Pathological Features and Genomic Characterization of an Actinobacillus equuli subsp. equuli Bearing Unique Virulence-Associated Genes from an Adult Horse with Pleuropneumonia

Abstract

Actinobacillus equuli subsp. equuli is the etiological agent of sleepy foal disease, an acute form of fatal septicemia in newborn foals. A. equuli is commonly found in the mucous membranes of healthy horses' respiratory and alimentary tracts and rarely causes disease in adult horses. In this study, we report a case of a 22-year-old American Paint gelding presenting clinical signs associated with an atypical pattern of pleuropneumonia subjected to necropsy. The gross and histopathological examinations revealed a unilateral fibrinosuppurative and hemorrhagic pleuropneumonia with an infrequent parenchymal distribution and heavy isolation of A. equuli. The whole genome sequence analysis indicated that the isolate shared 95.9% homology with the only other complete genome of A. equuli subsp. equuli available in GenBank. Seven virulence-associated genes specific to the isolate were identified and categorized as iron acquisition proteins, lipopolysaccharides (LPS), and capsule polysaccharides. Moreover, four genes (glf, wbaP, glycosyltransferase family 2 protein, and apxIB) shared higher amino acid similarity with the invasive Actinobacillus spp. than the reference A. equuli subsp. equuli genome. Availability of the whole genome sequence will allow a better characterization of virulence determinants of A. equuli subsp. equuli, which remain largely elusive.

Keywords: Actinobacillus equuli subsp. equuli; equine actinobacillosis; whole genome sequencing.

Figure 1

Gross findings in the lungs of the Actinobacillus equuli-infected horse. (A) The left lung is unilaterally affected by an extensive, well-demarcated dark red to black area of hemorrhage and necrosis coated by fibrinous exudate (hemorrhagic and fibrinous pleuropneumonia). (B) Left lung, cut surface. The cut surface shows an extensive and relatively well-delimited area of necrosis and hemorrhage.

Figure 2

Microscopic features of Actinobacillus equuli-associated pleuropneumonia. (A) The pleura is extensively coated by a thick layer of polymerized fibrin (arrows) and myriad coccobacillary bacteria underlying the fibrinous exudate and extending into the subpleural interstitium (arrowheads). The underlying pulmonary parenchyma is affected by hemorrhage with loss of alveolar septa and replacement by numerous degenerate leukocytes. (B) The bacteria underlying the fibrinous exudate are gram-negative (arrowheads). (C,D) The pulmonary parenchyma is affected by extensive hemorrhage, fibrinous exudation, and necrosis and is infiltrated by numerous degenerate leukocytes. Within areas of less pronounced necrosis, alveolar spaces are filled with degenerate leukocytes (D), and the pulmonary vasculature is multifocally occluded by fibrin thrombi ((D), arrows). (E,F) Within affected areas of the parenchyma, there are multifocal extracellular and intracellular colonies of gram-negative (F) coccobacilli (arrows). (A,C,D,E); H&E, 200×, 100×, 100×, and 400×, respectively. (B,F); Gram stain, 1000×.

Figure 3

Maximum likelihood phylogenetic tree of the complete genome sequences of isolate 4524 and other Actinobacillus spp. based on REALPY. Numbers at nodes represent the percentages of occurrence of nodes in 1000 bootstrap trials. The Haemophilus influenzae PittGG (CP000672) was used as an outgroup.

Figure 4

Circular map of the genome of A. equuli subsp. equuli 4524 strain generated using the CGView Server. From the outside to the center: coding sequences (CDSs) in the positive strand, CDSs in the reverse strand, BLASTn vs. A. equuli subsp. equuli ATCC 19392 (NZ_CP007715.1), GC skew, and GC content.

Figure 5

Venn diagram of the complete genomes of A. equuli subsp. equuili 4524 and ATCC 19392 strains.