scholarly journals Initiation of primary cell cultures from embryonic Haemaphysalis bispinosa ticks 

2017 ◽  
Vol 22 (3) ◽  
pp. 323 ◽  
Author(s):  
Fang-shiang Lim ◽  
Jing-jing Khoo ◽  
Fezshin Chen ◽  
Lesley Bell-sakyi ◽  
Chee-sieng Khor ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cell Cultures ◽  
Embryonic Cell ◽  
Rrna Gene ◽  
Hard Tick ◽  
Tick Cell ◽  
Haemaphysalis Bispinosa ◽  
Ornithodoros Moubata ◽  
Floating Cell ◽  
Axenic Conditions

Tick cell cultures have been widely used as an important tool for the study of tick-associated microorganisms, specifically for medically important bacteria or viruses that may be difficult to isolate or culture in axenic conditions. In this study, primary embryonic tick cell cultures were initiated separately from each of the egg batches laid by 10 female ticks belonging to the hard tick genus Haemaphysalis. All cultures were maintained at 28°C. After 10 months, 4 healthy cultures were identified with the potential for developing into continuous tick cell lines. These cultures comprise large cells predominantly forming floating cell clumps with multicellular vesicles, which are morphologically similar to cell lines derived from the soft tick Ornithodoros moubata. Subculture has not yet been performed due to the low cell density at the time of writing. Amplification and sequencing of a fragment of the 16S rRNA gene from DNA extracted from the parent ticks showed 99%-100% similarity to published sequences of Haemaphysalis bispinosa. This is the first report of the initiation of embryonic cell cultures from Haemaphysalis ticks found in Malaysia. Such tick cell cultures will be useful for studies of tick-borne pathogens in this region, where recent studies have shown that Haemaphysalis ticks are highly represented and harbor medically important bacteria. 

scholarly journals Analysis on the prokaryotic microbiome in females and embryonic cell cultures of Rhipicephalus sanguineus tropical and temperate lineages from two specific localities in Brazil

2021 ◽  
Vol 30 (3) ◽  
Author(s):  
Mayara de Cassia Luzzi ◽  
Lucas Amoroso Lopes de Carvalho ◽  
Daniel Guariz Pinheiro ◽  
Leidiane Lima-Duarte ◽  
Jaqueline Valéria Camargo ◽  
...  
Keyword(s):  
Cell Cultures ◽  
Rhipicephalus Sanguineus ◽  
Distribution Patterns ◽  
Embryonic Cell ◽  
Tick Cell ◽  
Illumina Platform ◽  
Region Gene ◽  
Host Environment ◽  
Canine Monocytic Ehrlichiosis ◽  
Vertebrate Hosts

Abstract Two lineages of Rhipicephalus sanguineus are known in Brazil: the temperate or southern and the tropical or northern populations. The distribution patterns of both lineages of R. sanguineus have epidemiological implications that can affect vectorial competence concerning Ehrlichia canis, the agent of canine monocytic ehrlichiosis. Intending to identify the microbiomes of both lineages and compare microorganisms in R. sanguineus, we used the 16S rRNA (V4-V5 region) gene-based metataxonomic approach, through NGS sequencing on the MiSeq Illumina platform. We selected specimens of females from the environment and samples of primary embryonic cell cultures, from both lineages, and this was the first study to investigate the prokaryotic microbiome in tick cell cultures. The results showed that many bacterial taxa detected in the samples were typical members of the host environment. A significant diversity of microorganisms in R. sanguineus females and in embryonic cell cultures from both lineages was found, with emphasis on the presence of Coxiella in all samples, albeit in different proportions. The Coxiella species present in the two lineages of ticks may be different and may have co-evolved with them, thus driving different patterns of interactions between ticks and the pathogens that they can harbor or transmit to vertebrate hosts.

Ultrastructural analysis of the invasion of tick cells by Lyme disease spirochetes (Borrelia burgdorferi) in vitro

1994 ◽  
Vol 72 (6) ◽  
pp. 977-994 ◽  
Author(s):  
T. J. Kurtti ◽  
U. G. Munderloh ◽  
S. F. Hayes ◽  
D. E. Krueger ◽  
G. G. Ahlstrand
Keyword(s):  
Electron Microscopy ◽  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Cell Lines ◽  
Cell Cultures ◽  
Dense Body ◽  
Tick Cell ◽  
Coated Pits ◽  
Intracellular Digestion

The association of Lyme disease spirochetes, Borrelia burgdorferi, with tick cell cultures was characterized by electron microscopy. These cells were active in endocytosis and intracellular digestion, containing coated vesicles, pits, and phagosomes. Borrelia burgdorferi in tick cell cultures resembled those described in tick tissues. In RAE25 cultures, isolated from Rhipicephalus appendiculatus embryos, invasion of cells was mediated by coated pits, indicating active host cell participation. The invading, tapered end of B. burgdorferi contained an electron-dense body that persisted throughout invasion. A host-derived membrane surrounded invading and intracellular borreliae as observed by transmission electron microscopy of cross and longitudinal sections, whereas degenerating ones lay inside secondary lysosomes. Borrelia burgdorferi cocultivated with cell lines from Ixodes scapularis were mainly found at the cell surface or within lysosomes. The differences seen in invasion of these cell lines are interpreted to reflect differences in cell types rather than species. Gemmae, indicative of degenerative changes in the spirochetes, were observed extra- and intra-cellularly. Membrane blebs were liberated by the spirochetes into the medium and onto the cells, and were avidly endocytosed. Tick cell cultures are a useful tool to elucidate spirochete – vector cell interactions that may be obscured in vivo.

scholarly journals Replication Kinetics of Rickettsia raoultii in Tick Cell Lines

2021 ◽  
Vol 9 (7) ◽  
pp. 1370
Author(s):  
Nurul Aini Husin ◽  
Jing Jing Khoo ◽  
Mulya Mustika Sari Zulkifli ◽  
Lesley Bell-Sakyi ◽  
Sazaly AbuBakar
Keyword(s):  
Cell Lines ◽  
Cell Cultures ◽  
Growth Kinetics ◽  
Growth Curves ◽  
Growth Characteristics ◽  
Tick Cell ◽  
Rickettsia Raoultii ◽  
Generation Times ◽  
Causative Agents ◽  
Kinetics Of

Rickettsia raoultii is one of the causative agents of tick-borne lymphadenopathy in humans. This bacterium was previously isolated and propagated in tick cell lines; however, the growth characteristics have not been investigated. Here, we present the replication kinetics of R. raoultii in cell lines derived from different tick genera (BME/CTVM23, RSE/PILS35, and IDE8). Tick cell cultures were infected in duplicate with cryopreserved R. raoultii prepared from homologous cell lines. By 12–14 days post infection, 100% of the cells were infected, as visualized in Giemsa-stained cytocentrifuge smears. R. raoultii growth curves, determined by rickettsiae-specific gltA qPCR, exhibited lag, exponential, stationary and death phases. Exponential phases of 4–12 days and generation times of 0.9–2.6 days were observed. R. raoultii in BME/CTVM23 and RSE/PILS35 cultures showed, respectively, 39.5- and 37.1-fold increases compared to the inoculum. In contrast, multiplication of R. raoultii in the IDE8 cultures was 110.1-fold greater than the inoculum with a 7-day stationary phase. These findings suggest variation in the growth kinetics of R. raoultii in the different tick cell lines tested, amongst which IDE8 cells could tolerate the highest levels of R. raoultii replication. Further studies of R. raoultii are needed for a better understanding of its persistence within tick populations.

Hepatoma Cell Cultures as In Vitro Models for the Hepatotoxicity of Xenobiotics

1988 ◽  
Vol 16 (1) ◽  
pp. 32-37
Author(s):  
Margherita Ferro ◽  
Anna Maria Bassi ◽  
Giorgio Nanni
Keyword(s):  
Cell Lines ◽  
Cell Cultures ◽  
Membrane Lipids ◽  
Hepatoma Cell ◽  
Positive Control ◽  
In Vitro Models ◽  
Low Concentrations ◽  
Formation Efficiency ◽  
Dose Dependent

Two hepatoma cell cultures were examined as in vitro models to be used in genotoxicity and cytotoxicity tests without the addition of bioactivating enzymes. The MH1C1, and HTC hepatoma lines were used in this study to establish their sensitivity to a number of xenobiotics, namely, cyclophosphamide (CP), the classical positive control in bioactivation tests; benzaldehyde (BA), a short-chain aldehyde; and 4-hydroxynonenal (HNE), a major toxic end-product of the peroxidative degradation of cell membrane lipids. As a first approach, we compared the following cytotoxicity tests: release of lactate dehydrogenase (LDH), and colony formation efficiency (CF). Colony-forming cells were exposed to the drugs according to different procedures, before or after the anchorage phase. The leakage of LDH into the medium following exposure of both cell lines to HNE, CP and BA for up to 24 hours was found not to be a good index of cytotoxicity. A better indicator of cytotoxicity was CF, as evaluated by exposure of the cells 24 hours after seeding. The effects were detectable at very low concentrations, corresponding to 10, 90 and 100μM for HNE, CP and BA, respectively. The impairment of CF efficiency was dose-dependent and time-dependent, and several differences between the two cell lines were observed.

scholarly journals Examination of the Transcriptional Response to LaMIR166a Overexpression in Larix kaempferi (Lamb.) Carr

Biology ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 576
Author(s):  
Yanru Fan ◽  
Wanfeng Li ◽  
Zhexin Li ◽  
Shaofei Dang ◽  
Suying Han ◽  
...  
Keyword(s):  
Cell Lines ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
De Novo ◽  
Pearson Correlation ◽  
Transcriptional Response ◽  
Correlation Coefficients ◽  
Embryonic Cell ◽  
Larix Kaempferi ◽  
Transgenic Cell

The study of somatic embryogenesis can provide insight into early plant development. We previously obtained LaMIR166a-overexpressing embryonic cell lines of Larix kaempferi (Lamb.) Carr. To further elucidate the molecular mechanisms associated with miR166 in this species, the transcriptional profiles of wild-type (WT) and three LaMIR166a-overexpressing transgenic cell lines were subjected to RNA sequencing using the Illumina NovaSeq 6000 system. In total, 203,256 unigenes were generated using Trinity de novo assembly, and 2467 differentially expressed genes were obtained by comparing transgenic and WT lines. In addition, we analyzed the cleaved degree of LaMIR166a target genes LaHDZ31–34 in different transgenic cell lines by detecting the expression pattern of LaHdZ31–34, and their cleaved degree in transgenic cell lines was higher than that in WT. The downstream genes of LaHDZ31–34 were identified using Pearson correlation coefficients. Yeast one-hybrid and dual-luciferase report assays revealed that the transcription factors LaHDZ31–34 could bind to the promoters of LaPAP, LaPP1, LaZFP5, and LaPHO1. This is the first report of gene expression changes caused by LaMIR166a overexpression in Japanese larch. These findings lay a foundation for future studies on the regulatory mechanism of miR166.

Isolation of embryonic cell‐lines from porcine blastocysts

1995 ◽  
Vol 6 (1) ◽  
pp. 1-14 ◽  
Author(s):  
R.W. Gerfen ◽  
M.B. Wheeler
Keyword(s):  
Cell Lines ◽  
Embryonic Cell ◽  
Embryonic Cell Lines

scholarly journals Growth of Nosema cuniculi in established cell lines

1975 ◽  
Vol 9 (1) ◽  
pp. 61-68 ◽  
Author(s):  
T. Waller
Keyword(s):  
Cell Lines ◽  
Cell Cultures ◽  
Growth Patterns ◽  
Rabbit Kidney ◽  
Kidney Cells ◽  
Scale Production ◽  
Simple Method ◽  
Established Cell Lines ◽  
Established Cell ◽  
Canine Kidney

Growth patterns of Nosema cuniculi ( Encephalitozoon cuniculi) in cell cultures of bovine kidney, canine kidney, feline lung, and rabbit kidney were studied. All cell cultures used were easy to manage and the last 3 are commercially-available established cell lines. The dog kidney cells were the most suitable for large-scale production of Nosema. When grown in plastic flasks with a bottom area of 75 cm2, the weekly yield from Nosema-infected canine kidney cells during the 10th to 17th week after inoculation was between 4·1 x 107 and 9·9 x 107 spores per flask. An equilibrium was obtained between the Nosema infection and the kidney cells during this time. A simple method for estimating the numbel of harvested spores is also described.

Virus-derived DNA forms mediate the persistent infection of tick cells by Hazara virus and Crimean-Congo hemorrhagic fever virus

2021 ◽  
Author(s):  
Maria Vittoria Salvati ◽  
Claudio Salaris ◽  
Vanessa Monteil ◽  
Claudia Del Vecchio ◽  
Giorgio Palù ◽  
...  
Keyword(s):  
Viral Replication ◽  
Cell Lines ◽  
Persistent Infection ◽  
Hemorrhagic Fever ◽  
Viral Reservoir ◽  
Tick Cell ◽  
Infected Tick ◽  
Crimean Congo Hemorrhagic Fever ◽  
Cchf Virus ◽  
Model Virus

Crimean-Congo hemorrhagic fever (CCHF) is a severe disease of humans caused by CCHF virus (CCHFV), a biosafety level (BSL)-4 pathogen. Ticks of the genus Hyalomma are the viral reservoir and they represent the main vector transmitting the virus to its hosts during blood feeding. We have previously shown that CCHFV can persistently infect Hyalomma -derived tick cell lines. However, the mechanism allowing the establishment of persistent viral infections in ticks is still unknown. Hazara virus (HAZV) can be used as a BSL-2 model virus instead of CCHFV to study virus/vector interactions. To investigate the mechanism behind the establishment of a persistent infection, we developed an in vitro model with Hyalomma -derived tick cell lines and HAZV. As expected, HAZV, like CCHFV, persistently infects tick cells without any sign of cytopathic effect, and the infected cells can be cultured for more than three years. Most interestingly, we demonstrated the presence of short viral-derived DNA forms (vDNAs) after HAZV infection. Furthermore, we demonstrated that the antiretroviral drug AZT could inhibit the production of vDNAs, suggesting that vDNAs are produced by an endogenous retrotranscriptase activity in tick cells. Moreover, we collected evidence that vDNAs are continuously synthesized, thereby downregulating viral replication to promote cell survival. Finally, vDNAs were also detected in CCHFV-infected tick cells. In conclusion, vDNA synthesis might represent a strategy to control the replication of RNA viruses in ticks allowing their persistent infection. IMPORTANCE Crimean-Congo hemorrhagic fever (CCHF) is an emerging tick-borne viral disease caused by CCHF virus (CCHFV). Ticks of the genus Hyalomma can be persistently infected with CCHFV representing the viral reservoir, and the main vector for viral transmission. Here we showed that tick cells infected with Hazara virus, a nonpathogenic model virus closely related to CCHFV, contained short viral-derived DNA forms (vDNAs) produced by endogenous retrotranscriptase activity. vDNAs are transitory molecules requiring viral RNA replication for their continuous synthesis. Interestingly, vDNA synthesis seemed to be correlated with downregulation of viral replication and promotion of tick cell viability. We also detected vDNAs in CCHFV-infected tick cells suggesting that they could represent a key element in the cell response to nairovirus infection and might represent a more general mechanism of innate immunity against RNA viral infection.

Antimigratory effect of berberine in T98G, U87MG and primary glioma cell culture.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e15045-e15045
Author(s):  
Irina V. Mezhevova ◽  
Svetlana Yu. Filippova ◽  
Sofia V. Timofeeva ◽  
Anastasia O. Sitkovskaya ◽  
Tatiana V. Shamova ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Lines ◽  
Cell Cultures ◽  
Glioma Cell ◽  
Glioma Cells ◽  
Surgical Removal ◽  
Experimental Sample ◽  
Wound Area ◽  
Migratory Activity ◽  
Primary Glioma

e15045 Background: Berberine is an alkaloid compound with a structure that is highly similar to that of intercalating agents. It affects numerous cell signaling pathways and is widely studied as potential anticancer drug. It is known that berberine affects cancer cells migration through metalloproteinase-2 inhibition, but this effect was never studied on glioma cells. Anti-migratory drugs are of special interest in brain cancer therapy since glioma's highly invasive nature makes total surgical removal of tumor practically impossible. The aim of the study was to evaluate berberine anti-migratory activity on glioma cells. Methods: Cell migration capacity of T98G and U87MG cell lines, as well as primary glioma cell culture established in our laboratory, was assessed via standard wound healing assay with automated image acquisition and analysis on Lionheart FX (BioTek) cell imager. Prior to assay setting up cell cultures were maintained in DMEM medium with L-glutamine (1 μM) (Gibco) and 10% FBS (Gibco) at 37C0 and 5.0% CO2. Cells were seeded at 250 000 cells per well on 24-well plates and incubated overnight in order to attach to plate bottom. After that a vertical wound was made manually in each well, and berberine was added to experimental wells to final concentration 50 mg/L. Plates with cells were continuously incubated and photographed in cell imager at 37C0 and 5.0% CO2. The extent of cells migration was measured as the percent of wound area decrease after 24 hours of incubation in relation to starting time point. Data are given as: Mean ± 95% confidence interval. Results: In our study we berberine exhibited anti-migratory activity in all cell cultures under study. In rather fast growing primary cell culture wound area decrease was 99.23%±0.62% in control sample and 91.75%±0.28% in experimental sample. The difference was small but significant at p < 0.001 level (df = 30). Popular permanent glioma cell lines T98G and U87MG showed more prominent decrease in studied parameter with higher degree of variance at the same time. In T98G wound area decrease was 71.6%±12.3% in control and 48.8%± 7.6% in experimental samples after 24 hours of cultivation in presence of 50 mg/L berberine. While U87MG demonstrated 60.28%±5.13% and 37.5%± 8.34% wound area decrease accordingly. The obtained difference between control and experimental groups in permanent cell cultures was statistically significant at the 0.05 level (df = 30). Conclusions: Our preliminary research proved berberine to be potent anti-migratory agent in glioma treatment. Further investigations are needed to evaluate its ability to inhibit glioma cell expansion in vivo.

Sign in / Sign up

Export Citation Format

Share Document