Screening for immune biomarkers associated with infection or protection against Ehrlichia ruminantium by RNA-sequencing analysis.

Heartwater is one of the most economically important tick-borne fatal diseases of livestock. The disease is caused by the bacteria Ehrlichia ruminantium transmitted by Amblyomma ticks. Although there is evidence that interferon-gamma controls E. ruminantium growth and that cellular immune responses are protective, an effective recombinant vaccine for this disease is lacking. Analyses of markers associated with infection as well as protection will lead to a better understanding of the E. ruminantium immune response and corresponding pathways induced in sheep peripheral blood mononuclear cells (PBMC) will assist in development of such a vaccine. In this study, Biomarkers of infection (BMI) were identified as uniquely expressed genes during primary infection and biomarkers of protection (BMP) associated with immune to heartwater were identified post challenge. Sheep were experimentally infected and challenged with E. ruminantium infected ticks. The immune phenotypic and transcriptome profile of their PBMC were compared to their own naïve PBMC collected before infection. The study revealed 305 differentially expressed genes (DEGs) as BMI, of these 17 were upregulated at all three time-points investigated. These DEGs, form part of the bacterial invasion of epithelial cells Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway, and others detected from day 1 post infection and are considered predictive markers for early heartwater infection in ruminants. Similarly, a total of 332 DEGs were identified as BMP, of these 100 were upregulated and 75 were downregulated at all three time-points investigated. However, at D1PC most DEGs were downregulated (n = 1312) that correlated with a reduction in the % CD4 and CD8 T cells detected with flow cytometry. KEGG pathway analyses showed complete down regulation of T cell specific pathways possibly due to homing of immune cells to the site of infection after acquired immunity developed. At D4PC, expression levels of most of these downregulated genes increased and by D6PC they were upregulated. This indicates that the sampling time-point for biomarker analyses is important when results for acquired immune responses are inferred. This data identified DEGs that could be considered as biomarkers of protective immunity that can be used for identification of vaccine antigens and provides a strong foundation to further development of heartwater recombinant vaccines.

Kinematic differences between left- and right-handed cricket fast bowlers during the bowling action.

Background: Despite differences between left- and right-handed athletes in other sports, minimal evidence exists regarding biomechanical similarities and differences between left- and right-handed cricket fast bowlers performing an equivalent task. Objectives: This study aimed to compare the kinematics between left and right-handed fast bowlers performing an equivalent task (i.e. bowling 'over the wicket' to a batter of the same handedness as the bowler). Methods: Full body, three-dimensional kinematic data for six left-handed and 20 right-handed adolescent, male, fast bowlers were collected using the Xsens inertial measurement system. Time-normalised joint and segment angle time histories from back foot contact to follow-through ground contacts were compared between groups via statistical parametric mapping. Whole movement and subphase durations were also compared. Results: Left-handed players displayed significantly more trunk flexion from 49%-56% of the total movement (ball release occurred at 54%; p = 0.037) and had shorter back foot contact durations on average (0.153 vs 0.177 s; p = 0.036) compared to right-handed players. Conclusion: Left- and right-handed bowlers displayed similar sagittal plane kinematics but appeared to use non-sagittal plane movements differently around the time of ball release. The kinematic differences identified in this study can inform future research investigating the effect of hand dominance on bowling performance and injury risk.

Predictors of 1-year survival in South African transcatheter aortic valve implant candidates.

BACKGROUND: Transcatheter aortic valve implantation (TAVI) has undergone rapid expansion internationally over the past 15 years. In view of resource constraints in developing countries, a major challenge in applying this technology lies in identifying patients most likely to benefit. The development of a risk prediction model for TAVI has proved elusive, with a reported area under the curve (AUC) of 0.6 - 0.65. The available models were developed in a First-World setting and may not be applicable to South Africa (SA). OBJECTIVES: To evaluate novel indicators and to develop a TAVI risk prediction model unique to the SA context. The current work represents the important initial steps of derivation cohort risk model development and internal validation. METHODS: Seven-year experience with 244 successive TAVI implants in three centres in Western Cape Province, SA, was used to derive risk parameters. All outcomes are reported in accordance with the Valve Academic Research Consortium definitions. Multiple preprocedural variables were assessed for their impact on 1-year survival using univariate and multivariate models. RESULTS: Factors found not to correlate with 1-year survival included age, renal function and aortic valve gradients. The commonly used surgical risk prediction models (Society of Thoracic Surgeons score and EuroSCORE) showed no correlation with outcomes. Factors found to correlate best with 1-year survival on multivariate analysis were preprocedural body mass index (BMI) (favouring higher BMI), preprocedural left ventricular end-diastolic dimension (LVED) and ejection fraction (EF) (favouring smaller LVED and higher EF), absence of atrial fibrillation, and three novel parameters: independent living, ability to drive a car, and independent food acquisition/cooking. Discriminant analysis of these factors yielded an AUC of 0.8 (95% confidence interval 0.7 - 0.9) to predict 1-year survival, with resubstitution sensitivities and specificities of 72% and 71%, respectively. CONCLUSIONS: Apart from existing predictors, we identified three novel risk predictors (independent living, ability to drive a car, and independent food acquisition/cooking) for 1-year survival in TAVI candidates. These novel parameters performed well in this early evaluation, with an AUC for predicting 1-year survival higher than the AUCs for many of the internationally derived parameters. The parameters are inexpensive and easy to obtain at the initial patient visit. If validated prospectively in external cohorts, they may be applicable to other resource-constrained environments.

Identification and characterisation of conserved epitopes of E. ruminantium that activate Th1 CD4+ T cells: Towards the development of a multi-epitope vaccine.

Several studies have shown that cytotoxic T lymphocytes (CTL) require CD4 + Th1 epitopes to generate strong immune responses to intracellular pathogens. However, not much is known about Ehrlichia ruminantium epitopes, particularly those that can be considered potential candidates for inclusion in a multi-epitope vaccine. In order to identify CD4+ Th1 epitopes that induce IFNγ, a number of proteins previously identified as immunogenic were first screened to determine if they induce cellular immunity in tick infected immune sheep PBMC. Significant IFN-γ production and other Th1 cytokines were evident for 10 recombinant proteins in all sheep tested. Secondly, peptides (n = 246) derived from the top 10 E. ruminantium vaccine candidate proteins were assayed using enzyme linked immunospot (ELISPOT) assay, quantitative real-time PCR and flow cytometry. Of the 246 peptides, 23 peptides, Erum0660 (p0660-42), Erum1150 (p1150-18, p1150-19), Erum2540 (p2540-6, p2540-16, p2540-19, p2540-20, p2540-21), Erum5420 (p5420-13, p5420-14), Erum7140 (p7140-6, p7140-7, p7140-12, p7140-13, p7140-20), Erum7320 (p7320-8, p7320-9, p7320-21), Erum7350 (p7350-9), Erum7360 (p7360-8), Erum7620 (p7620-2, p7620-12) and Erum8010 (p8010-8) were identified that stimulate the best and different cell mediated immune responses. Amino acid sequences of these peptides except for p7140-12, p7140-13, p7140-20, and p7350-9 were conserved between 13 different local strains. These peptides could efficiently induce memory CD4+ T cells to rapidly proliferate and significantly increase IFN-γ production in immune sheep PBMC. The upregulation of pro-inflammatory cytokines, which include, IL-1α, IL-2, IL-12p40, TNF-α, IFN-γ, inducible nitric oxide synthase (iNOS) and granulocyte-macrophage colony stimulating factor (GM-CSF) was also detected. Our results show that these peptides could serve as promising candidates for a multi-epitope vaccine against E. ruminantium.

Ehrlichia ruminantium antigens and peptides induce cytotoxic T cell responses in vitro.

Since CD8+ T cells play an important role in resistance to infection with heartwater, effective vaccines against this disease will likely require identification of antigens that contain CD8+ T cell epitopes responsible for cytotoxic T lymphocyte (CTL) responses. With the use of the fluorescent antigen-transfected target cell (FATT)-CTL assay, IFN-γ ELISPOT and flow cytometry, peptides that induce CTL, proliferation of CD8 + T cells and IFN-γ production were identified as possible target antigens for vaccine development. Of particular relevance was the finding that different peptides from different antigens were able to elicit varied cytotoxic activities by immune peripheral blood mononuclear cells (PBMC) from heartwater immune tick-infected sheep. Several peptides derived from Erum0660, Erum2330, Erum2540, Erum2580 and Erum5000 induced CTL in immune sheep PBMC. Peptide Erum2540-6 was the only peptide that induced significant CTL, CD8+CD45RO+ and CD8+IFN-γ+ by PBMC from all three sheep, and Erum2540 and p2540-20 induced the highest % CTL response in all three outbred sheep. These results suggest that these epitopes may be of major importance in heartwater recombinant vaccine development.

Skeletal dimensions as predictors for the shape of the nose in a South African sample: A cone-beam computed tomography (CBCT) study.

The profile of the nose is an important feature for facial approximations. Although several manual and semi-automated prediction guidelines exist for estimating the shape of the nose, the reliability and applicability of these methods to South Africans groups are unknown. The aim of this study was to predict the displacements of capulometric landmarks from hard-tissue planes to facilitate nasal soft-tissue reconstruction in a South African sample. Cone beam computed tomography (CBCT) scans of 120 adult South Africans were selected from the Oral and Dental Hospital, University of Pretoria, South Africa. Measurements involving craniometric and capulometric landmarks of the nose were obtained as plane-to-plane distances. Correlation coefficients between hard- and soft-tissue measurements were determined, and regression equations computed to assist in the prediction of the most probable shape and size of the nose. All hard- and soft-tissue measurements appeared significantly different between groups, except for the distance between the pronasale and nasion in the transverse plane and for the distance between the alare and the nasion in the coronal plane. The nasal height, nasal bone length and the nasal bone projection were significant predictors of the pronasale, subnasale and alare positions. More precisely, the nasal height and the nasal bone length were significant predictors of the pronasale position in both groups. Nasal bone projection was only useful for predicting shape in white South Africans. The variation in the skeletal predictors of the external shape of the nose noted between black and white South Africans and the results of the cross-validation testing emphasize the need for population specific guidelines.

Innate immune transcriptomic evaluation of PBMC isolated from sheep after infection with E. ruminantium Welgevonden strain.

Heartwater is a tick-borne non-infectious fatal disease of wild and domestic ruminants caused by the bacterium Ehrlichia ruminantium, transmitted by Amblyomma ticks. Although there is evidence that interferon-gamma (IFN-γ) controls E. ruminantium growth and that cellular immune responses could be protective, an effective recombinant vaccine for this disease is lacking. An overall analysis of which immune pathways are up- or down-regulated in sheep peripheral blood mononuclear cells is expected to lead to a better understanding of the global immune response of sheep to E. ruminantium infection. Therefore, a systems biology oriented approach following the infection with E. ruminantium was investigated from peripheral blood mononuclear cells to aid recombinant vaccine development. In this study, heartwater naïve sheep were infected and challenged by allowing E. ruminantium infected ticks to feed on them. After primary infection, all the animals were treated with antibiotic during the resulting febrile response. Blood was collected daily for E. ruminantium detection by qPCR (pCS20 assay). The pCS20 assay only detected the pathogen in the blood one day prior to and during the febrile stage of infection confirming infection of the sheep. IFN-γ real-time PCR indicated that this cytokine was expressed at specific time points: post infection, during the febrile stage of the disease and after challenge. These were used as a guide to select samples for transcriptome sequencing. This paper focuses on transcripts that are associated with innate activating pathways that were identified to be up- and down-regulated after primary infection and the subsequent challenge. These included the CD14 monocyte marker, toll-like receptor (TLR), nod-like receptor, chemokine, cytosolic and cytokine-cytokine interaction receptor pathways. In particular, TLR4, TLR9 and CD14 were activated together with DNA detection pathways, suggesting that vaccine formulations may be improved if CpG motifs and lipopolysaccharides are included. This data indicates that innate immune activation, perhaps by using adjuvants, should be an important component for consideration during future heartwater recombinant vaccine development.

Fatalities involving illicit drug use in Pretoria, South Africa, for the period 2003 - 2012.

BACKGROUND: Globally, illicit drugs are responsible for many fatalities annually, yet accurate data on the nature and extent of these deaths in South Africa (SA) are lacking. OBJECTIVES: To investigate the presence and profile of illicit drugs detected in deceased persons who were subjected to medicolegal autopsies and upon whom analyses were carried out in search of illicit drugs in their body fluids at the Pretoria Medico-Legal Laboratory (PMLL), SA, over a 10-year period. METHODS: A retrospective descriptive case audit was conducted for the period 2003 - 2012. RESULTS: Screening for illicit drugs was requested in 385 out of 22 566 medicolegal autopsies. Results were available for only 281 of these cases, with 154 cases showing the presence of one or more illicit drugs. The demographic profile of positive cases indicated the majority to be male (90.3%) and white (85.1%). Decedents who tested positive for illicit drugs were predominantly aged between 20 and 30 years (51.9%). The most frequently detected drug was heroin, the presence of which was confirmed in 35.2% of cases, followed by cocaine in 19.9%. Alcohol in combination with an illicit drug or drugs was detected in 56 cases (36.4%). CONCLUSIONS: Results from this study indicate that illicit drugs were implicated in a considerable number of fatalities in Pretoria. However, it is believed that the figures are a gross under-representation of the actual number of drug users who died during this period. It is therefore recommended that further research be conducted and that drug screening be requested routinely when unnatural deaths are investigated at medicolegal mortuaries, not only to ensure the administration of justice but also to obtain more accurate data for purposes of public health programmes and improve insight into the burden of illicit drug use in SA.

Flea (Ctenocephalides felis) control efficacy of topical indoxacarb on dogs subsequently bathed with a chlorhexidine-ketoconazole shampoo.

OBJECTIVE: An evaluation of the effect of chlorhexidine/ketoconazole shampoo baths on the flea control efficacy of indoxacarb applied topically to dogs. METHODS AND RESULTS: We randomly allocated 18 healthy mixed-breed dogs to 3 groups: shampoo only; indoxacarb treated and medicated shampoo; and indoxacarb treated but not shampooed. Indoxacarb was administered on day 0 and dogs were shampooed on days 9 and 23. Dogs were infested with 100 adult Ctenocephalides felis initially 2 days before treatment and then weekly from days 7 to 28. Fleas were removed and counted 48 h post-infestation. CONCLUSION: Medicated shampoo use did not significantly reduce indoxacarb efficacy against C. felis.

Identification of Ehrlichia ruminantium proteins that activate cellular immune responses using a reverse vaccinology strategy.

Ehrlichia ruminantium is an obligate intracellular bacterial pathogen which causes heartwater, a serious tick-borne disease of ruminants throughout sub-Saharan Africa. The development of promising recombinant vaccines has been reported previously, but none has been as effective as immunisation with live organisms. In this study we have used reverse vaccinology to identify proteins that elicit an in vitro cellular immune response similar to that induced by intact E. ruminantium. The experimental strategy involved four successive steps: (i) in silico selection of the most likely vaccine candidate genes from the annotated genome; (ii) cloning and expression of the selected genes; (iii) in vitro screening of the expressed proteins for their ability to induce interferon-gamma (IFN-γ) production in E. ruminantium-immune lymphocytes; and (iv) further examination of the cytokine response profiles of those lymphocytes which tested positive for IFN-γ induction. Based on their overall cytokine induction profiles the recombinant proteins were divided into four distinct groups. Eleven recombinant proteins induced a cytokine profile that was similar to the recall immune response induced by immune peripheral blood mononuclear cells (PBMC) stimulated with intact E. ruminantium. This response comprised the upregulation of cytokines associated with adaptive cellular immune responses as well as innate immunity. A successful vaccine may therefore need to contain a combination of recombinant proteins which induce both immune pathways to ensure protection against heartwater.

In vitro and in vivo evaluation of five low molecular weight proteins of Ehrlichia ruminantium as potential vaccine components.

Low molecular weight (LMW) proteins of E. ruminantium can induce proliferation of immune peripheral blood mononuclear cells (PBMCs) and the production of interferon-gamma (IFN-gamma) by CD4+-enriched T-cells. In this study, a reverse vaccinology approach was applied to identify additional vaccine candidates focusing on genes that encode LMW proteins smaller than 20 kDa. Five open reading frames (ORFs) were selected from the E. ruminantium genome and their corresponding recombinant (r) proteins were produced in a bacterial expression system. Their ability to induce proliferative responses and IFN-gamma production was evaluated in vitro using lymphocyte proliferation and ELISPOT assays. All five recombinant proteins induced proliferation of immune PBMCs and IFN-gamma production by these cells. The corresponding five genes were each individually incorporated into pCMViUBs, a mammalian expression vector and tested as a potential vaccine in sheep using a DNA prime-protein boost immunisation regimen. A cocktail of these DNA constructs protected one out of five sheep against a virulent E. ruminantium (Welgevonden) needle challenge. Three of the five vaccinated sheep showed an increase in their proliferative responses and production of IFN-gamma before challenge. This response decreased after challenge in the sheep that succumbed to the challenge and increased in the sheep that survived. This finding indicates that sustained IFN-gamma production is likely to be involved in conferring protective immunity against heartwater.

Studies of a polymorphic Ehrlichia ruminantium gene for use as a component of a recombinant vaccine against heartwater.

A previously identified polymorphic Ehrlichia ruminantium gene, Erum2510, was investigated to determine its ability to induce protective immunity in ruminants following two different DNA immunisation strategies; DNA-only and a DNA prime/recombinant protein (rprotein) boost immunisation. The DNA-only vaccine was also compared to a cocktail of three polymorphic E. ruminantium (Welgevonden) open reading frames (ORFs) adjacent to Erum2510 in the genome. Weak protection was observed in animals immunised with the pCMViUBs_Erum2510 construct alone, while none of the animals immunised with the DNA cocktail were protected. In contrast, all five animals immunised using a DNA prime/rprotein boost strategy survived challenge, thereby indicating that Erum2510 is a good candidate for inclusion in a recombinant vaccine against heartwater. One drawback of using polymorphic genes is a possible lack of cross-protection between genotypes, therefore the genetic diversity of Erum2510 was investigated to establish the degree of polymorphism among different E. ruminantium stocks. Three distinct genotypes were identified indicating that if this gene is used as a vaccine (prime/boost strategy) the vaccine should include a representative Erum2510 gene from each genotype.

Improved strategies for sequence-independent amplification and sequencing of viral double-stranded RNA genomes.

This paper reports significant improvements in the efficacy of sequence-independent amplification and quality of sequencing of viruses with segmented double-stranded RNA (dsRNA) genomes. We demonstrate that most remaining bottlenecks in dsRNA virus genome characterization have now been eliminated. Both the amplification and sequencing technologies used require no previous sequence knowledge of the viral dsRNA, there is no longer a need to separate genome segments or amplicons and the sequence-determined bias observed in cloning has been overcome. Combining very efficient genome amplification with pyrophosphate-based 454 (GS20/FLX) sequencing enabled sequencing of complete segmented dsRNA genomes and accelerated the sequence analysis of the amplified viral genomes. We report the complete consensus sequence of seven viruses from four different dsRNA virus groups, which include the first complete sequence of the genome of equine encephalosis virus (EEV), the first complete sequence of an African horsesickness virus (AHSV) genome determined directly from a blood sample and a complete human rotavirus genome determined from faeces. We also present the first comparison between the complete consensus sequence of a virulent and an attenuated strain of AHSV1. Ultra-deep sequencing (>400-fold coverage) of the AHSV1 reference and attenuated strains revealed different ratios of reassortants in the reference strain and allowed quasispecies detection in the plaque-purified attenuated strain of AHSV1. This approach amounts to a paradigm shift in dsRNA virus research, since it is sensitive and specific enough for comprehensive investigations of the evolution and genetic diversity in dsRNA virus populations.