Limits...
Evaluation of the in vitro expression of ATP binding-cassette (ABC) proteins in an Ixodes ricinus cell line exposed to ivermectin.

Mangia C, Vismarra A, Kramer L, Bell-Sakyi L, Porretta D, Otranto D, Epis S, Grandi G - Parasit Vectors (2016)

Bottom Line: Cell viability ranged between 84% and 92% with no significant differences between untreated and treated cells. qRT-PCR showed that ABC pump expression was not significantly modulated by ivermectin treatment.ABCB6 and ABCB10 gene expression was not modulated by ivermectin treatment and ABCB1 expression was not detected.Development of an in vitro model for the study of acaricide resistance mechanisms would greatly facilitate screening for drug resistance in ticks.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Sciences, University of Parma, 43126, Parma, Italy.

ABSTRACT

Background: Ticks are among the most important vectors of pathogens causing human and animal disease. Acaricides are used to control tick infestation, although there are increasing reports of resistance. Recently, over-expression of ATP-binding cassette (ABC) transporter proteins (P-glycoproteins, PgP) has been implicated in resistance to the acaricide ivermectin in the ticks Rhipicephalus (Boophilus) microplus and Rhipicephalus sanguineus sensu lato. Ixodid tick cell lines have been used to investigate drug resistance mechanisms. The aim of the present study was to evaluate expression of several PgPs in the Ixodes ricinus-derived cell line IRE/CTVM19 and to determine modulation of expression following treatment with ivermectin.

Findings: IRE/CTVM19 cells were treated with different concentrations of ivermectin (0, 11, 22 or 33 μM) and incubated for 10 days. Evaluation of viability and relative expression of ABCB1, ABCB6, ABCB8 and ABCB10 genes were carried out at day 10 post treatment. Cell viability ranged between 84% and 92% with no significant differences between untreated and treated cells. qRT-PCR showed that ABC pump expression was not significantly modulated by ivermectin treatment. Expression of the ABCB8 PgP subfamily revealed a biphasic trend, based on the ivermectin concentration. ABCB6 and ABCB10 gene expression was not modulated by ivermectin treatment and ABCB1 expression was not detected.

Conclusions: This is the first report of PgP expression in an I. ricinus-derived tick cell line. Development of an in vitro model for the study of acaricide resistance mechanisms would greatly facilitate screening for drug resistance in ticks.

No MeSH data available.


Related in: MedlinePlus

IRE/CTVM19 cell viability on day 5 of cultivation either untreated (L-15), treated with 0.1% DMSO alone (DMSO 0.1 %) or treated with ivermectin (IVM) in 0.1 % DMSO at concentrations of 11, 22 or 33 μM. Cells were evaluated by flow cytometry following Live vs. Dead® staining and data represents the mean of four replicate tubes ± S.E.M. Viability measured by Trypan Blue exclusion on days 5 and 10 was comparable (data not shown)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4835901&req=5

Fig3: IRE/CTVM19 cell viability on day 5 of cultivation either untreated (L-15), treated with 0.1% DMSO alone (DMSO 0.1 %) or treated with ivermectin (IVM) in 0.1 % DMSO at concentrations of 11, 22 or 33 μM. Cells were evaluated by flow cytometry following Live vs. Dead® staining and data represents the mean of four replicate tubes ± S.E.M. Viability measured by Trypan Blue exclusion on days 5 and 10 was comparable (data not shown)

Mentions: Growth curve analysis revealed a doubling-time of approximately ten days for IRE/CTVM19 cells under all conditions. IVM treatment modified cell morphology and adherance to the plastic tube, but did not alter cell viability (Fig. 2). As measured by Trypan Blue exclusion assay (data not shown) and flow-cytometry (Fig. 3), viability was 92 % in the untreated control cells and between 84 % and 88 % in cells treated with DMSO alone or with IVM. Differences between groups were not significant.Fig. 2


Evaluation of the in vitro expression of ATP binding-cassette (ABC) proteins in an Ixodes ricinus cell line exposed to ivermectin.

Mangia C, Vismarra A, Kramer L, Bell-Sakyi L, Porretta D, Otranto D, Epis S, Grandi G - Parasit Vectors (2016)

IRE/CTVM19 cell viability on day 5 of cultivation either untreated (L-15), treated with 0.1% DMSO alone (DMSO 0.1 %) or treated with ivermectin (IVM) in 0.1 % DMSO at concentrations of 11, 22 or 33 μM. Cells were evaluated by flow cytometry following Live vs. Dead® staining and data represents the mean of four replicate tubes ± S.E.M. Viability measured by Trypan Blue exclusion on days 5 and 10 was comparable (data not shown)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4835901&req=5

Fig3: IRE/CTVM19 cell viability on day 5 of cultivation either untreated (L-15), treated with 0.1% DMSO alone (DMSO 0.1 %) or treated with ivermectin (IVM) in 0.1 % DMSO at concentrations of 11, 22 or 33 μM. Cells were evaluated by flow cytometry following Live vs. Dead® staining and data represents the mean of four replicate tubes ± S.E.M. Viability measured by Trypan Blue exclusion on days 5 and 10 was comparable (data not shown)
Mentions: Growth curve analysis revealed a doubling-time of approximately ten days for IRE/CTVM19 cells under all conditions. IVM treatment modified cell morphology and adherance to the plastic tube, but did not alter cell viability (Fig. 2). As measured by Trypan Blue exclusion assay (data not shown) and flow-cytometry (Fig. 3), viability was 92 % in the untreated control cells and between 84 % and 88 % in cells treated with DMSO alone or with IVM. Differences between groups were not significant.Fig. 2

Bottom Line: Cell viability ranged between 84% and 92% with no significant differences between untreated and treated cells. qRT-PCR showed that ABC pump expression was not significantly modulated by ivermectin treatment.ABCB6 and ABCB10 gene expression was not modulated by ivermectin treatment and ABCB1 expression was not detected.Development of an in vitro model for the study of acaricide resistance mechanisms would greatly facilitate screening for drug resistance in ticks.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Sciences, University of Parma, 43126, Parma, Italy.

ABSTRACT

Background: Ticks are among the most important vectors of pathogens causing human and animal disease. Acaricides are used to control tick infestation, although there are increasing reports of resistance. Recently, over-expression of ATP-binding cassette (ABC) transporter proteins (P-glycoproteins, PgP) has been implicated in resistance to the acaricide ivermectin in the ticks Rhipicephalus (Boophilus) microplus and Rhipicephalus sanguineus sensu lato. Ixodid tick cell lines have been used to investigate drug resistance mechanisms. The aim of the present study was to evaluate expression of several PgPs in the Ixodes ricinus-derived cell line IRE/CTVM19 and to determine modulation of expression following treatment with ivermectin.

Findings: IRE/CTVM19 cells were treated with different concentrations of ivermectin (0, 11, 22 or 33 μM) and incubated for 10 days. Evaluation of viability and relative expression of ABCB1, ABCB6, ABCB8 and ABCB10 genes were carried out at day 10 post treatment. Cell viability ranged between 84% and 92% with no significant differences between untreated and treated cells. qRT-PCR showed that ABC pump expression was not significantly modulated by ivermectin treatment. Expression of the ABCB8 PgP subfamily revealed a biphasic trend, based on the ivermectin concentration. ABCB6 and ABCB10 gene expression was not modulated by ivermectin treatment and ABCB1 expression was not detected.

Conclusions: This is the first report of PgP expression in an I. ricinus-derived tick cell line. Development of an in vitro model for the study of acaricide resistance mechanisms would greatly facilitate screening for drug resistance in ticks.

No MeSH data available.


Related in: MedlinePlus