Phylogeny of Theileria buffeli genotypes identified in the South African buffalo (Syncerus caffer) population

Theileria buffeli/orientalis is a group of benign and mildly pathogenic species of cattle andbuffalo in various parts of the world. In a previous study, we identified T. buffeli in blood sam-ples originating from the African buffalo (Syncerus caffer) in the Hluhluwe–iMfolozi GamePark (HIP) and the Addo Elephant Game Park (AEGP) in South Africa. The aim of this studywas to characterise the 18S rRNA gene and complete internal transcribed spacer (ITS1-5.8S-ITS2) region of T. buffeli samples, and to establish the phylogenetic position of this speciesbased on these loci. The 18S rRNA gene and the complete ITS region were amplified fromDNA extracted from blood samples originating from buffalo in HIP and AEGP. The PCR prod-ucts were cloned and the resulting recombinants sequenced. We identified novel T. buffeli-like 18S rRNA and ITS genotypes from buffalo in the AEGP, and novel Theileria sinensis-like18S rRNA genotypes from buffalo in the HIP. Phylogenetic analyses indicated that the T.buffeli-like sequences were similar to T. buffeli sequences from cattle and buffalo in Chinaand India, and the T. sinensis-like sequences were similar to T. sinensis 18S rRNA sequencesof cattle and yak in China. There was extensive sequence variation between the novel T.buffeli genotypes of the African buffalo and previously described T. buffeli and T. sinensisgenotypes. The presence of organisms with T. buffeli-like and T. sinensis-like genotypes inthe African buffalo could be of significant importance, particularly to the cattle industry inSouth Africa as these animals might act as sources of infections to naïve cattle. This is thefirst report on the characterisation of the full-length 18S rRNA gene and ITS region of T.buffeli and T. sinensis genotypes in South Africa. Our study provides invaluable informationtowards the classification of this complex group of benign and mildly pathogenic species.South African National Research Foundation (NRFICD2006072000009) and UP Research Development Programme.http://www.elsevier.com/locate/vetparhb201

Sequence variation identified in the 18S rRNA gene of Theileria mutans and Theileria velifera from the African buffalo (Syncerus caffer)

The African buffalo (Syncerus caffer) is a natural reservoir host for both pathogenic and nonpathogenic Theileria species. These often occur naturally as mixed infections in buffalo. Although the benign and mildly pathogenic forms do not have any significant economic importance, their presence could complicate the interpretation of diagnostic test results aimed at the specific diagnosis of the pathogenic T. parva in cattle and buffalo in South Africa. The 18S rRNA gene has been used as the target in a quantitative real-time PCR (qPCR) assay for the detection of T. parva infections. However, the extent of sequence variation within this gene in the non-pathogenic Theileria spp. of the Africa buffalo is not well known. The aim of this study was, therefore, to characterize the full-length 18S rRNA genes of T. mutans, Theileria sp. (strain MSD) and T. velifera and to determine the possible influence of any sequence variation on the specific detection of T. parva using the 18S rRNA qPCR. The reverse line blot (RLB) hybridization assay was used to select samples which either tested positive for several different Theileria spp., or which hybridized only with the Babesia/Theileria genus-specific probe and not with any of the Babesia or Theileria speciesspecific probes. The full-length 18S rRNA genes from 14 samples, originating from 13 buffalo and one bovine from different localities in South Africa, were amplified, cloned and the resulting recombinants sequenced. Variations in the 18S rRNA gene sequences were identified in T. mutans, Theileria sp. (strain MSD) and T. velifera, with the greatest diversity observed amongst the T. mutans variants. This variation possibly explained why the RLB hybridization assay failed to detect T. mutans and T. velifera in some of the analysed samples.South African National Research Foundation (NRF ICD2006072000009) and UP Research Development Programme.http://www.elsevier.com/locate/vetparhb2013ab201

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.The South African Department of Agriculture OV9/23/C167 grant and the FP6 EU INCO-DEV EPIGENEVAC FP6-003713 grant.http://www.elsevier.com/locate/vetimmab201

Selection and evaluation of housekeeping genes as endogenous controls for quantification of mRNA transcripts in Theileria parva using quantitative real-time polymerase chain reaction (qPCR)

The reliability of any quantitative real-time polymerase chain reaction (qPCR) experiment can be seriously compromised by variations between samples as well as between PCR runs. This usually result from errors in sample quantification, especially with samples that are obtained from different individuals and tissues and have been collected at various time intervals. Errors also arise from differences in qPCR efficiency between assays performed simultaneously to target multiple genes on the same plate. Consequently, the derived quantitative data for the target genes become distorted. To avoid this grievous error, an endogenous control, with relatively constant transcription levels in the target individual or tissue, is included in the qPCR assay to normalize target gene expression levels in the analysis. Several housekeeping genes (HKGs) have been used as endogenous controls in quantification studies of mRNA transcripts; however, there is no record in the literature of the evaluation of these genes for the tick-borne protozoan parasite, Theileria parva. Importantly, the expression of these genes should be invariable between different T. parva stocks, ideally under different experimental conditions, to gain extensive application in gene expression studies of this parasite. Thus, the expression of several widely used HKGs was evaluated in this study, including the genes encoding β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 28S rRNA, cytochrome b and fructose-2.6-biphosphate aldolase (F6P) proteins. The qPCR analysis revealed that the expression of genes encoding cytochrome b, F6P and GAPDH varied considerably between the two T. parva stocks investigated, the cattlederived T. parva Muguga and the buffalo-derived T. parva 7014. 28S rRNA and β-actin gene expression was the most stable; thus, these genes were considered suitable candidates to be used as endogenous control genes for mRNA quantification studies in T. parva.This work was funded by the Genomics Research Institute of the University of Pretoria, (http://www.up.ac.za/the-genomics-research- institute/home) to KPS-M.http://www.plosone.orgam2018Veterinary Tropical Disease

Molecular detection and phylogenetic analysis of Anaplasma marginale and Anaplasma centrale amongst transhumant cattle in north-eastern Uganda

There is little molecular data from Anaplasma marginale and Anaplasma centrale isolates from cattle in Uganda. Between November 2013 and January 2014, blood was collected from 240 cattle in 20 randomly-selected herds in two districts of the Karamoja Region in north-eastern Uganda. A duplex quantitative real-time polymerase chain reaction (qPCR) assay was used to detect and determine the prevalence of A. marginale (targeting the msp1β gene) and A. centrale (targeting the groEL gene). The qPCR assay revealed that most cattle (82.9%; 95% confidence interval [CI] 78.2–87.7%) were positive for A. marginale DNA, while fewer cattle (12.1%; 95% CI 7.9–16.2%) were positive for A. centrale DNA. A mixed effects logistic regression model showed that the age of cattle was significantly associated with A. centrale infection, while the prevalence of A. marginale varied significantly according to locality. The near full-length 16S ribosomal RNA (16S rRNA) gene and the heat shock protein gene, groEL, for both Anaplasma species were amplified from a selection of samples. The amplicons were cloned and the resulting recombinants sequenced. We found three novel A. marginale 16S rRNA variants, seven A. marginale groEL gene sequence variants and two A. centrale groEL gene sequence variants. Phylogenetic trees were inferred from sequence alignments of the 16S rRNA gene and GroEL amino acid sequences determined here and published sequences using maximum likelihood, Bayesian inference and parsimony methods Phylogenetic analyses classified the 16S rRNA gene and GroEL amino acid sequences into one clade for A. marginale and a separate clade for A. centrale. This study reveals a high prevalence and sequence variability of A. marginale and A. centrale, and is the first report on the phylogenetic characterisation of A. marginale and A. centrale from cattle in Uganda using molecular markers. Sequence variation can be attributed to mobile pastoralism, communal grazing and grazing with wildlife. These data support future epidemiological investigations for bovine anaplasmosis in Uganda.The National Agricultural Research Organisation (NARO), Uganda (P.109224) and University of Pretoria, South Africa (Postgraduate bursary 13399650).http://www.elsevier.com/locate/ttbdis2019-03-01hj2018Veterinary Tropical Disease

The XMM Cluster Survey: Evidence for energy injection at high redshift from evolution of the X-ray luminosity-temperature relation

We measure the evolution of the X-ray luminosity-temperature (L_X-T) relation since z~1.5 using a sample of 211 serendipitously detected galaxy clusters with spectroscopic redshifts drawn from the XMM Cluster Survey first data release (XCS-DR1). This is the first study spanning this redshift range using a single, large, homogeneous cluster sample. Using an orthogonal regression technique, we find no evidence for evolution in the slope or intrinsic scatter of the relation since z~1.5, finding both to be consistent with previous measurements at z~0.1. However, the normalisation is seen to evolve negatively with respect to the self-similar expectation: we find E(z)^{-1} L_X = 10^{44.67 +/- 0.09} (T/5)^{3.04 +/- 0.16} (1+z)^{-1.5 +/- 0.5}, which is within 2 sigma of the zero evolution case. We see milder, but still negative, evolution with respect to self-similar when using a bisector regression technique. We compare our results to numerical simulations, where we fit simulated cluster samples using the same methods used on the XCS data. Our data favour models in which the majority of the excess entropy required to explain the slope of the L_X-T relation is injected at high redshift. Simulations in which AGN feedback is implemented using prescriptions from current semi-analytic galaxy formation models predict positive evolution of the normalisation, and differ from our data at more than 5 sigma. This suggests that more efficient feedback at high redshift may be needed in these models.Comment: Accepted for publication in MNRAS; 12 pages, 6 figures; added references to match published versio

Protection against heartwater by DNA immunisation with four Ehrlichia ruminantium open reading frames

We have reported previously that a recombinant DNA vaccine consisting of four Ehrlichia ruminantium (Welgevonden) open reading frames (ORFs) known as the 1H12 cocktail provided protection against a virulent E. ruminantium (Welgevonden) needle challenge in sheep. In this study, we have investigated the vaccine effectiveness of two other cocktails of E. ruminantium (Welgevonden) ORFs, as well as single ORFs from the 1H12 cocktail, to protect sheep against a virulent needle challenge with the homologous strain. Each individual 1H12 ORF provided protection, but all the animals vaccinated with the other cocktails succumbed to the challenge.The authors thank Stephen Johnston (University of Texas Southwestern Medical Center) for supplying the pCMViUBs vector and Dr. Erich Zweygarth and Antoinette Josemans (ARC-Onderstepoort Veterinary Institute, SA) for providing E. ruminantium tissue culture material for DNA extraction. This work was supported by the South African Department of Science and Technology LEAD biotechnology grant and the EU INCO-ICA4-CT-2000-30026 grant

Cowdria ruminantium DNA is unstable in a SuperCos1 library

A Cowdria ruminantium genomic library was constructed in a cosmid vector to serve as a source of easily accessible and pure C. ruminantium DNA for molecular genetic studies. The cosmid library contained 846 clones which were arrayed into microtitre plates. Restriction enzyme digestion patterns indicated that these clones had an average insert size of 35 kb. Probing of the arrays did not detect any bovine clones and only one of the known C. ruminantium genes, pCS20, was detected. Due to the high AT content and the fact that C. ruminantium genes are active in the Escherichia coli host, the C. ruminantium clones were unstable in the SuperCos 1 vector and most clones did not grow reproducibly. The library was contaminated with E. coli clones and these clones were maintained with greater fidelity than the C. ruminantium clones, resulting in a skewed representation over time. We have isolated seven C. ruminantium clones which we were able to serially culture reproducibly; two of these clones overlap. These clones constitute the first large regions of C. ruminantium DNA to be cloned and represent almost 10% of the C. ruminantium genome.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. Adobe Acrobat v.9 was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.mn201

Occurrence of blood-borne tick-transmitted parasites in common tsessebe (Damaliscus lunatus) antelope in Northern Cape Province, South Africa

Blood samples were collected from 71 tsessebes relocated from the deproclaimed Vaalbos National Park to Mokala National Park, South Africa. DNA was extracted from the samples and the reverse line blot (RLB) hybridization technique was used to detect and identify any haemoparasites present. Six samples hybridized to the Theileria/Babesia genus-specific probe, the Theileria genus-specific probe and the Theileria sp. (sable) probe, while 3/6 also hybridized to the Theileria separata probe. Full-length 18S rRNA genes of the Theileria spp. detected were amplified, cloned and sequenced. Two novel Theileria 18S rRNA gene sequences were identified which are phylogenetically very closely related to both Theileria sp. (sable) and T. separata. All animals appeared to be in good health. It seems likely, therefore, that these Theileria spp. do not cause disease under normal circumstances. Nevertheless, care should be taken when translocating wild animals, as introduction of novel piroplasm parasites into new areas could cause clinical disease and losses in naïve wildlife and domestic animals, and new parasite species could become established in areas in which they previously did not occur.National Research Foundation grant UID 44403 to B.L. Penzhorn.http://www.elsevier.com/locate/vetparab201

Right at home: living with dementia and multi-morbidities

Dementia is recognised as the biggest health crisis of our time in terms of high personal and social costs and wider impact on health and social care systems. Increases in people living with dementia and multimorbidities presents critical challenges for homecare worldwide. Healthcare systems struggle to provide adequate home-care services, delivering limited care restricted to a single-condition focus. This study explored the experiences and expectations of homecare from the multiple perspectives of people living with dementia and multimorbidities and homecare workers providing support. Findings draw from qualitative semi-structured interviews with people with dementia (n=2), their partners (n=2), other partners or family carers (n=6) and homecare workers (n=26). Three themes are identified: (a) the preference for and value of home; (b) inadequate homecare provision and enhanced care-burden; (c) limited training and education. Despite continued calls for homecare investment, the focus on reduction in costs hides key questions and further dialogue required exploring how people with dementia can be supported to live independently and flourish at-home. This study considers these complex experiences and care requirements through the prism of disability and human rights frameworks. This paper concludes with consideration of more recent human social rights debate. We critically discuss what this may mean for people living with dementia and consider the implications for corequisite policy development to optimise available homecare support. Keywords: dementia, multimorbidities, homecare, independent-living, social right