Seqman v 5

The chromatograms of all the obtained sequences were manually observed and trimmed for purification purposes to remove the contaminated and poor reading regions through SeqMan V. 5 (DNASTAR, Inc., Madison, WI, USA). Final trimmed sequences were subjected to Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990 (link)) at National Center for Biotechnology Information to get the high identity sequences in FASTA format. ClustalW multiple alignments (Thompson et al., 1994 (link)) were used to align all the downloaded sequences along with the obtained and selected outgroup sequences in BioEdit Sequence Alignment Editor V.7.0.5 (Raleigh, NC, USA) (Hall et al., 2011 ). The phylogenetic trees based on partial fragments of cox1 and GroEL were constructed in MEGA-X (Molecular Evolutionary Genetics Analysis) (Kumar et al., 2018 (link)) through the neighbor-joining method (Tamura-Nei model) and the Maximum Parsimony method (Tamura-Nei model) (Tamura and Nei, 1993 (link)) with support of 1000 bootstrapping replicons, respectively. The coding fragments (cox1 and GroEL) were aligned using MUSCLE (Edgar, 2004 (link)).

All amplified amplicons of cox1 (5: 1 male, 2 adult females, and 2 nymphs) for ticks, 18S rDNA (2: 1 adult female and 1 nymph) for Theileria spp. and 16S rDNA (4: 2 adult females and 2 nymphs) for Anaplasma spp. were sequenced (Macrogen Inc., Seoul, Republic of Korea) by Sanger sequencing. The obtained sequences were trimmed/edited via SeqMan v. 5 (DNASTAR, Inc., Madison/WI, USA) for the removal of poor reading sequences and subjected to Basic Local Alignment Search Tool (BLAST, https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on: 10 July 2022) at the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/, accessed on: 10 July 2022). After BLAST, maximum identity sequences of the most similar/subgenus species were downloaded in FASTA format from the NCBI. Obtained sequences were aligned with the downloaded sequences using ClustalW multiple alignments in BioEdit Sequence Alignment Editor v. 7.0.5 [39 ]. The phylogenies were constructed individually for each DNA sequence of tick and associated pathogens through the Maximum Likelihood statistical method and Kimura 2-parameter model in Molecular Evolutionary Genetics Analysis (MEGA-X) with a bootstrapping value of 1000 [40 (link)]. The coding sequences like cox1 sequences were aligned by using MUSCLE algorithms [41 (link)].

Twelve amplicons, four of each 16S rRNA, cox I, and ITS-2, and portions of all amplified rickettsial genes (gltA, ompA, ompB) of the positive samples mentioned in Table 1, were sequenced bi-directionally (Macrogen, Seoul, Republic of Korea). The obtained sequences were assembled and trimmed to eliminate the poor nucleotide regions using SeqMan V. 5 (DNASTAR, Madison, WI, USA). Trimmed sequences were subjected to BLAST (Basic Local Alignment Search Tool) at NCBI (National Center for Biotechnological Information) to download identical sequences [61 (link)]. Downloaded sequences, along with obtained sequences, and out group were aligned using ClustalW multiple alignment in BioEdit V.7.0.5 [62 ]. Aligned sequences were used to construct phylogenetic trees through the Tamura-Nei model and maximum-likelihood statistical method, keeping bootstrap value 1000 in MEGA-11 [63 (link)].

All amplified amplicons of cox1 and 16S rDNA partial fragments were sequenced bidirectionally (Macrogen Inc., Seoul, South Korea) using the Sanger sequencing method. The obtained sequences were cropped through SeqMan v. 5 (DNASTAR, Inc., Madison/WI, United States) to remove poor reading sequences and subjected to Basic Local Alignment Search Tool (BLAST, https://blast.ncbi.nlm.nih.gov/Blast.cgi) at the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/). After BLAST, high identity sequences were downloaded in FASTA format from the NCBI. The obtained sequences were aligned with the downloaded sequences using ClustalW multiple alignments in BioEdit Sequence Alignment Editor v. 7.0.5 (49 ). The phylogenetic trees were constructed individually for each gene sequence of the tick, using the maximum likelihood method with the Tamura-Nei model in Molecular Evolutionary Genetics Analysis (MEGA-X), with a bootstrapping value of 1,000 (50 (link)). The coding sequences were aligned using MUSCLE alignments (51 (link)).

Amplified amplicons of 16S rDNA of Anaplasma spp. from different tick species infesting Holstein-Friesian, Jersey, and Asian water buffaloes were sequenced in both directions (Macrogen, Inc., Seoul, South Korea). The obtained sequences were trimmed to remove the contaminated and poor reading regions via SeqMan V. 5 (DNASTAR, Inc., Madison, WI, USA). The obtained sequences were subjected to the Basic Local Alignment Search Tool (BLAST) [30 (link)] at National Center for Biotechnology Information (NCBI). The homologous sequences were downloaded in FASTA format from NCBI based on their high percentage identity. The downloaded sequences were aligned with the obtained sequences and an outgroup sequence using ClustalW multiple alignments [31 (link)] in BioEdit alignment editor V.7.0.5 (Raleigh, NC, USA) [32 (link)]. The phylogenetic tree was constructed through the Maximum-Likelihood statistical method and Tamura-Nei model [33 (link)] with a 1000 bootstrapping value in Molecular evolutionary genetics analysis (MEGA-X) [34 (link)].

PCR products of 16S rDNA and cox1 for ticks, as well as 16S rDNA, groEL, dsb, 18S rDNA, gltA, ompA, and ompB for associated tick-borne pathogens, were sent for capillary bidirectional sequencing (Macrogen Inc., Seoul, South Korea). The obtained sequences were trimmed and assembled using SeqMan v. 5 (DNASTAR, Inc., Madison/WI, United States) to remove poor reading sequences. The obtained multiple identical sequences for each gene were considered a consensus sequence and subjected to the basic local alignment search tool (BLAST) at the National Center for Biotechnology Information (NCBI). Sequences with maximum identities were downloaded in FASTA format from the NCBI. The obtained sequences were aligned with the downloaded sequences using ClustalW multiple alignments in BioEdit Sequence Alignment Editor v. 7.0.5 (36 ). The coding nucleotide sequences were aligned using MUSCLE (37 (link)). Phylogenetic trees were constructed individually for each partial sequence of tick and associated tick-borne pathogens, by employing the maximum likelihood (ML) method with Kimura 2-parameter in Molecular Evolutionary Genetics Analysis (MEGA-X), accompanied by a bootstrapping value of 1,000 (38 (link)).

Amplicons were purified with GeneClean II Kit (Qbiogene, Illkirch, France) following the manufacturer’s protocol and sequenced bi-directionally (Macrogen, Inc., Seoul, South Korea) via the Sanger-based sequencing method. The obtained sequences were trimmed by removing the poor-quality regions followed by obtaining a consensus sequence in SeqMan v 5.00 (DNASTAR, Inc., Madison, WI, USA). Maximum identity sequences were retrieved from GenBank using the Basic Local Alignment Search Tool (BLAST) [28 (link)] on the National Center for Biotechnology Information (NCBI) user interface. They were aligned with the obtained sequences in BioEdit Sequence Alignment Editor v. 7.0.5 [29 ] using CLUSTAL W multiple alignments [30 (link)]. The Neighbor-Joining method with the Kimura 2-parameter model was applied to construct phylogenetic trees with 1000 bootstrap replicates in Molecular Evolutionary Genetic Analysis (MEGA-X) software [31 (link)]. The coding (cox, gltA, ompA, and ompB) nucleotide sequences were aligned by MUSCLE [32 (link)]. The final positions in the dataset comprised the obtained sequence of each fragment.