Tick-borne pathogens were screened using sets of primers, including the 18S ribosomal RNA (rRNA) gene of piroplasms (Theileria/Babesia spp.), Rickettsial gltA and Anaplasma spp. 16S rRNA gene as previously described (Table 1). In each PCR reaction, PCR water was used as a negative control while Theileria annulata, Rickettsia massiliae and Anaplasma marginale DNA were used as the positive control for the screening of tick-borne pathogens. To amplify the full-length open reading frame (ORF) sequences of SUB and P0 from R. microplus and those of FER2 from H. anatolicum, primers were designed based on the retrieved homologous sequences from the GenBank for SUB (KM115651, EU301808, JQ922399, JX431507-09, JQ713774-77, JQ713779-80, JQ713782-83, JQ713785), FER2 (KT924235-47) and P0 (KC845304, KP087926). Degenerated primers were used in SUB and P0 cloning (Table 1).

Primers for tick-borne pathogens screening, and amplification of tick open reading frame coding genes

OrganismPrimer sequenceAmplicon (bp)References
Piroplasms (Theileria/Babesia spp.)/18S rRNA gene

F: ACCGTGCTAATTGTAGGGCTAATAC

R: GAACCCAAAGACTTTGATTTCTCTC

897[33 (link)]
Rickettsial gltA

F: GCAAGTATCGGTGAGGATGTAAT

R:CTTCCTTAAAATTCAATAAATCAGGAT

401[34 (link)]
Anaplasma spp./16S rRNA gene

F: GGTACCYACAGAAGAAGTCC

R: TGCACTCATCGTTTACAG

345[35 (link)]
Tick’s ORF coding genes
 Subolesin

F: ATGGCTTGYGCRACATTAAAGCG

R: TTACGACAAATAGCTGGGCG

486This study
 P0

F1: ATGGTCAGGGAGGATAAGAC

F2: ATTGTGAACGGCCTGAAAAACCTGA

R: YYTAGTCGAAGAGTCCGAAGCCCAT

957This study
234This study
 Ferritin 2

F: ATGGGCAACAACCTGAACGAACAG

R: TTAGGTACGCAGCTGCTGATCCAG

531This study
 Actin

F: TCAGGTCATCACCATCGGCAAC

R: GTACATGGTGGTGCCGCCG

184[36 (link)]

Degenerated nucleotides are underlined

F Forward, R reverse, PO 60S acidic ribosomal protein, rRNA ribosomal RNA

PCR and thermocycling conditions for screening the tick-borne pathogens were as previously described [33 (link)–35 (link)]. For the amplification of full-length ORF sequences, a total reaction volume of 25 µl was prepared containing a template cDNA (500 ng/μl), 1× PCR buffer, 0.2 mM dNTPs, 3 mM MgCl2, 1 U Taq DNA polymerase, nuclease-free water (Thermo Fisher Scientific) and 0.5 mM each forward and reverse primers. The thermocycling conditions were: an initial denaturation at 94 °C for 4 min; followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C (SUB and FER2) and 50 °C (P0) for 30 s and extension at 72 °C for 60 s; with a final extension at 72 °C for 10 min. A similar PCR assay was prepared, and a targeted partial 234-bp sequence of the amplified P0 gene was further amplified using a new forward primer with the previously used reverse primer (Table 1). A negative control without cDNA and a positive control containing cDNA and a specific set of actin primers were prepared to check the integrity of cDNA [36 (link)]. The PCR assays were performed in a PCR thermocycler (model T100; Bio-Rad Laboratories Inc., Hercules, CA, USA), and the obtained PCR products were analyzed in a 1% agarose gel stained with ethidium bromide.
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