Genome sequences of Pasteurellaceae involved in bovine respiratory disease

ABSTRACT

We present closed circular genome sequences of Histophilus somni, Mannheimia haemolytica, and Pasteurella multocida associated with bovine respiratory disease in feedlot cattle. The data will contribute to better understanding of disease and the development of potential mitigation strategies.

ANNOUNCEMENT

Bovine respiratory disease (BRD) is the primary cause of animal treatment and loss at beef feedlots (1–3). Opportunistic bacterial pathogens, including three Pasteurellaceae family members, Histophilus somni, Mannheimia haemolytica, and Pasteurella multocida, commonly contribute to BRD, along with viral co-infection and social and environmental stressors (4).

Seven distinct bacteria – H. somni, three P. multocida, and three M. haemolytica – were isolated from cattle suffering from or lost due to BRD at a feedlot in Alberta, Canada, in November 2020. They originated from six febrile animals, living in four different pens (A–D; Table 1), sampled by deep nasopharyngeal swab (DNS), or necropsy (University of Saskatchewan, Animal Use Protocol 20170021). DNSs were used directly in streak plating on blood agar (tryptic soy agar;TSB, with 5% sheep’s blood) supplemented with 15 μg/mL bacitracin (Dalynn Biological, Calgary, AB, Canada; PB07). A post-mortem lung sample (~2 cm²), obtained from the interface between consolidated and unconsolidated area of a lesion, was stomached in liquid media prior to plating on blood agar. Cultivation for isolation was carried out at 37°C in a 5% CO₂ environment.

TABLE 1.

Genome sequencing and assembly statistics of the genomes^a

ID [origin], Genbank, SRR no.	ST	Pen	Size (Mbp)	reads	read N50	Coverage	GC (%)	Gene count: coding/RNA/pseudo/ARG
H. somni
CCR1 [DNS],  CP186878.1,  SRR32971702	1	A	2.27	74,783	12,285	385	38	2,020/70/42/9
M. haemolytica
CCR2 [DNS],  CP186707.1,  SRR32971701	1	A	2.71	59,034	12,207	252	41	2,613/89/86/1
CCR3 [DNS],  CP187427.1,  SRR32971700	1	B	2.70	79,586	11,853	336	41	2,602/89/90/1
CCR4 [DNS]  CP187428.1,  SRR32971699	1	B	2.70	68,820	10,850	233	41	2,609/89/87/1
P. multocida
CCR5 [DNS]  CP189725.1,  SRR32971698	1	B	2.45	93,808	11,719	431	40	2,192/80/32/7
CCR6 [Lung],  CP187520.1,  SRR32971697	7	C	2.51	70,773	11,385	306	40	2,346/78/29/1
CCR7 [DNS]  CP187521.1,  SRR32971696	7	D	2.51	49,410	12,760	241	40	2,345/79/28/1

^a ST, sequence type.

To prepare DNA for sequencing, M. haemolytica and P. multocida were cultured aerobically overnight at 37°C in TSB whereas H. somni was cultivated in TSB supplemented with 5% horse serum (v/v) in an oxygen-deprived environment (a sealed culture tube). Cells were harvested by centrifugation at 4,000 g and DNA was isolated using the Purelink Microbiome DNA purification kit (Waltham, MA, USA). DNA (1 μg) was sheared by a Megaruptor (Diagenode, NJ, USA) and size selected using a Femto Pulse system (Agilent, CA, USA). Sequencing libraries were constructed and barcoded using the SMRTbell Prep Kit 3.0 and SMRTbell Barcoded Adapter Plate 3.0 before size selection using a Sage Science BluePippin system (Beverly, MA, USA). A PacBio Sequel IIe system and SMRT Cell 8M was employed to generate HiFi reads. Assemblies were performed using Flye (5) for pacbiohifi data and default parameters (v.2.9.5-b1801; Table 1), resulting in circularized, non-rotated genomes (a single chromosome) for each isolate with one exception. Manual curation to generate the final circular, non-rotated CCR5 assembly was required: a 48.7 kb phage-encoding contig was found to have homologous regions (19.8 and 16.8 kb) to the termini of a second 2.4 Mb contig, and the two were merged accordingly. Genes were defined by the NCBI prokaryotic genome annotation pipeline (6), antimicrobial resistance genes (ARGs) were identified (>80% identity match; Table 1) using the Comprehensive Antibiotic Resistance Database (v.4.0.1) through the Resistance Gene Identifier (RGI v.6.0.5) web portal tool (7) and sequence typing (Table 1) was performed using 7-gene PubMLST schemes (8).

Notably, H. somni CCR1 encoded for nine ARGs, including floR, msrE, mphE, and tet(H), which are known to be involved in resistance to commonly used metaphylactic antibiotics.

DATA AVAILABILITY

Genome assemblies have been deposited in the NCBI (PRJNA1246242; CP186878.1, CP186707.1, CP187427.1, CP187428.1, CP189725.1, CP187520.1, CPI87521.1). Raw reads were deposited in the Sequence Read Archive (SRP576414; SRR32971702, SRR32971701 SRR32971700, SRR32971699, SRR32971698, SRR32971697, SRR32971696).