Spirochaetes as intestinal pathogens: Lessons from a Brachyspira genome

Anaerobic spirochaetes of the genus Brachyspira have long been known as important gut pathogens of pigs, but increasingly they are recognised as causing disease in birds and other animal species, including human beings. The genome sequence of the major swine pathogen Brachyspira hyodysenteriae was recently published, and this revealed extensive genome optimisation that leads to adaptation to the complex environment of the colon. The genome sequences of other pathogenic and non-pathogenic Brachyspira species are becoming available, and this data will help to reveal how these species have evolved and adapted to varied lifestyles in the large intestines of different species, and why some but not others can induce colitis and diarrhoea

The use of ELISAs for monitoring exposure of pig herds to Brachyspira hyodysenteriae

Background Swine dysentery (SD), a mucohaemorrhagic diarrhoeal disease of pigs, results from infection of the large intestine with the spirochaete Brachyspira hyodysenteriae. ELISA systems using whole spirochaete cells (WC) and the B. hyodysenteriae outer membrane lipoprotein Bhlp29.7 previously have been established as potential diagnostic tools for SD. However, their true value in identifying infected herds remains unclear. The present study aimed to compare the performance of whole-cell and Bhlp29.7 based ELISAs in detecting specific immunoglobulin class IgG and IgM to B. hyodysenteriae in growing pigs, and additionally evaluated whether meat juice could serve as a source of specific antibodies. Results Levels of circulating IgG and IgM reacting with WC spirochaete preparations and recombinant Bhlp29.7 peaked 4-6 weeks post-infection in the experimentally challenged pigs, and remained elevated in the present study. In a cohort of pigs on an infected farm levels of antibody directed against both antigens showed a progressive increase with time. However, other than for the level of IgG against WC antigen, a significant increase in antibody levels also was observed in a cohort of pigs on a non-infected farm. In addition, assays using meat juice had 100% specificity and equivalent sensitivity to those based on serum, and likewise the best performance was achieved using the WC IgG ELISA. Conclusions IgG ELISAs using either WC or Bhlp29.7 as plate-coating antigens were shown to be useful for monitoring the dynamics of B. hyodysenteriae infection in grower pigs. Of the two antigens, the WC preparation tended to give better discrimination between pigs from infected and non-infected farms. Testing of meat juice was shown to have potential for identifying infected herds

Potential for Zoonotic Transmission of Brachyspira pilosicoli

Anaerobic intestinal spirochetes of the genus Brachyspira colonize the large intestine (1). Most Brachyspira species have a restricted host range, whereas Brachyspira (formerly Serpulina) pilosicoli colonizes a variety of animal and bird species and humans. B. pilosicoli is an important colonic pathogen of pigs and chickens (2). It occurs at high prevalence rates in humans in developing countries and in male homosexuals and HIV-positive persons in industrialized countries (3). Its potential as a human pathogen was emphasized after its identification in the bloodstream of a series of debilitated persons (4)

The Intestinal Spirochete Brachyspira pilosicoli Attaches to Cultured Caco-2 Cells and Induces Pathological Changes

BACKGROUND:Brachyspira pilosicoli is an anaerobic spirochete that has received relatively little study, partly due to its specialized culture requirements and slow growth. This bacterium colonizes the large intestine of various species, including humans; typically, a dense layer of spirochete cells may be found intimately attached by one cell end to the surface of colonic enterocytes. Colonized individuals may develop colitis, but the mechanisms involved are not understood. The current study aimed to develop an in vitro model to investigate this process. METHODOLOGY/PRINCIPAL FINDINGS:Four strains of B. pilosicoli were incubated at a high multiplicity of infection with monolayers of a human colonic adenocarcinoma cell line (Caco-2 cells). One strain isolated from a pig (95/1000) and one from a human (WesB) attached to the monolayers. Colonization increased with time, with the Caco-2 cell junctions being the initial targets of attachment. By electron microscopy, individual spirochete cells could be seen to have one cell end invaginated into the Caco-2 cell membranes, with the rest of the spirochete draped over the Caco-2 cell surface. After 6 h incubation, the monolayer was covered with a layer of spirochetes. Colonized monolayers demonstrated a time-dependent series of changes: staining with labelled phalloidin identified accumulation of actin at the cell junctions; ZO-1 staining revealed a loss of Caco-2 tight junction integrity; and Hoechst staining showed condensation and fragmentation of nuclear material consistent with apoptosis. Using quantitative reverse transcription PCR, the colonized monolayers demonstrated a significant up-regulation of interleukin-1beta (IL-1beta) and IL-8 expression. B. pilosicoli sonicates caused significant up-regulation of IL-1beta, TNF-alpha, and IL-6, but culture supernatants and non-pathogenic Brachyspira innocens did not alter cytokine expression. CONCLUSIONS/SIGNIFICANCE:The changes induced in the Caco-2 cells provide evidence that B. pilosicoli has pathogenic potential, and give insights into the likely in vivo pathogenesis

Intracortical Remodeling Parameters Are Associated With Measures of Bone Robustness

Prior work identified a novel association between bone robustness and porosity, which may be part of a broader interaction whereby the skeletal system compensates for the natural variation in robustness (bone width relative to length) by modulating tissue‐level mechanical properties to increase stiffness of slender bones and to reduce mass of robust bones. To further understand this association, we tested the hypothesis that the relationship between robustness and porosity is mediated through intracortical, BMU‐based (basic multicellular unit) remodeling. We quantified cortical porosity, mineralization, and histomorphometry at two sites (38% and 66% of the length) in human cadaveric tibiae. We found significant correlations between robustness and several histomorphometric variables (e.g., % secondary tissue [R 2  = 0.68, P  < 0.004], total osteon area [R 2  = 0.42, P  < 0.04]) at the 66% site. Although these associations were weaker at the 38% site, significant correlations between histological variables were identified between the two sites indicating that both respond to the same global effects and demonstrate a similar character at the whole bone level. Thus, robust bones tended to have larger and more numerous osteons with less infilling, resulting in bigger pores and more secondary bone area. These results suggest that local regulation of BMU‐based remodeling may be further modulated by a global signal associated with robustness, such that remodeling is suppressed in slender bones but not in robust bones. Elucidating this mechanism further is crucial for better understanding the complex adaptive nature of the skeleton, and how interindividual variation in remodeling differentially impacts skeletal aging and an individuals' potential response to prophylactic treatments. Anat Rec, 297:1817–1828, 2014. © 2014 Wiley Periodicals, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/108629/1/ar22962.pd

Dissemination of Clonal Groups of Brachyspira hyodysenteriae amongst Pig Farms in Spain, and Their Relationships to Isolates from Other Countries

Background: Swine dysentery (SD) is a widespread diarrhoeal disease of pigs caused by infection of the large intestine with the anaerobic intestinal spirochaete Brachyspira hyodysenteriae. Understanding the dynamics of SD, and hence being able to develop more effective measures to counter its spread, depends on the ability to characterise B. hyodysenteriae variants and trace relationships of epidemic strains. Methodology/Principal Findings: A collection of 51 Spanish and 1 Portuguese B. hyodysenteriae isolates was examined using a multilocus sequence typing (MLST) scheme based on the sequences of seven conserved genomic loci. The isolates were allocated to 10 sequence types (STs) in three major groups of descent. Isolates in four of the STs were widely distributed in farms around Spain. One farm was infected with isolates from more than one ST. Sequence data obtained from PubMLST for 111 other B. hyodysenteriae strains from other countries then were included in the analysis. Two of the predominant STs that were found in Spain also were present in other European countries. The 73 STs were arranged in eleven clonal complexes (Cc) containing between 2 and 26 isolates. A population snapshot based on amino acid types (AATs) placed 75% of the isolates from 32 of the 48 AATs into one major cluster. The founder type AAT9 included 22 isolates from 10 STs that were recovered in Spain, Australia, Sweden, Germany, Belgium, the UK, Canada, and the USA. Conclusions/Significance: This MLST scheme provided sufficient resolution power to unambiguously characterise B. hyodysenteriae isolates, and can be recommended as a routine typing tool that rapidly enables comparisons of isolates. Using this method it was shown that some of the main genetic lineages of B. hyodysenteriae in Spain also occurred in other countries, providing further evidence for international transmission. Finally, analysis of AATs appeared useful for deducing putative ancestral relationships between strains

P-Rex1 Controls Sphingosine 1-Phosphate Receptor Signalling, Morphology, and Cell-Cycle Progression in Neuronal Cells.

P-Rex1 is a guanine-nucleotide exchange factor (GEF) that activates Rac-type small G proteins in response to the stimulation of a range of receptors, particularly G protein-coupled receptors (GPCRs), to control cytoskeletal dynamics and other Rac-dependent cell responses. P-Rex1 is mainly expressed in leukocytes and neurons. Whereas its roles in leukocytes have been studied extensively, relatively little is known about its functions in neurons. Here, we used CRISPR/Cas9-mediated P-Rex1 deficiency in neuronal PC12 cells that stably overexpress the GPCR S1PR1, a receptor for sphingosine 1-phosphate (S1P), to investigate the role of P-Rex1 in neuronal GPCR signalling and cell responses. We show that P-Rex1 is required for the S1P-stimulated activation of Rac1 and Akt, basal Rac3 activity, and constitutive cAMP production in PC12-S1PR1 cells. The constitutive cAMP production was not due to increased expression levels of major neuronal adenylyl cyclases, suggesting that P-Rex1 may regulate adenylyl cyclase activity. P-Rex1 was required for maintenance of neurite protrusions and spreading in S1P-stimulated PC12-S1PR1 cells, as well as for cell-cycle progression and proliferation. In summary, we identified novel functional roles of P-Rex1 in neuronal Rac, Akt and cAMP signalling, as well as in neuronal cell-cycle progression and proliferation

Reducing the risk of transmission of critical antimicrobial resistance determinants from contaminated pork products to humans in South-East Asia

Antimicrobial resistance (AMR) is a critical challenge worldwide as it impacts public health, especially via contamination in the food chain and in healthcare-associated infections. In relation to farming, the systems used, waste management on farms, and the production line process are all determinants reflecting the risk of AMR emergence and rate of contamination of foodstuffs. This review focuses on South East Asia (SEA), which contains diverse regions covering 11 countries, each having different levels of development, customs, laws, and regulations. Routinely, here as elsewhere antimicrobials are still used for three indications: therapy, prevention, and growth promotion, and these are the fundamental drivers of AMR development and persistence. The accuracy of detection of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARG) depends on the laboratory standards applicable in the various institutes and countries, and this affects the consistency of regional data. Enterobacteriaceae such as Escherichia coli and Klebsiella pneumoniae are the standard proxy species used for indicating AMR-associated nosocomial infections and healthcare-associated infections. Pig feces and wastewater have been suspected as one of the hotspots for spread and circulation of ARB and ARG. As part of AMR surveillance in a One Health approach, clonal typing is used to identify bacterial clonal transmission from the production process to consumers and patients – although to date there have been few published definitive studies about this in SEA. Various alternatives to antibiotics are available to reduce antibiotic use on farms. Certain of these alternatives together with improved disease prevention methods are essential tools to reduce antimicrobial usage in swine farms and to support global policy. This review highlights evidence for potential transfer of resistant bacteria from food animals to humans, and awareness and understanding of AMR through a description of the occurrence of AMR in pig farm food chains under SEA management systems. The latter includes a description of standard pig farming practices, detection of AMR and clonal analysis of bacteria, and AMR in the food chain and associated environments. Finally, the possibility of using alternatives to antibiotics and improving policies for future strategies in combating AMR in a SEA context are outlined