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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

Primer couples tested in traditional PCR. All fragments were approximately 101–157 bp long. The no-template control (NTC) presented a spot due to primer dimerization
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Fig1: Primer couples tested in traditional PCR. All fragments were approximately 101–157 bp long. The no-template control (NTC) presented a spot due to primer dimerization

Mentions: On day 10 following the start of ivermectin treatment, RNA was extracted from samples of resuspended cells from each replicate culture using an RNeasy Mini Kit (Qiagen) following the manufacturer’s instructions. RNA was measured by spectrophotometric analysis for quality and content and then converted into cDNA using a QuantiTect Reverse Transcription Kit (Qiagen). The resultant cDNAs were used as templates for molecular analysis. To date, there is no published information about I. ricinus sequences for any of the pumps under investigation (those encoded by the ABCB1, ABCB6, ABCB8 and ABCB10 genes); thus primers were designed (Table 1) based on conserved regions of sequences of selected ABC transporters of I. scapularis available in VectorBase (ABCB1: ISCW004310; ABCB6: ISCW021257; ABCB8: ISCW005908; ABCB10: ISCW008199) [14]. As an endogenous control, the I. ricinus β-actin gene was chosen and primers were designed based on the partial sequence available in Genbank (HQ682101). Primers were first tested in a traditional PCR using cDNA derived from an untreated control IRE/CTVM19 culture and reactions were run on a 2 % agarose gel stained with SYBR Safe Gel and examined under UV light (UView mini Transilluminator, Biorad) (Fig. 1). The amplification fragments, obtained using standard PCR conditions and the thermal profile indicated below, were sequenced in order to confirm the specificity of the amplification. The resultant sequences were deposited in the EMBL Nucleotide Sequence Database (ABCB6: LT222036; ABCB8: LT222037; ABCB10: LT222038).Table 1


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)

Primer couples tested in traditional PCR. All fragments were approximately 101–157 bp long. The no-template control (NTC) presented a spot due to primer dimerization
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Primer couples tested in traditional PCR. All fragments were approximately 101–157 bp long. The no-template control (NTC) presented a spot due to primer dimerization
Mentions: On day 10 following the start of ivermectin treatment, RNA was extracted from samples of resuspended cells from each replicate culture using an RNeasy Mini Kit (Qiagen) following the manufacturer’s instructions. RNA was measured by spectrophotometric analysis for quality and content and then converted into cDNA using a QuantiTect Reverse Transcription Kit (Qiagen). The resultant cDNAs were used as templates for molecular analysis. To date, there is no published information about I. ricinus sequences for any of the pumps under investigation (those encoded by the ABCB1, ABCB6, ABCB8 and ABCB10 genes); thus primers were designed (Table 1) based on conserved regions of sequences of selected ABC transporters of I. scapularis available in VectorBase (ABCB1: ISCW004310; ABCB6: ISCW021257; ABCB8: ISCW005908; ABCB10: ISCW008199) [14]. As an endogenous control, the I. ricinus β-actin gene was chosen and primers were designed based on the partial sequence available in Genbank (HQ682101). Primers were first tested in a traditional PCR using cDNA derived from an untreated control IRE/CTVM19 culture and reactions were run on a 2 % agarose gel stained with SYBR Safe Gel and examined under UV light (UView mini Transilluminator, Biorad) (Fig. 1). The amplification fragments, obtained using standard PCR conditions and the thermal profile indicated below, were sequenced in order to confirm the specificity of the amplification. The resultant sequences were deposited in the EMBL Nucleotide Sequence Database (ABCB6: LT222036; ABCB8: LT222037; ABCB10: LT222038).Table 1

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