Absolute quantification of the host-to-parasite DNA ratio in Theileria parva-infected lymphocyte cell lines

Theileria parva is a tick-transmitted intracellular apicomplexan pathogen of cattle in sub-Saharan Africa that causes East Coast fever (ECF). ECF is an acute fatal disease that kills over one million cattle annually, imposing a tremendous burden on African small-holder cattle farmers. The pathology and level of T. parva infections in its wildlife host, African buffalo (Syncerus caffer), and in cattle are distinct. We have developed an absolute quantification method based on quantitative PCR (qPCR) in which recombinant plasmids containing single copy genes specific to the parasite (apical membrane antigen 1 gene, ama1) or the host (hypoxanthine phosphoribosyltransferase 1, hprt1) are used as the quantification reference standards. Our study shows that T. parva and bovine cells are present in similar numbers in T. parva-infected lymphocyte cell lines and that consequently, due to its much smaller genome size, T. parva DNA comprises between 0.9% and 3% of the total DNA samples extracted from these lines. This absolute quantification assay of parasite and host genome copy number in a sample provides a simple and reliable method of assessing T. parva load in infected bovine lymphocytes, and is accurate over a wide range of host-to-parasite DNA ratios. Knowledge of the proportion of target DNA in a sample, as enabled by this method, is essential for efficient high-throughput genome sequencing applications for a variety of intracellular pathogens. This assay will also be very useful in future studies of interactions of distinct host-T. parva stocks and to fully characterize the dynamics of ECF infection in the field

The impact of within-herd genetic variation upon inferred transmission trees for foot-and-mouth disease virus

Full-genome sequences have been used to monitor the fine-scale dynamics of epidemics caused by RNA viruses. However, the ability of this approach to confidently reconstruct transmission trees is limited by the knowledge of the genetic diversity of viruses that exist within different epidemiological units. In order to address this question, this study investigated the variability of 45 foot-and-mouth disease virus (FMDV) genome sequences (from 33 animals) that were collected during 2007 from eight premises (10 different herds) in the United Kingdom. Bayesian and statistical parsimony analysis demonstrated that these sequences exhibited clustering which was consistent with a transmission scenario describing herd-to-herd spread of the virus. As an alternative to analysing all of the available samples in future epidemics, the impact of randomly selecting one sequence from each of these herds was used to assess cost-effective methods that might be used to infer transmission trees during FMD outbreaks. Using these approaches, 85% and 91% of the resulting topologies were either identical or differed by only one edge from a reference tree comprising all of the sequences generated within the outbreak. The sequence distances that accrued during sequential transmission events between epidemiological units was estimated to be 4.6 nucleotides, although the genetic variability between viruses recovered from chronic carrier animals was higher than between viruses from animals with acute-stage infection: an observation which poses challenges for the use of simple approaches to infer transmission trees. This study helps to develop strategies for sampling during FMD outbreaks, and provides data that will guide the development of further models to support control policies in the event of virus incursions into FMD free countries

Barro's fertility equations: the robustness of the role of female education and income

Barro and Lee (1994) and Barro and Sala-i-Martin (1995) find that real per-capita GDP and both male and female education have important effects on fertility in their cross-country empirical studies. In order to assess the robustness of their results, their estimated models are subjected to specification and diagnostic testing, the effects on the model of using the improved Barro and Lee (1996) cross-country data on educational attainment of the population aged 15 and over are examined, and the different specifications used by Barro and Lee and by Barro and Sala-i-Martin compared. The results obtained suggest that their fertility equations do not perform well in terms of diagnostic testing, and are very sensitive to the use of different vintages of the educational attainment proxies and of the Summers-Heston cross-country income data. A robust explanation of fertility, to link with empirical growth equations, has, therefore, not yet been found; further work is required in this area

TNFα and GM-CSF-induced activation of the CAEV promoter is independent of AP-1

AbstractCaprine arthritis encephalitis virus transcription is under the control of the viral promoter within the long terminal repeat. Previous studies with the closely related maedi visna lentivirus have indicated that viral transcription is dependent upon the AP-1 transcription factor. Other studies have indicated a potential role for the cytokines TNFα and GM-CSF in CAEV pathogenesis by increasing viral loads in infected tissues. The hypotheses that AP-1 transcription factors are necessary for transcriptional activation of the CAEV promoter and that CAEV transcriptional activation results from treatment with the cytokines GM-CSF and TNFα were tested with a stably transduced U937 cell line. Here, we found that TNFα and GM-CSF activated CAEV transcription in U937 cells. However, this activation effect was not blocked by SP600125, an inhibitor of Jun N-terminal kinase. SP600125 effectively prevented Jun phosphorylation in cells subsequently treated with cytokines. The cytokines TNFα and GM-CSF therefore activate CAEV transcription, and this effect occurs independently of AP-1. A set of progressive deletion mutants was utilized to show that TNFα-induced expression depends on an element or elements within the U3 70-bp repeat

CD21-Positive Follicular Dendritic Cells A Possible Source of PrP\u3csup\u3eSc\u3c/sup\u3e in Lymph Node Macrophages of Scrapie-Infected Sheep

Natural sheep scrapie is a prion disease characterized by the accumulation of PrPSc in brain and lymphoid tissues. Previous studies suggested that lymph node macrophages and follicular dendritic cells (FDC) accumulate PrPSc. In this study, lymph nodes were analyzed for the presence of PrPSc and macrophage or FDC markers using dual immunohistochemistry. A monoclonal antibody (mAb) to the C-terminus of PrP reacted with CD172a+ macrophages and CD21+ FDC processes in secondary follicles. However, a PrP N-terminus- specific mAb reacted with CD21+ FDC processes but not CD172a+ macrophages in secondary follicles. Neither the PrP N-terminus nor C-terminus-specific mAb reacted with CD172a+ macrophages in the medulla. These results indicate that lymph node follicular macrophages acquire PrPSc by phagocytosis of CD21+ FDC processes. The results also suggest that follicular macrophages have proteases that process full-length PrPSc to N-terminally truncated PrPSc

CD21-Positive Follicular Dendritic Cells A Possible Source of PrP\u3csup\u3eSc\u3c/sup\u3e in Lymph Node Macrophages of Scrapie-Infected Sheep

Natural sheep scrapie is a prion disease characterized by the accumulation of PrPSc in brain and lymphoid tissues. Previous studies suggested that lymph node macrophages and follicular dendritic cells (FDC) accumulate PrPSc. In this study, lymph nodes were analyzed for the presence of PrPSc and macrophage or FDC markers using dual immunohistochemistry. A monoclonal antibody (mAb) to the C-terminus of PrP reacted with CD172a+ macrophages and CD21+ FDC processes in secondary follicles. However, a PrP N-terminus- specific mAb reacted with CD21+ FDC processes but not CD172a+ macrophages in secondary follicles. Neither the PrP N-terminus nor C-terminus-specific mAb reacted with CD172a+ macrophages in the medulla. These results indicate that lymph node follicular macrophages acquire PrPSc by phagocytosis of CD21+ FDC processes. The results also suggest that follicular macrophages have proteases that process full-length PrPSc to N-terminally truncated PrPSc

PrP-C and PrP-Sc at the Fetal-Maternal Interface

Scrapie is a naturally occurring prion (PrP) disease causing a fatal neurodegenerative disorder in sheep and goats. Previous studies suggest that scrapie is transmitted naturally through exposure to the scrapie agent in wasted placentas of infected ewes. This study determined the distribution and biochemical properties of PrP cellular (PrP-C) and the distribution of PrP scrapie (PrP-Sc) in reproductive, placental, and selected fetal tissues and fetal fluids in sheep. Glycosylated, N-terminally truncated, proteinase K-sensitive PrP-C with apparent molecular masses of 23–37 kDa was present in reproductive, placental, and fetal tissues and fetal fluids. PrP-C was low or undetectable in intercotyledonary chorioallantois, amnion, urachus, amniotic fluid, and fetal urine. In pregnant ewes, cotyledonary chorioallantois, allantoic fluid, and caruncular endometrium contained higher levels of PrP-C than did intercaruncular endometrium, myometrium, oviduct, ovary, fetal bladder, or fetal kidney. Caruncular endometrial PrP-C was up-regulated during pregnancy. Despite the wide distribution of PrP-C in reproductive, placental, and selected fetal tissues and fetal fluid, PrP-Sc was detected only in caruncular endometrium and cotyledonary chorioallantois of pregnant scrapie-infected ewes. The embryo/fetus may not be exposed to scrapie in utero because it is separated physically from PrP-positive allantois and chorioallantois by PrP-negative amnion

BoHV-4-based vector delivering Ebola virus surface glycoprotein

Background: Ebola virus (EBOV) is a Category A pathogen that is a member of Filoviridae family that causes hemorrhagic fever in humans and non-human primates. Unpredictable and devastating outbreaks of disease have recently occurred in Africa and current immunoprophylaxis and therapies are limited. The main limitation of working with pathogens like EBOV is the need for costly containment. To potentiate further and wider opportunity for EBOV prophylactics and therapies development, innovative approaches are necessary. Methods: In the present study, an antigen delivery platform based on a recombinant bovine herpesvirus 4 (BoHV-4), delivering a synthetic EBOV glycoprotein (GP) gene sequence, BoHV-4-syEBOVgD106TK, was generated. Results: EBOV GP was abundantly expressed by BoHV-4-syEBOVgD106TK transduced cells without decreasing viral replication. BoHV-4-syEBOVgD106TK immunized goats produced high titers of anti-EBOV GP antibodies and conferred a long lasting (up to 6 months), detectable antibody response. Furthermore, no evidence of BoHV-4-syEBOVgD106TK viremia and secondary localization was detected in any of the immunized animals. Conclusions: The BoHV-4-based vector approach described here, represents: an alternative antigen delivery system for vaccination and a proof of principle study for anti-EBOV antibodies generation in goats for potential immunotherapy applications

Development and evaluation of tailored specific real-time RT-PCR assays for detection of foot-and-mouth disease virus serotypes circulating in East Africa

AbstractRapid, reliable and accurate diagnostic methods provide essential support to programmes that monitor and control foot-and-mouth disease (FMD). While pan-specific molecular tests for FMD virus (FMDV) detection are well established and widely used in endemic and FMD-free countries, current serotyping methods mainly rely either on antigen detection ELISAs or nucleotide sequencing approaches. This report describes the development of a panel of serotype-specific real-time RT-PCR assays (rRT-PCR) tailored to detect FMDV lineages currently circulating in East Africa. These assays target sequences within the VP1-coding region that share high intra-lineage identity, but do not cross-react with FMD viruses from other serotypes that circulate in the region. These serotype-specific assays operate with the same thermal profile as the pan-diagnostic tests making it possible to run them in parallel to produce CT values comparable to the pan-diagnostic test detecting the 3D-coding region. These assays were evaluated alongside the established pan-specific molecular test using field samples and virus isolates collected from Tanzania, Kenya and Ethiopia that had been previously characterised by nucleotide sequencing. Samples (n=71) representing serotype A (topotype AFRICA, lineage G-I), serotype O (topotypes EA-2 and EA-4), serotype SAT 1 (topotype I (NWZ)) and serotype SAT2 (topotype IV) were correctly identified with these rRT-PCR assays. Furthermore, FMDV RNA from samples that did not contain infectious virus could still be serotyped using these assays. These serotype-specific real-time RT-PCR assays can detect and characterise FMDVs currently circulating in East Africa and hence improve disease control in this region