The impact of mutation and gene conversion on the local diversification of antigen genes in African trypanosomes

Patterns of genetic diversity in parasite antigen gene families hold important information about their potential to generate antigenic variation within and between hosts. The evolution of such gene families is typically driven by gene duplication, followed by point mutation and gene conversion. There is great interest in estimating the rates of these processes from molecular sequences for understanding the evolution of the pathogen and its significance for infection processes. In this study, a series of models are constructed to investigate hypotheses about the nucleotide diversity patterns between closely related gene sequences from the antigen gene archive of the African trypanosome, the protozoan parasite causative of human sleeping sickness in Equatorial Africa. We use a hidden Markov model approach to identify two scales of diversification: clustering of sequence mismatches, a putative indicator of gene conversion events with other lower-identity donor genes in the archive, and at a sparser scale, isolated mismatches, likely arising from independent point mutations. In addition to quantifying the respective probabilities of occurrence of these two processes, our approach yields estimates for the gene conversion tract length distribution and the average diversity contributed locally by conversion events. Model fitting is conducted using a Bayesian framework. We find that diversifying gene conversion events with lower-identity partners occur at least five times less frequently than point mutations on variant surface glycoprotein (VSG) pairs, and the average imported conversion tract is between 14 and 25 nucleotides long. However, because of the high diversity introduced by gene conversion, the two processes have almost equal impact on the per-nucleotide rate of sequence diversification between VSG subfamily members. We are able to disentangle the most likely locations of point mutations and conversions on each aligned gene pair

IL-10-conditioned dendritic cells, decommissioned for recruitment of adaptive immunity, elicit innate inflammatory gene products in response to danger signals

Dendritic cells (DCs) are the professional APCs of the immune system, enabling T cells to perceive and respond appropriately to potentially dangerous microbes, while also being able to maintain T cell tolerance toward self. In part, such tolerance can be determined by IL-10 released from certain types of regulatory T cells. IL-10 has previously been shown to render DCs unable to activate T cells and it has been assumed that this process represents a general block in maturation. Using serial analysis of gene expression, we show that IL-10 pretreatment of murine bone marrow-derived DCs alone causes significant changes in gene expression. Furthermore, these cells retain the ability to respond to Toll-like receptor agonists, but in a manner skewed toward the selective induction of mediators known to enhance local inflammation and innate immunity, among which we highlight a novel CXCR2 ligand, DC inflammatory protein-1. These data suggest that, while the presence of a protolerogenic and purportedly anti-inflammatory agent such as IL-10 precludes DCs from acquiring their potential as initiators of adaptive immunity, their ability to act as initiators of innate immunity in response to Toll-like receptor signaling is enhanced

Co-receptor and co-stimulation blockade for mixed chimerism and tolerance without myelosuppressive conditioning

BACKGROUND: A major challenge in the application of marrow transplantation as a route to immunological tolerance of a transplanted organ is to achieve hematopoietic stem cell (HSC) engraftment with minimal myelosuppressive treatments. RESULTS: We here describe a combined antibody protocol which can achieve long-term engraftment with clinically relevant doses of MHC-mismatched bone marrow, without the need for myelosuppressive drugs. Although not universally applicable in all strains, we achieved reliable engraftment in permissive strains with a two-stage strategy: involving first, treatment with anti-CD8 and anti-CD4 in advance of transplantation; and second, treatment with antibodies targeting CD4, CD8 and CD40L (CD154) at the time of marrow transplantation. Long-term mixed chimerism through co-receptor and co-stimulation blockade facilitated tolerance to donor-type skin grafts, without any evidence of donor-antigen driven regulatory T cells. CONCLUSION: We conclude that antibodies targeting co-receptor and co-stimulatory molecules synergise to enable mixed hematopoietic chimerism and central tolerance, showing that neither cytoreductive conditioning nor 'megadoses' of donor bone marrow are required for donor HSC to engraft in permissive strains

Comparison of antimicrobial resistance phenotypes and genotypes in enterotoxigenic Escherichia coli isolated from Australian and Vietnamese pigs

This study aimed to compare the antibiogram phenotype and carriage of antimicrobial resistance genes (ARGs) of 97 porcine multidrug-resistant (MDR) enterotoxigenic Escherichia coli (ETEC) isolates obtained from Vietnam and 117 porcine MDR-ETEC obtained from Australia, two countries with different antimicrobial regulation systems. An antimicrobial resistance index (ARI) was calculated to quantify their potential significance to public health. Both Vietnamese and Australian isolates had moderate to high levels of resistance to commonly used antibiotics (ampicillin, tetracycline and sulphonamides). None of the Australian isolates were resistant to fluoroquinolones or third-generation cephalosporins and none possessed associated plasmid-mediated ARGs. However, 23.1% of Australian isolates were resistant to gentamicin owing to ARGs associated with apramycin or neomycin resistance [e.g. aac(3)-IV] that impart cross-resistance to gentamicin. Whilst Vietnamese isolates carried aminoglycoside ARGs, 44.4% of commercial pig isolates were resistant to gentamicin in comparison with 0% of village pig isolates. The plasmid-mediated fluoroquinolone ARG qnrB was commonly detected in Vietnamese isolates (52.3% commercial, 44.1% village), but phenotypic resistance was low (3.2% and 11.8%, respectively). The mean ARI for Vietnamese isolates (26.0) was significantly different (P < 0.001) from the mean ARI for Australian isolates (19.8), primarily reflecting fluoroquinolone resistance in the former collection. This comparison suggests the effectiveness of regulations that slow the dissemination of 'critical' resistance by restricting the availability of important classes of antimicrobials

An Optimized Protocol for Molecular Screening of Avian Pathogenic Escherichia Coli From Broiler Chickens in South East Queensland, Australia

Avian pathogenic Escherichia coli (APEC) is the causative agent of avian colibacillosis and causes localized and/or systemic infections in poultry. The presence of various virulence genes (VGs) may be a useful marker for the detection of APEC directly from fecal samples. The objectives of this study were to evaluate and compare 3 different DNA extraction methods from cloacal swabs and fecal samples of broiler chickens and determine if APEC can be detected directly from feces. The DNA extraction methods were assessed by measuring DNA yield and purity, absence of DNA shearing, 16S ribosomal DNA amplification, and reproducibility. Repeated bead beating plus column (RBB+C) was the preferred extraction method, as it yielded an adequate amount of quality DNA for PCR directly from feces. The DNA extracted from feces, with RBB+C method and DNA extracted from E. coli isolates of organs and feces, taken from 23 broiler chickens (10 healthy, 9 with colibacillosis, and 4 unhealthy with other infections), were screened with a pentaplex-PCR for the prevalence of APEC-associated VGs: iroN, ompT, iutA, iss, and hlyF. There was a statistically significant correlation between the presence of the 5 VGs in E. coli cultured from the cloaca, fecal, and organs samples from chicken affected with colibacillosis. However, screening extracted DNA from the feces for the selected VGs was not an effective diagnostic tool to detect APEC as all of the VGs were detected in the extracted fecal DNA from all chickens

Veterinary Students’ Knowledge and Perceptions About Antimicrobial Stewardship and Biosecurity—A National Survey

A better understanding of veterinary students’ perceptions, attitudes, and knowledge about antimicrobial stewardship and biosecurity could facilitate more effective education of future veterinarians about these important issues. A multicenter cross-sectional study was performed by administering a questionnaire to veterinary students expected to graduate in 2017 or 2018 in all Australian veterinary schools. Four hundred and seventy-six of 1246 students (38%) completed the survey. Many students were unaware of the high importance of some veterinary drugs to human medicine, specifically enrofloxacin and cefovecin (59% and 47% of responses, respectively). Fewer than 10% of students would use appropriate personal protective equipment in scenarios suggestive of Q fever or psittacosis. Students expected to graduate in 2018 were more likely to select culture and susceptibility testing in companion animal cases (OR 1.89, 95% CI 1.33–2.69, p < 0.001), and were more likely to appropriately avoid antimicrobials in large animal cases (OR 1.75, 95% CI 1.26–2.44, p = 0.001) than those expected to graduate in 2017. However, 2018 graduates were less likely to correctly identify the importance rating of veterinary antimicrobials for human health (OR 0.48, 95% CI 0.34–0.67, p < 0.001) than 2017 graduates. Students reported having a good knowledge of antimicrobial resistance, and combating resistance, but only 34% thought pharmacology teaching was adequate and only 20% said that teaching in lectures matched clinical teaching. Efforts need to be made to harmonize preclinical and clinical teaching, and greater emphasis is needed on appropriate biosecurity and antimicrobial stewardship

Health-related Quality of Life in Patients With Nonalcoholic Fatty Liver Disease: A Prospective Multi-center UK Study

BACKGROUND & AIMS: It is unclear whether health-related quality of life (HRQoL) is impaired in patients with nonalcoholic fatty liver disease (NAFLD) without advanced fibrosis and how this compares with the general population. We aimed to assess HRQoL in patients with NAFLD in comparison to the general population and any associations of fibrosis severity and metabolic comorbidities with impairments in HRQoL. METHODS: We prospectively enrolled 513 consecutive patients with NAFLD who completed the EuroQol 5-dimensional questionnaire (EQ-5D) and Chronic Liver Disease Questionnaires (CLDQ). Demographic and clinical information, liver biopsy results, and/or liver stiffness (LS) by transient elastography were recorded. A general population sub-cohort of the Health Survey for England 2018 was used as a comparator (n = 5483), and a 1:1 propensity-score (PS) matching was performed, according to age, sex, body mass index, and type 2 diabetes mellitus (T2DM). RESULTS: EQ-5D-5L utility was significantly lower in 466 PS-matched patients with NAFLD compared with PS-matched controls (0.77 ± 0.27 vs 0.84 ± 0.19; P < .001), even in those without advanced fibrosis (F ≤2 or LS <8kPa) (0.80 ± 0.24 vs 0.84 ± 0.19; P = .024). HRQoL measures (EQ-5D-5L, EQ-VAS, CLDQ) did not differ between patients with NAFLD with and without advanced fibrosis. LS was independently associated with lower EQ-5D-5L in all patients with NAFLD but not in those without advanced fibrosis. In the latter, lower EQ-5D-5L was associated with female sex, T2DM, and depression. CONCLUSIONS: Patients with NAFLD, even those without advanced fibrosis, have worse HRQoL compared with the general population. In patients with NAFLD without advanced fibrosis, HRQoL is independently associated with non-liver comorbidities but not LS. Multi-disciplinary management is therefore required in NAFLD, irrespective of fibrosis severity

Phylogenetic grouping, antibiotic resistance profile, fluoroquinolone susceptibility and ST131 status of canine extra intestinal Escherichia Coli isolated from submissions to a veterinary diagnostic laboratory 2005-08

Fluoroquinolones (FQs) are a recommended treatment for Escherichia coli infections in companion animals, particularly in cases of resistance to other drug classes. In a retrospective study, 162 canine clinical E. coli isolates, obtained from veterinary diagnostic submissions (January 2005 - June 2008), were analyzed for phylogenetic group and antibiogram phenotype, using nine antimicrobials and enrofloxacin, ciprofloxacin, moxifloxacin and pradofloxacin minimum inhibitory concentrations (MICs), either in the absence or presence of an efflux pump inhibitor. The isolate susceptibility distribution was bimodal; a high proportion (141/162;87%) showed a sensitivity equivalent to wildtype E. coli (enrofloxacin MIC 0.004 - 0.06 μg/mL), while a minority (4/162;2%) showed reduced susceptibility (enrofloxacin MICs of 0.125 - 0.5 μg/mL), and the remainder (17/162;10%) yielding enrofloxacin MICs in the highlevel resistance range of ≥16 μg/mL. All FQ-resistant isolates were also multidrug-resistant. The majority of FQsensitive isolates belonged to phylogenetic group B2 (101/162;62%), and the majority of resistant isolates to group D (8/17;47%). A single resistant B2 isolate and three FQ-sensitive isolates were identified as ST131. Efflux pump activity contributed significantly to MICs for all FQs, except for ciprofloxacin, which may be attributable to its higher polarity compared to the other FQs. These findings confirm a low prevalence of FQ resistance in Australian canine E. coli isolates. Detection of a high moxifloxacin: low ciprofloxacin MIC efflux-associated phenotype (102/162;63%) amongst canine strains may indicate previous exposure to moxifloxacin selective pressure, providing more evidence of exchange of E. coli strains between humans and dogs. The presence of sensitive ST131 strains in the isolate collection does suggest, however, that resistant ST131 strains could potentially emerge under both human and veterinary antimicrobial selection pressure, a risk that could be mitigated by using the most active fluoroquinolone (i.e. pradofloxacin in dogs) against wild-type E. coli at mutant prevention concentrations.Joanne L, Platell, Darren J. Trott, Heinz-Georg Wetzstein, Micheal Leitner and Rowland N. Cobbol

Non-canonical metabolic pathways in the malaria parasite detected by isotope-tracing metabolomics

The malaria parasite, Plasmodium falciparum, proliferates rapidly in human erythrocytes by actively scavenging multiple carbon sources and essential nutrients from its host cell. However, a global overview of the metabolic capacity of intraerythrocytic stages is missing. Using multiple