The genome of a pathogenic rhodococcus: cooptive virulence underpinned by key gene acquisitions.

We report the genome of the facultative intracellular parasite Rhodococcus equi, the only animal pathogen within the biotechnologically important actinobacterial genus Rhodococcus. The 5.0-Mb R. equi 103S genome is significantly smaller than those of environmental rhodococci. This is due to genome expansion in nonpathogenic species, via a linear gain of paralogous genes and an accelerated genetic flux, rather than reductive evolution in R. equi. The 103S genome lacks the extensive catabolic and secondary metabolic complement of environmental rhodococci, and it displays unique adaptations for host colonization and competition in the short-chain fatty acid-rich intestine and manure of herbivores--two main R. equi reservoirs. Except for a few horizontally acquired (HGT) pathogenicity loci, including a cytoadhesive pilus determinant (rpl) and the virulence plasmid vap pathogenicity island (PAI) required for intramacrophage survival, most of the potential virulence-associated genes identified in R. equi are conserved in environmental rhodococci or have homologs in nonpathogenic Actinobacteria. This suggests a mechanism of virulence evolution based on the cooption of existing core actinobacterial traits, triggered by key host niche-adaptive HGT events. We tested this hypothesis by investigating R. equi virulence plasmid-chromosome crosstalk, by global transcription profiling and expression network analysis. Two chromosomal genes conserved in environmental rhodococci, encoding putative chorismate mutase and anthranilate synthase enzymes involved in aromatic amino acid biosynthesis, were strongly coregulated with vap PAI virulence genes and required for optimal proliferation in macrophages. The regulatory integration of chromosomal metabolic genes under the control of the HGT-acquired plasmid PAI is thus an important element in the cooptive virulence of R. equi.

A demographic survey of unwanted horses in Ireland in 2011 and totals for 2012 and a comparison with 2010.

This report compiles the available information on unwanted horses in Ireland for 2011 and 2012 and builds upon the previous report for the period 2005 to 2010. Similar trends are present in the high value responsible ownership category and the practicing veterinary profession although extensively involved in horse welfare, euthanises a small proportion of Ireland's unwanted horses. Welfare groups have limited resources and a limited ability to deal with such an extensive problem, which has involved very large numbers of horses. Local authorities continue to have to devote significant efforts and calls on public finances to deal with unwanted horses. Those that they have to deal with are, in the main, not identifiable by either passports or microchips. Category 2 plants and abattoirs continue to provide the principal means of disposal of unwanted horses. The need for abattoirs continues to increase and it is essential that these facilities remain in operation. They processed more than 49,000 horses between 2010 and 2012. The samples they have to submit for Trichinella testing are the most sensitive indicator of the extent of the unwanted horse problem and the most immediate source of information on when it may begin to abate. Trichinella sample numbers and this by inference, horses ponies and donkeys sent to slaughter have fallen by some 35% from 2012 numbers, in the year to date (2013). This may reflect the commercial decision to cease horse slaughter by two slaughterhouses that had hitherto provided this service. Their commercial decision was not in any way related to the identification of fraudulent mislabeled beef in other plants.

Effects of Feeding Coenzyme Q10-Ubiquinol on Plasma Coenzyme Q10 Concentrations and Semen Quality in Stallions.

Although coenzyme Q10 (CoQ10) serves as an antioxidant and energy source for spermatozoa when added to stallion semen before cooling or freezing, the effects of feeding CoQ10 on semen quality have not been studied. We assessed the effects of daily oral ingestion of CoQ10-ubiquinol by stallions on their plasma CoQ10 concentrations and semen quality. Seven mature Andalusian stallions ate 1g ubiquinol/day for 4 weeks followed by a 4-week washout period. Four horses initially completed an additional 4-week control period without ubiquinol. Blood was sampled weekly for determination of plasma CoQ10 concentrations. Ejaculates were collected every two weeks and assessed for total motility (TM), progressive motility (PM), and viability (V) after cooling for 24hours (T1), immediate cryopreservation (T2), and cryopreservation after 24hours cooling (T3). Ingesting ubiquinol resulted in an increase in plasma CoQ10 concentration (P < .001). Two weeks of CoQ10-ubiquinol resulted in improved V with all treatments (T1: P = .007; T2: P = .05; T3: P = .01) and PM with T3 (P = .04). In five stallions, TM and PM were also improved for T1 (P = .01 and P = .02, respectively) and TM increased with T2 (P = .03). Overall, semen quality parameters increased within the first 2 weeks of supplementation, plateaued at the end of the 4-week supplementation period and persisted after discontinuing ubiquinol until the end of the sampling period (8 weeks). Feeding 1 g CoQ10-ubiquinol for 4 weeks to breeding stallions improved semen quality after cooling and freezing in 5 of 7 stallions. This could be important for improving reproductive efficiency in stallions.

Investigating the presence of equine piroplasmosis in Ireland.

BACKGROUND: Equine piroplasmosis (EP) is a notifiable disease in Ireland and a significant concern to domestic and international equine industries. Information regarding EP presence in Ireland is currently limited. This retrospective surveillance study describes a serological and molecular analysis of blood samples submitted to the Irish Equine Centre for EP testing between January 2013 and April 2016. METHODS: Following serological testing, seropositive samples were screened using a PCR targeting the 18S ribosomal RNA gene. Amplicon sequences were bioinformatically analysed to identify the parasite species and to assess genetic diversity. RESULTS: From 2099 screened equine blood samples, 2.5 per cent and 1 per cent were seropositive for Theileria equi and Babesia caballi, respectively. T equi DNA was detected in 9 per cent of the seropositive samples while B caballi DNA was not detected in any sample. The T equi DNA sequences displayed no genetic diversity at this locus, in contrast to samples from the UK and from endemic areas. CONCLUSION: Detection of EP-seropositive and parasitaemic horses in Ireland indicates a clear and present health risk to the equine population. It is recommended that owners adopt appropriate biosecurity measures and that clinicians are mindful of this disease as a differential diagnosis.

Evolution of the Rhodococcus equi vap pathogenicity island seen through comparison of host-associated vapA and vapB virulence plasmids.

The pathogenic actinomycete Rhodococcus equi harbors different types of virulence plasmids associated with specific nonhuman hosts. We determined the complete DNA sequence of a vapB(+) plasmid, typically associated with pig isolates, and compared it with that of the horse-specific vapA(+) plasmid type. pVAPB1593, a circular 79,251-bp element, had the same housekeeping backbone as the vapA(+) plasmid but differed over an approximately 22-kb region. This variable region encompassed the vap pathogenicity island (PAI), was clearly subject to selective pressures different from those affecting the backbone, and showed major genetic rearrangements involving the vap genes. The pVAPB1593 PAI harbored five different vap genes (vapB and vapJ to -M, with vapK present in two copies), which encoded products differing by 24 to 84% in amino acid sequence from the six full-length vapA(+) plasmid-encoded Vap proteins, consistent with a role for the specific vap gene complement in R. equi host tropism. Sequence analyses, including interpolated variable-order motifs for detection of alien DNA and reconstruction of Vap family phylogenetic relationships, suggested that the vap PAI was acquired by an ancestor plasmid via lateral gene transfer, subsequently evolving by vap gene duplication and sequence diversification to give different (host-adapted) plasmids. The R. equi virulence plasmids belong to a new family of actinobacterial circular replicons characterized by an ancient conjugative backbone and a horizontally acquired niche-adaptive plasticity region.

Epidemiologic study of results of pulsed-field gel electrophoresis of isolates of Rhodococcus equi obtained from horses and horse farms.

OBJECTIVE: To compare isolates of Rhodococcus equi on the basis of geographic source and virulence status by use of pulsed-field gel electrophoresis (PFGE). SAMPLE POPULATION: 290 isolates of R equi (218 virulent isolates from foals and 72 avirulent isolates from feces, soil, and respiratory tract samples) obtained between 1985 and 2000 from horses and horse farms from 4 countries. PROCEDURE: DNA from isolates was digested with the restriction enzyme Asel and tested by use of PFGE. Products were analyzed for similarities in banding patterns by use of dendrograms. A similarity matrix was constructed for isolates, and the matrix was tested for nonrandom distributions of similarity values with respect to groupings of interest. RESULTS: There was little grouping of isolates on the basis of country, virulence status, or region within Texas. Isolates of R equi were generally < 80% similar, as determined by use of PFGE. Isolates from the same farm generally were rarely of the same strain. CONCLUSIONS AND CLINICAL RELEVANCE: Considerable chromosomal variability exists among isolates of R equiobtained from the same farm, sites withinTexas, or among countries from various continents. Only rarely will it be possible to link infections to a given site or region on the basis of analysis of isolates by use of PFGE of chromosomal DNA.

Molecular epidemiology of Rhodococcus equi based on traA, vapA, and vapB virulence plasmid markers.

Molecular typing of the actinomycete Rhodococcus equi is insufficiently developed, and little is known about the epidemiology and transmission of this multihost pathogen. We report a simple, reliable polymerase chain reaction typing system for R. equi based on 3 plasmid gene markers: traA from the conserved conjugal transfer machinery and vapA and vapB, found in 2 different plasmid subpopulations. This "TRAVAP" typing scheme classifies R. equi into 4 categories: traA(+)/vapA(+)B(-), traA(+)/vapA(-)B(+), traA(+)/vapAB(-), and traA(-)/vapAB(-) (plasmidless). A TRAVAP survey of 215 R. equi strains confirmed the strong link between vapA (traA(+)/vapA(+)B(-) plasmids) and horse isolates and revealed other host-related plasmid associations: between traA(+)/vapA(-)B(+) and pigs and between traA(+)/vapAB(-)--a new type of R. equi plasmid--and cattle. Plasmidless strains were more frequent among isolates from nonpathological specimens. All plasmid categories were common in human isolates, which possibly reflects the predominantly opportunistic nature of R. equi infection in this host and a zoonotic origin.