Complete Genome Sequence of Campylobacter fetus Isolated from a Sheep

ANNOUNCEMENT

Campylobacter species are Epsilonproteobacteria adapted to vertebrate hosts. Many of them cause disease in a wide range of livestock species and have extensive reservoirs in wildlife. Campylobacter fetus is an important animal pathogen and an opportunistic human pathogen. It produces considerable economic losses as a major reproductive pathogen of cattle and sheep. This species is currently divided into C. fetus subsp. fetus, C. fetus subsp. venerealis (1), and C. fetus subsp. testudinum (2). C. fetus subsp. fetus causes abortion mainly in sheep (3) and, to a lesser extent, cattle.

Here, we describe the closed whole genome of a strain of C. fetus subsp. fetus isolated from a sheep, representing an important resource for evolutionary and epidemiological studies of the species.

Fresh placenta was received at the Instituto Nacional de Investigación Agropecuaria (INIA) Animal Health Platform (Uruguay) for bacteriological analyses. The sample was spiked into Skirrow agar and incubated in a microaerobic atmosphere for 48 h at 37°C using CampyGen (Oxoid) (4). Biochemical testing was performed, including catalase, oxidase, and hydrogen sulfide production (triple-sugar iron medium), growth in the presence of 3.5% sodium chloride or 1% glycine, and growth at 25 and 42°C in Skirrow agar (4). Results were consistent with those of Campylobacter fetus subsp. fetus.

Bacterial colonies from five petri dishes of pure culture were suspended in 500 μl of phosphate-buffered saline solution (pH 7.4), and DNA was extracted using the QIAamp DNA minikit (Qiagen, Inc., Valencia, CA). Whole-genome sequencing (WGS) was performed with Illumina and Pacific Biosciences (PacBio) sequencing technologies. An Illumina library was prepared with the Nextera XT library prep kit without modifications. Illumina reads were obtained using a MiSeq platform at Institut Pasteur de Montevideo, obtaining 1,443,064 2 × 150-bp paired-end (PE) reads. All bioinformatic analysis software was set to default values. Read quality was assessed using FastQC v0.11.7 (5). Bases at the ends of the reads with a quality score lower than 20 and reads shorter than 50 bases were trimmed using Trimmomatic v0.39 (TRAILING:20 MINLEN:50) (6). A total of 1,421,381 PE reads remained after filtration (mean length, 145 bases) (Table 1).

Long reads were obtained using PacBio RS II single-molecule real-time (SMRT) technology with P6-C4 chemistry (Pacific Biosciences) at Macrogen, Inc. (Seoul, South Korea). A library was prepared with the 20-kb SMRTbell template library kit without modifications. DNA was sheared, and 20-kb fragments were selected. Preprocessing was done using the SMRT Portal (smrtanalysis_2.3.0.140936.p5.167094) RS Subreads 1 protocol. It obtained 1,608,855,393 bases from 150,292 reads, with an average read length of 10,704 bp and an N ₅₀ value of 16,155 bp. After filtering, 97% of these data remained (Table 1).

A hybrid assembly approach was performed with Unicycler v0.4.7 assembler (7), using bold mode. The quality of the assembly was addressed using Quast v4.3 (8).

Assembly resulted in a unique 1,849,237-bp contig with an average G+C content of 33.3%, as expected for C. fetus subsp. fetus genomes.

The sequences were annotated with NCBI Prokaryotic Genome Annotation Pipeline (PGAP v4.13) (9). The genome contains 1,884 coding sequences: 1,811 hypothetical proteins, 44 tRNA genes, 3 rRNA operons as in all other Campylobacter species, and 2 CRISPR arrays.