The haematological, proinflammatory cytokines and IgG changes during an ovine experimental theileriosis

Malignant ovine theileriosis is caused by Theileria lestoquardi, which is highly pathogenic in sheep. Theileriosis involves different organs in ruminants. Little is known about the role of proinflammatory cytokines in the pathogenesis of T. lestoquardi infection. The aim of this study was to measure concentration changes of proinflammatory cytokines and immunoglobulin G (IgG) during an ovine experimental theileriosis and correlate it with clinical and haematological parameters. During an experimental study, seven healthy Baluchi sheep (four females and three males) about 6–8 months old were infected with T. lestoquardi by feeding of infected unfed ticks on the sheep’s ears. The infected sheep were clinically examined during the study and blood samples were collected on days 0, 2, 5, 7, 10, 12, 14, 17 and 21. The haematological parameters were analysed by an automatic veterinary haematology cell counter and the inflammatory cytokines interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and IgG were measured by enzyme-linked immunosorbent assay. All infected sheep had temperatures above 40 °C on days 3–4 post infection (PI). The maximum temperature was noted on day 7, and it remained high until day 21. The parasitaemia of T. lestoquardi infection increased from 0.01% (day 7 PI) to 3.3% (day 21 PI). The mean white blood cell (WBC), red blood cell (RBC), lymphocyte, neutrophil and platelet values slightly increased on day 2 PI and decreased by day 17 and day 21 PI. The percentage parasitaemia and fever had a negative correlation with the numbers of WBCs, RBCs, lymphocytes, neutrophils and platelets. The serum concentration of IL-6, TNF-α and IFN-γ cytokines increased and peaked on day 12 and thereafter decreased to levels lower than 0. Out of all tested cytokines, the concentration of IL-6 was significantly higher, as early as day 2 PI. No significant changes were observed for the IgG levels during the course of disease. A significant and strong correlation was observed between IL-6, TNF-α and IFN-γ values and a moderate correlation between IL-6 and the numbers of lymphocytes in the present study. A strong correlation was determined between the percentage parasitaemia and haematological parameters in T. lestoquardi-infected sheep. In addition, preliminary results indicate that the measurement of the serum concentrations of IL-6 in combination with haematological parameters could be considered a good marker to estimate the pathogenicity of T. lestoquardi strain

Dual RNA-seq to catalogue host and parasite gene expression changes associated with virulence of T. annulata-transformed bovine leukocytes: towards identification of attenuation biomarkers

The apicomplexan parasite Theileria annulata is transmitted by Hyalomma ticks and causes an acute lymphoproliferative disease that is invariably lethal in exotic cattle breeds. The unique ability of the schizont stage of T. annulata to transform infected leukocytes to a cancer-like phenotype and the simplicity of culturing and passaging T. annulata-transformed cells in vitro have been explored for live vaccine development by attenuating the transformed cells using lengthy serial propagation in vitro. The empirical in vivo evaluation of attenuation required for each batch of long-term cultured cells is a major constraint since it is resource intensive and raises ethical issues regarding animal welfare. As yet, the molecular mechanisms underlying attenuation are not well understood. Characteristic changes in gene expression brought about by attenuation are likely to aid in the identification of novel biomarkers for attenuation. We set out to undertake a comparative transcriptome analysis of attenuated (passage 296) and virulent (passage 26) bovine leukocytes infected with a Tunisian strain of T. annulata termed Beja. RNA-seq was used to analyse gene expression profiles and the relative expression levels of selected genes were verified by real-time quantitative PCR (RT-qPCR) analysis. Among the 3538 T. annulata genes analysed, 214 were significantly differentially expressed, of which 149 genes were up-regulated and 65 down-regulated. Functional annotation of differentially expressed T. annulata genes revealed four broad categories of metabolic pathways: carbon metabolism, oxidative phosphorylation, protein processing in the endoplasmic reticulum and biosynthesis of secondary metabolites. It is interesting to note that of the top 40 genes that showed altered expression, 13 were predicted to contain a signal peptide and/or at least one transmembrane domain, suggesting possible involvement in host-parasite interaction. Of the 16,514 bovine transcripts, 284 and 277 showed up-regulated and down-regulated expression, respectively. These were assigned to functional categories relevant to cell surface, tissue morphogenesis and regulation of cell adhesion, regulation of leucocyte, lymphocyte and cell activation. The genetic alterations acquired during attenuation that we have catalogued herein, as well as the accompanying in silico functional characterization, do not only improve understanding of the attenuation process, but can also be exploited by studies aimed at identifying attenuation biomarkers across different cell lines focusing on some host and parasite genes that have been highlighted in this study, such as bovine genes (CD69, ZNF618, LPAR3, and APOL3) and parasite genes such as TA03875

miR-34c-3p Regulates Protein Kinase A Activity Independent of cAMP by Dicing prkar2b Transcripts in Theileria annulata-Infected Leukocytes

MicroRNAs (miRNAs) are small noncoding RNAs that can play critical roles in regulating various cellular processes, including during many parasitic infections. Here, we report a regulatory role for miR-34c-3p in cAMP-independent regulation of host cell protein kinase A (PKA) activity in Theileria annulata-infected bovine leukocytes. We identified prkar2b (cAMP-dependent protein kinase A type II-beta regulatory subunit) as a novel miR-34c-3p target gene and demonstrate how infection-induced upregulation of miR-34c-3p repressed PRKAR2B expression to increase PKA activity. As a result, the disseminating tumorlike phenotype of T. annulata-transformed macrophages is enhanced. Finally, we extend our observations to Plasmodium falciparum-parasitized red blood cells, where infection-induced augmentation in miR-34c-3p levels led to a drop in the amount of prkar2b mRNA and increased PKA activity. Collectively, our findings represent a novel cAMP-independent way of regulating host cell PKA activity in infections by Theileria and Plasmodium parasites. IMPORTANCE Small microRNA levels are altered in many diseases, including those caused by parasites. Here, we describe how infection by two important animal and human parasites, Theileria annulata and Plasmodium falciparum, induce changes in infected host cell miR-34c-3p levels to regulate host cell PKA kinase activity by targeting mammalian prkar2b. Infection-induced changes in miR-34c-3p levels provide a novel epigenetic mechanism for regulating host cell PKA activity independent of fluxes in cAMP to both aggravate tumor dissemination and improve parasite fitness

Theileria highjacks JNK2 into a complex with the macroschizont GPI-anchored surface protein p104.

Constitutive JNK activity characterizes bovine T and B cells infected with Theileria parva, and B cells and macrophages infected with T. annulata. Here, we show that T. annulata infection of macrophages manipulates JNK activation by recruiting JNK2 and not JNK1 to the parasite surface, whereas JNK1 is found predominantly in the host cell nucleus. At the parasite's surface JNK2 forms a complex with p104 a GPI-anchored T. annulata plasma membrane protein. Sequestration of JNK2 depended on PKA-mediated phosphorylation of a JNK-binding motif common to T. parva and a cell penetrating peptide harbouring the conserved p104 JNK-binding motif competitively ablated binding, whereupon liberated JNK2 became ubiquitinated and degraded. Cytosolic sequestration of JNK2 suppressed small mitochondrial ARF-mediated autophagy, whereas it sustained nuclear JNK1 levels, c-Jun phosphorylation and matrigel traversal. Therefore, T. annulata sequestration of JNK2 contributes to both survival and dissemination of Theileria-transformed macrophages

Rapid and Specific Action of Methylene Blue against Plasmodium Transmission Stages

Methylene blue (MB) is the oldest synthetic anti-infective. Its high potency against asexual and sexual stages of malaria parasites is well documented. This study aimed to investigate possible additional activities of MB in interfering with parasite transmission and determine target stages in Anopheles vectors and humans. MB’s transmission-blocking activity was first evaluated by an ex vivo direct membrane feeding assay (DMFA) using Plasmodium falciparum field isolates. To investigate anti-mosquito stage activity, Plasmodium berghei-infected Anopheles stephensi mosquitoes were fed a second blood meal on mice that had been treated with methylene blue, 3, 6- and 15-days after the initial infectious blood meal. Anti-sporozoite and liver stage activities were evaluated in vitro and in vivo via sporozoite invasion and liver stage development assays, respectively. MB exhibited a robust inhibition of P. falciparum transmission in An. gambiae, even when added shortly before the DMFA but only a moderate effect against P. berghei oocyst development. Exposure of mature P. berghei and P. falciparum sporozoites to MB blocked hepatocyte invasion, yet P. berghei liver stage development was unaffected by MB. Our results indicate previously underappreciated rapid specific activities of methylene blue against Plasmodium transmission stages, preventing the establishment of both mosquito midgut and liver infections as the first essential steps in both hosts

Interaction between transforming Theileria parasites and their host bovine leukocytes.

Theileria are tick-transmitted parasites that cause often fatal leuko-proliferative diseases in cattle called tropical theileriosis (T. annulata) and East Coast fever (T. parva). However, upon treatment with anti-theilerial drug-transformed leukocytes die of apoptosis indicating that Theileria-induced transformation is reversible making infected leukocytes a powerful example of how intracellular parasites interact with their hosts. Theileria-transformed leukocytes disseminate throughout infected cattle causing a cancer-like disease and here, we discuss how cytokines, noncoding RNAs and oncometabolites can contribute to the transformed phenotype and disease pathology

Establishment of an Artificial Tick Feeding System to Study <i>Theileria lestoquardi</i> Infection

<div><p>The establishment of good experimental models for <i>Theileria</i> sp. infection is important for theileriosis research. Routinely, infection of ticks is accomplished by feeding on parasite-infected animals (sheep, cows and horses), which raises practical and ethical problems, driving the search for alternative methods of tick infection. Artificial tick feeding systems are based mainly on rearing ticks on host-derived or hand-made artificial membranes. We developed a modified feeding assay for infecting nymphal stages of <i>Hyalomma anatolicum</i> ticks with <i>Theileria lestoquardi</i>, a highly pathogenic parasite of sheep. We compared two different membranes: an artificial silicone membrane and a natural alternative using mouse skin. We observed high attachment rates with mouse skin, whereas <i>in vitro</i> feeding of <i>H</i>. <i>anatolicum</i> nymphs on silicone membranes was unsuccessful. We could infect <i>H</i>. <i>anatolicum</i> nymphs with <i>T</i>. <i>lestoquardi</i> and the emerging adult ticks transmitted infective parasites to sheep. In contrast, similar infections with <i>Rhipicephalus bursa</i>, a representative tick with short mouth-parts that was proposed as a vector for <i>T</i>. <i>lestoquardi</i>, appeared not to be a competent vector tick species. This is the first report of an experimentally controlled infection of <i>H</i>. <i>anatolicum</i> with <i>T</i>. <i>lestoquardi</i> and opens avenues to explore tick-parasite dynamics in detail.</p></div

<i>In vitro</i> tick feeding and detection of <i>T</i>. <i>lestoquardi</i> in both animal and tick samples.

<p>(A) <i>H</i>. <i>anatolicum</i> nymphs clustering on the mouse skin membrane on day 6 post-tick-infestation of a small feeding unit (SFU). A small amount of blood leak (*) was observed, but was controlled by application of filter papers. (B) 3-day fed <i>R</i>. <i>bursa</i>. (C) Agarose gel electrophoresis of <i>T</i>. <i>lestoquardi</i>-specific PCR products from sheep blood samples and adult tick salivary glands. To avoid repetition, data from selected batches of ticks are presented. Positive signals were detected for the initial donor sheep and also for the two experimentally infected sheep. The alphabet letters in parenthesis in front of the samples refers to the experiments defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169053#pone.0169053.g001" target="_blank">Fig 1</a>. All <i>H</i>. <i>anatolicum</i> ticks fed either naturally or artificially from infected blood, showed <i>Theileria</i> infections in their salivary glands. Parasite DNA was not amplified from any of the <i>R</i>. <i>bursa</i> groups. The <i>Theileria</i>-free sheep served as a non-infected negative control. The size of the amplicon is 785 bp. (D) Azure staining of unfed adult tick salivary glands emerged from nymphs that were engorged with <i>T</i>. <i>lestoquardi</i> infected blood meals. (Left panel) Salivary gland of a <i>H</i>. <i>anatolicum</i> fed from sheep in experiment {<i>b</i>}. <i>T</i>. <i>lestoquardi</i>-infected acini are indicated by thin black arrows. Middle panel, Magnified view of an infected acinus adjacent to uninfected acini. Numerous <i>Theileria</i> particles (small arrows) are visible inside the infected acinar cell that has a hypertrophied nucleus. Right panel, Acini of a <i>R</i>. <i>bursa</i> salivary gland were all normal and free from parasite.</p

Summary of the experiments conducted in this project.

<p>Test group (experiment {<i>a</i>}): Nymphs of <i>H</i>. <i>anatolicum</i> were fed <i>in vitro</i> with infected blood obtained from the initial donor sheep. Emerged adults were tested for <i>T</i>. <i>lestoquardi</i> infection by staining the salivary glands and PCR detection of the parasite DNA. When infection was confirmed in a cohort of ticks, the rest were used to infect a healthy sheep that was monitored clinically. Test group (experiment {<i>b</i>}): This was a replicate of experiment {<i>a</i>} in which nymphs were artificially infected with blood taken from the sheep that was infected by <i>in vitro</i> fed ticks in experiment <i>(a)</i> and adults resulting from these instars were transmission fed on another sheep. Test group (experiment {<i>e</i>}): <i>T</i>. <i>lestoquardi</i> infected blood was provided for <i>R</i>. <i>bursa</i> nymphs feeding under artificial conditions. Negative control groups (experiments {<i>c</i>} and {<i>f</i>}): <i>H</i>. <i>anatolicum and R</i>. <i>bursa</i> nymphs were engorged with uninfected blood of a healthy sheep. Positive control groups (experiments {<i>d</i>} and {<i>g</i>}): Experiments were performed simultaneously. Nymphs of <i>H</i>. <i>anatolicum</i> and <i>R</i>. <i>bursa</i> fed on ears of the donor sheep. Here, <i>H</i>. <i>anatolicum</i> ticks only acted as positive controls for <i>T</i>. <i>lestoquardi</i> acquisition.</p

<i>In vitro</i> feeding performance of <i>H</i>. <i>anatolicum</i> and <i>R</i>. <i>bursa</i> nymphs fed on skin membranes and detection of <i>T</i>. <i>lestoquardi</i>.

<p><i>In vitro</i> feeding performance of <i>H</i>. <i>anatolicum</i> and <i>R</i>. <i>bursa</i> nymphs fed on skin membranes and detection of <i>T</i>. <i>lestoquardi</i>.</p