhibited reverse transcriptase (RT) activity that banded at 1.15 g/ml in sucrose density gradients. Infection of concentrated JSRV21 into ovine choroid plexus (CP), testes (OAT-T3), turbinate (FLT), and intestinal carcinoma (ST6) cell lines resulted in establishment of infection as measured by PCR amplification. Evidence that this reflected genuine infection included the fact that heat inactivation of the virus eliminated it, the levels of viral DNA increased with passage of the infected cells, and the infected cells released active RT as measured by the sensitive product enhancement RT assay. The RT activity released from the infected cells banded at 1.15 g/ml, and JSRV21 provirus was transmitted from infected cells to uninfected ones by cocultivation. However, the amount of virus released from infected cells was low. These results suggest that the JSRV receptor is present on many ovine cell types and that the observed restriction of JSRV expression in vivo to tumor cells might be controlled by factors other than the viral receptor. Finally we tagged the U3 of pJSRV21 with the bacterial supF gene, an amber suppressor tRNA gene. The resulting clone, termed pJSRV supF , is infectious in vitro. It may be a useful tool for future studies on viral DNA integration, since the normal sheep genome contains 15 to 20 copies of highly JSRV-related endogenous sequences that cross-react with many JSRV hybridization probes.

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Pyocyanin induced in vitro oxidative damage and its toxicity level in human, fish and insect cell lines for its selective biological applications

Cytotechnology ◽

10.1007/s10616-014-9765-5 ◽

2014 ◽

Vol 68 (1) ◽

pp. 143-155 ◽

Cited By ~ 11

Author(s):

P. Priyaja ◽

P. Jayesh ◽

Rosamma Philip ◽

I. S. Bright Singh

Keyword(s):

Oxidative Damage ◽

Cell Lines ◽

Insect Cell ◽

Biological Applications ◽

Toxicity Level ◽

Insect Cell Lines

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In vitro infection of Wolbachia in insect cell lines

Applied Entomology and Zoology ◽

10.1303/aez.2008.519 ◽

2008 ◽

Vol 43 (4) ◽

pp. 519-525 ◽

Cited By ~ 3

Author(s):

Seiichi Furukawa ◽

Kohjiro Tanaka ◽

Takema Fukatsu ◽

Tetsuhiko Sasaki

Keyword(s):

Cell Lines ◽

Insect Cell ◽

Insect Cell Lines

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Infection, growth and maintenance of Wolbachia pipientis in clonal and non-clonal Aedes albopictus cell cultures

Bulletin of Entomological Research ◽

10.1017/s0007485312000648 ◽

2012 ◽

Vol 103 (3) ◽

pp. 251-260 ◽

Cited By ~ 4

Author(s):

C.C.H. Khoo ◽

C.M.P. Venard ◽

Y. Fu ◽

D.R. Mercer ◽

S.L. Dobson

Keyword(s):

Cell Line ◽

Cell Lines ◽

Aedes Albopictus ◽

Cell Types ◽

Infection Level ◽

Wolbachia Pipientis ◽

Albopictus Cell ◽

Host Cell Interactions ◽

Insect Cell Lines

AbstractInsect cell lines provide useful in vitro models for studying biological systems, including interactions between mosquitoes and obligate intracellular endosymbionts such as Wolbachia pipientis. The Aedes albopictus Aa23 cell line was the first cell line developed to allow examination of Wolbachia infections. However, Wolbachia studies using Aa23 can be complicated by the presence of different cell types in the cell line and the substantial temporal variation in infection level. Two approaches were examined to ameliorate infection variability. In the first approach, multiple Aa23 passaging regimes were tested for an effect on infection variability. Fluorescence in situ hybridization (FISH) staining was used to characterize Wolbachia infection level over time. The results demonstrate an impact of passaging method on Wolbachia infection level, with some methods resulting in loss of infection. None of the passaging methods succeeded in effectively mitigating infection level variation. In a second approach, the clonal C7-10 A. albopictus cell line was infected with Wolbachia from Aa23 cells and Drosophila simulans (Riverside), resulting in cell lines designated C7-10B and C7-10R, respectively. Characterization via FISH staining showed greater stability and uniformity of Wolbachia infection in C7-10R relative to the infection in C7-10B. Characterization of the Aa23, C7-10B and C7-10R lines is discussed as a tool for the study of Wolbachia-host cell interactions.

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