In vitro infection of bovine erythrocytes with Theileria annulata merozoites as a key step in completing the T. annulata life cycle in vitro.

Theileria annulata is a protozoan parasite with a complex life cycle involving a bovine host and a tick vector. It is transmitted by Hyalomma ticks and is the causative agent of tropical theileriosis, a debilitating and often fatal disease in southern Europe, northern Africa and large parts of Asia. Understanding the biology of different life cycle stages is critical for the control of tropical theileriosis and requires the use of experimental animals which poses an ethical concern. We present for the first time the in vitro infection of red blood cells (RBCs) with T. annulata differentiated schizonts. The Ankara cell line of T. annulata was cultured at 41 °C for nine days to induce merogony and subsequently incubated with purified RBCs for one to three days. Percentage of parasitized erythrocyte (PPE) over the short culture period was estimated by Giemsa staining (0.007-0.01%), Flow cytometry activated sorting (FACS) (0.02-1.1%) and observation of FACS sorted cells by confocal microscopy (0.05-0.4%). There was a significant difference in the PPE between FACS and the two other techniques (one-way ANOVA followed by Tukey test, P = 0.004) but no significant difference was observed between the confocal imaging and Giemsa staining methods (ANOVA one-way followed by Tukey test, P = 0.06). Importantly, all three complementary methods confirmed the invasion of RBCs by T. annulata merozoites in vitro. Although the experimental conditions will require further optimization to increase the PPE, the in vitro infection of RBCs by T. annulata merozoites is pivotal in paving the way for the eventual completion of the T. annulata life cycle in vitro when combined with artificial tick feeding.

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.

Preliminary Study on Artificial versus Animal-Based Feeding Systems for Amblyomma Ticks (Acari: Ixodidae).

Hard ticks pose a threat to animal and human health. Active life stages need to feed on a vertebrate host in order to complete their life cycle. To study processes such as tick-pathogen interactions or drug efficacy and pharmacokinetics, it is necessary to maintain tick colonies under defined laboratory conditions, typically using laboratory animals. The aim of this study was to test a membrane-based artificial feeding system (AFS) applicable for Amblyomma ticks using Amblyomma tonelliae as a biological model. Adult ticks from a laboratory colony were fed in a membrane-based AFS. For comparison, other A. tonelliae adults were fed on calf and rabbit. The proportions of attached (AFS: 76%; calf/rabbit: 100%) and engorged females (AFS: 47.4%; calf/rabbit: 100%) in the AFS were significantly lower compared to animal-based feeding (p = 0.0265). The engorgement weight of in vitro fed ticks (x¯ = 658 mg; SD ± 259.80) did not significantly differ from that of ticks fed on animals (p = 0.3272, respectively 0.0947). The proportion of females that oviposited was 100% for all three feeding methods. However, the incubation period of eggs (x¯ = 54 days; SD ± 7) was longer in the AFS compared to conventional animal-based feeding (p = 0.0014); x¯ = 45 days; SD ± 2 in the rabbit and (p = 0.0144). x¯ = 48 days; SD ± 2 in the calf). Egg cluster hatching (x¯ = 41%; SD ± 44.82) was lower in the AFS than in the other feeding methods (rabbit: x¯ = 74%; SD ± 20; p = 0.0529; calf: x¯ = 81%; SD ± 22; p = 0.0256). Although the attachment, development, and the hatching of AFS ticks were below those from animal-based feeding, the method may be useful in future experiments. Nevertheless, further experiments with a higher number of tick specimens (including immature life stages) and different attractant stimuli are required to confirm the preliminary results of this study and to evaluate the applicability of AFS for Amblyomma ticks as an alternative to animal-based feeding methods.

Sequence polymorphisms in a Theileria annulata surface protein (TaSP) known to augment the immunity induced by live attenuated cell line vaccine.

Theileria annulata is a tick-borne protozoan causing tropical theileriosis in cattle. The use of attenuated cell line vaccines in combination with subunit vaccines has been relatively successful as a control method, as exemplified by a recent study in which immunization with a local cell line followed by booster vaccinations with recombinant T. annulata surface protein (TaSP) resulted in 100% protection upon field challenge in Sudan. However, these findings cannot be directly extrapolated to other countries as culture-attenuated live vaccines are generated using local strains and no systematic evaluation of genotype differences between countries has been undertaken. In this study, we sequenced the TaSP gene from T. annulata cell lines and field isolates from Tunisia (n = 28) and compared them to genotypes from Sudan (n = 25) and Morocco (n = 1; AJ316259.1). Our analyses revealed 20 unique TaSP genotypes in the Tunisian samples, which were all novel but similar to genotypes found in Asia. The impact of these polymorphisms on the ability of the TaSP antigen to boost the immunity engendered by live cell line vaccines, especially in Tunisia where studies with TaSP have not been conducted, remains to be examined. Interestingly, phylogenetic analyses of publicly available TaSP sequences resolved the sequences into two clusters with no correlation to the geographical origin of the isolates. The availability of candidate vaccines that were recently attenuated using local strains from Sudan, Tunisia, Egypt and Morocco should be exploited to generate a comprehensive catalogue of genetic variation across this regional collection of attenuated live vaccines.

Morphological and phylogenetic analyses of Rhipicephalus microplus ticks from Bangladesh, Pakistan and Myanmar.

Ticks of the Boophilus subgenus, classified in the genus Rhipicephalus, are widespread in subtropical and tropical regions, but knowledge on their distribution in Asia is fragmentary. The most important representatives belong to the Rhipicephalus (Boophilus) microplus complex, which is composed of species that share many morphological similarities and are therefore difficult to distinguish. In this study, adult Rhipicephalus (Boophilus) ticks from Bangladesh, Myanmar and Pakistan were subjected to phylogenetic and Scanning Electron Microscopy (SEM) analyses. Phylogenetic analyses of cytochrome oxidase 1 (cox1), internal transcribed spacer 2 (ITS2) and 12S rRNA gene sequences confirmed that the R. microplus complex consists of at least five taxa: R. annulatus, R. australis, and R. microplus clades A-C. Ticks from Bangladesh, Myanmar and Pakistan were assigned to R. microplus clade C. SEM images revealed a wide range of variation in the morphology of the clade C adults, including morphological features previously identified as critical for distinguishing R. microplus from R. australis, which is illustrative for the complications in identifying species within the R. microplus complex using morphology only. Further morphological, genomic and crossbreeding studies are required to elucidate the species status of R. microplus clades A-C.

A comparison of DNA extraction protocols from blood spotted on FTA cards for the detection of tick-borne pathogens by Reverse Line Blot hybridization.

An essential step in the molecular detection of tick-borne pathogens (TBPs) in blood is the extraction of DNA. When cooled storage of blood under field conditions prior to DNA extraction in a dedicated laboratory is not possible, the storage of blood on filter paper forms a promising alternative. We evaluated six DNA extraction methods from blood spotted on FTA Classic® cards (FTA cards), to determine the optimal protocol for the subsequent molecular detection of TBPs by PCR and the Reverse Line Blot hybridization assay (RLB). Ten-fold serial dilutions of bovine blood infected with Babesia bovis, Theileria mutans, Anaplasma marginale or Ehrlichia ruminantium were made by dilution with uninfected blood and spotted on FTA cards. Subsequently, DNA was extracted from FTA cards using six different DNA extraction protocols. DNA was also isolated from whole blood dilutions using a commercial kit. PCR/RLB results showed that washing of 3mm discs punched from FTA cards with FTA purification reagent followed by DNA extraction using Chelex® resin was the most sensitive procedure. The detection limit could be improved when more discs were used as starting material for the DNA extraction, whereby the use of sixteen 3mm discs proved to be most practical. The presented best practice method for the extraction of DNA from blood spotted on FTA cards will facilitate epidemiological studies on TBPs. It may be particularly useful for field studies where a cold chain is absent.

Hd86, the Bm86 tick protein ortholog in Hyalomma scupense (syn. H. detritum): expression in Pichia pastoris and analysis of nucleotides and amino acids sequences variations prior to vaccination trials.

The genus Hyalomma includes the most frequent tick species infesting livestock in North Africa, one of these species, Hyalomma scupense (syn. H. detritum) is particularly important due to its role in the transmission of tropical theileriosis to cattle (Theileria annulata infection). We have cloned and characterized the orthologs of the Bm86 gene from H. scupense strains collected over Tunisia in 2006 and 2009. The recombinant protein rHd86 was expressed in Pichia pastoris for vaccination purpose using a transcript from the 2006 strain. The rHd86 was then purified from the yeast culture supernatant by a filtration and a size exclusion process. It was recognized by specific anti-Bm86 antisera. An important extent of inter-specific diversity ranging from 35 to 40% was recorded between Hd86 and Bm86/Bm95 proteins whilst a very limited level of intra-specific diversity (1.7%) occurred between the Hd86 vaccine candidate protein and its homologues from H. scupense strains collected in 2009. These results emphasise the need for assessing the efficacy against H. scupense and others important cattle Hyalomma species in Tunisia of our Hd86 vaccine candidate alongside with a Bm86 vaccine.