Spiroplasma

Moyaba, Percy, Ouedraogo, Gisele Ms, Pagabeleguem, Soumaïla, Njokou, Flobert, Freitas, Ngambia, Vreysen, Marc Jb & Abd-Alla, Adly Mm, 2023, Prevalence of Spiroplasma and interaction with wild Glossina tachinoides microbiota, Parasite (Paris, France) 30 (62), pp. 1-14 : 3

Prevalence of Spiroplasma and Trypanosoma

To detect Spiroplasma infection, PCR amplification of an approximately 455 bp fragment of the 16S rRNA gene was performed [ 21]. The PCR was carried out in 25 µ L reaction mixtures containing 22.5 µ L of 1.1 × Pre-Aliquoted PCR Master Mix (0.625 units Thermoprime Plus DNA Polymerase, 75 mM Tris–HCl (pH 8.8 at 25 ° C), 20 mM (NH 4)2SO4, 2.0 mM MgCl 2, 0.01% (v/v) Tween-20 and 0.2 mM each of the dNTPs (ABgene, UK), and 1.5 µ L of template DNA plus 1 µ L of Spiroplasma 16S RNA primers (63F and TKSS) (Supplementary Table 2) to a final concentration of 0.2 mM per primer. PCR conditions were 95 ° C for 5 min, followed by 34 cycles of 95 ° C for 30 s, 58 ° C for 30 s, 72 ° C for 30 s, and final extension 72 ° C for 10 min. PCR products were electrophoresed in 2% molecular grade agarose (Fisher Biotech) stained with Safe- Green. DNA from the IPCL colony of G. f. fuscipes , which is known to be infected with Spiroplasma , and sterilized distilled water were included in each PCR test as positive and negative controls, respectively. As described previously [ 19], the Glossina species microsatellite GpCAG133 was used to control the quality of the extracted DNA and only validated samples were considered for Spiroplasma or Trypanosoma infection status. To confirm that the amplified PCR products obtained with G. pallidipes , G. m. morsitans , and G. p. gambiensis were Spiroplasma- specific sequences, two approaches were used: the first was to confirm the specificity of the amplification by performing PCR using the MLST primers shown in Supplementary Table 2. The second was to sequence the PCR product obtained by the 16S rRNA gene primers. For the sequencing, PCR products were purified using the High Pure PCR Cleanup Micro Kit (Roche, Basel, Switzerland) and ligated to the pGEM-T vector (Promega, Madison, WI, USA), following the supplier’ s instructions. The recombinant plasmids were transformed into DH5 Oi -competent bacteria (Invitrogen, Carlsbad, CA, USA), following the supplier’ s instructions. The recombinant plasmids and the inserted sequences were confirmed by Sanger sequencing (Eurofins Genomics, Ebersberg, Germany) with the universal vector primers M13F_uni (-21) (5 0 –TGT AAA ACG GCC AGT– 3 0) and M13 R _rev (-29) (5 0 –CAG GAA ACA GCT ATG ACC– 3 0). For the other tsetse species including G. f. fuscipes , G. brevipalpis and G. tachinoides , the amplified PCR products were purified with the ZR-96 DNA Clean & Concentrator ® -5 (Zymo Research, Irvine, CA, USA), following the manufacturer’ s protocol and submitted directly to sequencing without cloning using the 63F and TKSS primers (Eurofins Genomics, Ebersberg, Germany). The resulting sequences were blasted against the non-redundant protein sequence (nr) database in the NCBI server using the BLAST tool https://blast.ncbi.nlm.nih.gov/ Blast.cgi to identify and annotate the sequence. The sequence was considered a Spiroplasma sequence if it matched with Spiroplasma sequence in the database. The prevalence of Trypanosoma was assessed as in Ouedraogo et al. [ 43].