Evaluation of oxidative/nitrosative stress biomarkers and DNA damage in buffaloes naturally infected with Theileria annulata.

Previously, we evaluated serum sialic acid (SA) levels in buffaloes naturally infected with T. annulata. In the current paper, we conducted a further study on oxidative/nitrosative stress biomarkers in erythrocyte lysate samples of the same buffaloes. DNA damage also was assessed. Additionally, we tested whether, there is any correlation between SA and the aforementioned indicators or not. To achieve these aims, several biomarkers including the activities of key antioxidant enzymes, superoxide dismutase, glutathione peroxidase and catalase, as well as malondialdehyde (MDA), protein carbonyl (PCO), nitric oxide contents (NO), total antioxidant capacity (TAC) and DNA damage levels were measured. The obtained results showed that the activities of the antioxidant enzymes and TAC levels decreased significantly as the percentage of parasitemia increased accordingly. Also, a significant increase in the levels of PCO, MDA, NO and DNA damage were recorded, depending on the degree of parasitemia. There was a significant correlation between oxidative/nitrosative stress indicators and SA. Conclusively, T. annulata infection in buffaloes is associated with a parasitic burden-dependent oxidative/nitrosative damages to erythrocytes and SA plays a crucial role in pathogenesis of the disease, as it is tightly correlated with oxidative/nitrosative indicators.

Optimization of a suspension culture for a Theileria annulata-infected bovine cell line.

Theileria annulata schizont transformed bovine lymphocytes show the feature of permanent proliferation in vitro culture. In this study, we optimized a suitable culture medium for transformed cells to ensure a high yield of quality cells in suspension culture. As the basis for the optimized medium, we combined 75% Gibco (GB) and 25% RPMI-1640 medium. Glucose, lactic acid, ammonia, growth factors and several kinds of amino acids at specific concentrations play important roles in maintaining the maximum growth rate and the quality of cells. The metabolic flow of 17 amino acids, glucose and nutrients was determined with high-performance liquid chromatography (HPLC) and cell viability analysis. The genetic stability of the TaSP and TaSE genes at different passages of the cell line in suspension culture was determined using PCR amplification. The optimal concentrations or tolerated levels of glucose, lactic acid and ammonia were 10-14, 2-5.5 and 3.5-5.5 mmol/L, respectively. Our data demonstrated that the potential utility of the medium optimized here to yield high quality cells compared with basal (normally used) medium. The medium also facilitated the easy maintenance of transformed cells with high yields and excellent quality for in vitro studies. This study also provides insight into the processes of optimization and vaccine development.

Discovery of a new Theileria sp. closely related to Theileria annulata in cattle from Sri Lanka.

Theileria annulata is a haemoprotozoan parasite that causes a cancer-like illness known as tropical theileriosis in cattle. In the course of analyzing the genetic diversity of T. annulata in Sri Lanka, we observed that merozoite-piroplasm surface antigen (tams1) and surface protein (tasp)-like gene sequences obtained from bovine blood DNA samples, which were PCR-positive for T. annulata, were conserved but shared low identity with T. annulata GenBank sequences. Moreover, the 18S rRNA sequences from the Sri Lankan samples contained ten unique single-nucleotide polymorphisms compared with all known T. annulata sequences. The cytochrome b (cob) gene sequences isolated from the Sri Lankan samples were highly conserved and shared low identity scores with similarly conserved T. annulata sequences from GenBank. Phylogenetic analysis showed that the Sri Lankan tams1-like, tasp-like, 18S rRNA, and cob sequences clustered together and formed sister clades to the common ancestors of all known T. annulata and Theileria lestoquardi sequences. These findings demonstrated that the Sri Lankan cattle were not infected with T. annulata but with a new Theileria sp. (designated as Theileria sp. Yokoyama) closely related to T. annulata.

Immuno-informatics Analysis to Identify Novel Vaccine Candidates and Design of a Multi-Epitope Based Vaccine Candidate Against Theileria parasites.

Theileriosis poses a serious threat to ruminants in tropical and subtropical countries. It is a tick-borne disease, caused by an apicomplexan parasite, Theileria. The high disease burden in animals causes huge economic losses to marginal farmers. Further, with increasing cases of resistance to commonly used drugs, it is highly desirable to develop better and cost-effective vaccines against theileriosis. The only available vaccine, live attenuated parasite vaccine, has many drawbacks and hence is unsuitable for controlling this disease. Immuno-informatics has emerged as a useful tool in down selection of potential molecules for vaccine development. In this study, we have used an immuno-informatics driven genome-wide screening strategy to identify potential vaccine targets containing important and effective dominant immunogens against Theileria. The proteome of Theileria annulata was screened for proteins with probability of plasma membrane localization or GPI anchor. The proteins non-homologous to the host (bovine) were selected and their antigenicity was analyzed. The B-cell epitopes were identified in the selected proteins and mapped in the modeled structure of the proteins. A total of 19 linear epitopes in 12 proteins, exposed in the extracellular space and having the potential to induce protective antibodies were obtained. Additionally, CTL epitopes which are peptides with 9-mer core sequence, were also identified, modeled and docked with bovine MHC-I structures. The CTL epitopes showing high binding energy with the bovine MHC-I were further engineered in silico to design a putative multi-epitope vaccine candidate against Theileria parasites. The docking studies and molecular dynamics studies with the predicted multi-epitope vaccine candidate and modeled bovine TLR4 exhibited strong binding energy, suggesting that the complex is stable and the putative multi-epitope vaccine candidate can be a potentially good candidate for vaccine development.

Exploring the TLR and NLR signaling pathway relevant molecules induced by the Theileria annulata infection in calves.

Theileria annulata is the pathogen of bovine tropical theileriosis. It is extremely harmful to the cattle industry, with huge economic losses. The toll-like receptor (TLR) and NOD-like receptor (NLR) signaling pathways are crucial for resistance to infection of the protozoa, such as Plasmodium falciparum, Toxoplasma gondii, and Trypanosoma cruzi. However, the role of these immune-related pathways is unclear during T. annulata infection. In the present study, peripheral blood mononuclear cells and serum were separated from blood samples of calves infected with homogenized tick supernatants carrying T. annulata sporozoites at 12 h, 24 h, 36 h, 48 h, 72 h, 96 h, 120 h, 144 h and 168 h postinoculation. The Custom RT2 Profiler PCR Array was used to explore the mRNA levels of 42 TLR and NLR signaling pathway relevant genes. The TLR1, TLR6, TLR10, NLRP1, and MyD88 genes and their downstream signaling molecules significantly differed after the T. annulata infection in comparison with that of preinfection from 72 h to 168 h postinoculation. The serum concentrations of IL-6, IL-1ß, and TNFα were significantly increased at 96 h and 168 h postinfection. These findings provided novel information to help determine the mechanisms of TLR and NLR signaling pathway involvement in protection against T. annulata infection.

Epidemiological study on tropical theileriosis (Theileria annulata infection) in the Egyptian Oases with special reference to the molecular characterization of Theileria spp.

Theileria annulata infection is a tick-borne disease known as Egyptian fever since 1947. It is a destructive obstacle for the livestock production in the Egyptian Oases (EL-Wady EL-Geded Province). The present study was conducted on 1068 cattle, ranged from below one year to more than eight years old; belonged to different farms and villages in EL-Wady EL-Geded Province. The infection was confirmed by blood smears, Tams-1 target based polymerase chain reaction (Tams-1 PCR), 18Ss rRNA polymerase chain reaction and semi nested-polymerase chain reaction (nPCR) followed by DNA sequencing and phylogenetic analyses, in addition to tick identification. Molecular techniques confirmed the infection in 63.6% (679/1068) of the examined animals while Giemsa-stained blood smears confirmed it in 36.8% (393/1062). Male and female animals showed molecular confirmed infection rates of 64.5 and 62.7%, respectively. Animals less than one year old were more infected (83.33%, 400/480) followed by animals less than three years (57.31%, 149/260) and animals less than five years (42.45%, 90/212), respectively. On the other hand, animal of five years old or above were less infected and the infection rate in this group was estimated to be 34.48% (40/116). Two tick species were identified during the present study: Hyalomma anatolicum and Rhipicephalus annulatus. Theileria annulata was the only Theileria species found in the Egyptian oases in respect to phylogenetic analysis of the obtained sequences.

Identification of candidate transmission-blocking antigen genes in Theileria annulata and related vector-borne apicomplexan parasites.

BACKGROUND: Vector-borne apicomplexan parasites are a major cause of mortality and morbidity to humans and livestock globally. The most important disease syndromes caused by these parasites are malaria, babesiosis and theileriosis. Strategies for control often target parasite stages in the mammalian host that cause disease, but this can result in reservoir infections that promote pathogen transmission and generate economic loss. Optimal control strategies should protect against clinical disease, block transmission and be applicable across related genera of parasites. We have used bioinformatics and transcriptomics to screen for transmission-blocking candidate antigens in the tick-borne apicomplexan parasite, Theileria annulata. RESULTS: A number of candidate antigen genes were identified which encoded amino acid domains that are conserved across vector-borne Apicomplexa (Babesia, Plasmodium and Theileria), including the Pfs48/45 6-cys domain and a novel cysteine-rich domain. Expression profiling confirmed that selected candidate genes are expressed by life cycle stages within infected ticks. Additionally, putative B cell epitopes were identified in the T. annulata gene sequences encoding the 6-cys and cysteine rich domains, in a gene encoding a putative papain-family cysteine peptidase, with similarity to the Plasmodium SERA family, and the gene encoding the T. annulata major merozoite/piroplasm surface antigen, Tams1. CONCLUSIONS: Candidate genes were identified that encode proteins with similarity to known transmission blocking candidates in related parasites, while one is a novel candidate conserved across vector-borne apicomplexans and has a potential role in the sexual phase of the life cycle. The results indicate that a 'One Health' approach could be utilised to develop a transmission-blocking strategy effective against vector-borne apicomplexan parasites of animals and humans.

Validation of Reference Genes for Quantitative Real-Time PCR in Bovine PBMCs Transformed and Non-transformed by Theileria annulata.

Theileria annulata is a tick-borne intracellular protozoan parasite that causes tropical theileriosis, a fatal bovine lymphoproliferative disease. The parasite predominantly invades bovine B lymphocytes and macrophages and induces host cell transformation by a mechanism that is not fully comprehended. Analysis of signaling pathways by quantitative real-time PCR (qPCR) could be a highly efficient means to understand this transformation mechanism. However, accurate analysis of qPCR data relies on selection of appropriate reference genes for normalization, yet few papers on T. annulata contain evidence of reference gene validation. We therefore used the geNorm and NormFinder programs to evaluate the stability of 5 candidate reference genes; 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ACTB (ß-actin), PRKG1 (protein kinase cGMP-dependent, type I) and TATA box binding protein (TBP). The results showed that 18S rRNA was the reference gene most stably expressed in bovine PBMCs transformed and non-transformed with T. annulata, followed by GAPDH and TBP. While 18S rRNA and GAPDH were the best combination, these 2 genes were chosen as references to study signaling pathways involved in the transformation mechanism of T. annulata.

Validation of Reference Genes for Quantitative Real-Time PCR in Bovine PBMCs Transformed and Non-transformed by Theileria annulata

Theileria annulata is a tick-borne intracellular protozoan parasite that causes tropical theileriosis, a fatal bovine lymphoproliferative disease. The parasite predominantly invades bovine B lymphocytes and macrophages and induces host cell transformation by a mechanism that is not fully comprehended. Analysis of signaling pathways by quantitative real-time PCR (qPCR) could be a highly efficient means to understand this transformation mechanism. However, accurate analysis of qPCR data relies on selection of appropriate reference genes for normalization, yet few papers on T. annulata contain evidence of reference gene validation. We therefore used the geNorm and NormFinder programs to evaluate the stability of 5 candidate reference genes; 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ACTB (β-actin), PRKG1 (protein kinase cGMP-dependent, type I) and TATA box binding protein (TBP). The results showed that 18S rRNA was the reference gene most stably expressed in bovine PBMCs transformed and non-transformed with T. annulata, followed by GAPDH and TBP. While 18S rRNA and GAPDH were the best combination, these 2 genes were chosen as references to study signaling pathways involved in the transformation mechanism of T. annulata.

Genetic Diversity and Population Structure of Theileria annulata in Oman.

BACKGROUND: Theileriosis, caused by a number of species within the genus Theileria, is a common disease of livestock in Oman. It is a major constraint to the development of the livestock industry due to a high rate of morbidity and mortality in both cattle and sheep. Since little is currently known about the genetic diversity of the parasites causing theileriosis in Oman, the present study was designed to address this issue with specific regard to T. annulata in cattle. METHODS: Blood samples were collected from cattle from four geographically distinct regions in Oman for genetic analysis of the Theileria annulata population. Ten genetic markers (micro- and mini-satellites) representing all four chromosomes of T. annulata were applied to these samples using a combination of PCR amplification and fragment analysis. The resultant genetic data was analysed to provide a first insight into the structure of the T. annulata population in Oman. RESULTS: We applied ten micro- and mini-satellite markers to a total of 310 samples obtained from different regions (174 [56%] from Dhofar, 68 [22%] from Dhira, 44 [14.5%] from Batinah and 24 [8%] from Sharqia). A high degree of allelic diversity was observed among the four parasite populations. Expected heterozygosity for each site ranged from 0.816 to 0.854. A high multiplicity of infection was observed in individual hosts, with an average of 3.3 to 3.4 alleles per locus, in samples derived from Batinah, Dhofar and Sharqia regions. In samples from Dhira region, an average of 2.9 alleles per locus was observed. Mild but statistically significant linkage disequilibrium between pairs of markers was observed in populations from three of the four regions. In contrast, when the analysis was performed at farm level, no significant linkage disequilibrium was observed. Finally, no significant genetic differentiation was seen between the four populations, with most pair-wise FST values being less than 0.03. Slightly higher FST values (GST' = 0.075, θ = 0.07) were detected when the data for T. annulata parasites in Oman was compared with that previously generated for Turkey and Tunisia. CONCLUSION: Genetic analyses of T. annulata samples representing four geographical regions in Oman revealed a high level of genetic diversity in the parasite population. There was little evidence of genetic differentiation between parasites from different regions, and a high level of genetic diversity was maintained within each sub-population. These findings are consistent with a high parasite transmission rate and frequent movement of animals between different regions in Oman.

Transforming growth factor ß2 promotes transcription of COX2 and EP4, leading to a prostaglandin E2-driven autostimulatory loop that enhances virulence of Theileria annulata-transformed macrophages.

Transforming growth factor beta (TGF-ß) is a pleiotropic cytokine known to regulate cell growth, differentiation, and motility and is a potent modulator of immune function. TGF-ß consequently plays a central role in carcinogenesis, and a dampened TGF-ß2 response by Theileria annulata-infected monocytes/macrophages underpins disease resistance to tropical theileriosis. Here, we show that concomitant with the loss of TGF-ß2 production, there is ablated expression of COX2 and EP4, which leads to a drop in cyclic AMP (cAMP) levels and, consequently, reduced activation of protein kinase A (PKA) and EPAC. This ablated phenotype can be rescued in attenuated macrophages by the addition of exogenous TGF-ß2, which reactivates the expression of COX2 and EP4 while repressing that of protein kinase inhibitor gamma (PKIG) to the levels in virulent macrophages. TGF-ß2 therefore promotes the adhesion and invasiveness of virulent macrophages by modulating COX2, EP4, and PKIG transcription to initiate a prostaglandin E2 (PGE2)-driven autostimulatory loop that augments PKA and EPAC activities. A virulence phenotype stemming from the double activation of PKA and EPAC is the induction of a CREB-mediated transcriptional program and the upregulation of JAM-L- and integrin 4αß1-mediated adhesion of Theileria-infected macrophages.

A survey of ixodid tick species and molecular identification of tick-borne pathogens.

This study was undertaken in two different climatic areas of Turkey to determine the presence of tick-borne pathogens of medical and veterinary importance. The ticks were removed from humans, pooled according to species and developmental stages, and analyzed by PCR, reverse line blot (RLB) and sequencing. Of the 2333 removed ticks from 10 species, 1238 (53.06%) were obtained from the arid cold zone, and the remaining 1095 (46.93%) were obtained from the humid zone. The removed ticks were identified as Hyalomma marginatum, Hyalomma detritum, Hyalomma excavatum, Rhipicephalus bursa, Rhipicephalus turanicus, Rhipicephalus sanguineus, Dermacentor marginatus, Haemaphysalis punctata, Haemaphysalis sulcata, Ixodes ricinus, Haemaphysalis and Ixodes spp. nymphs. The dominant species was I. ricinus (61.27%) in the humid zone, whereas the Haemaphysalis spp. nymph dominated (30.29%) in the arid zone. Infection rates were calculated as the maximum likelihood estimation (MLE) with 95% confidence intervals (CI). Of the 169 pools tested, 49 (28.99%) were found to be infected with the pathogens, and the overall MLE of the infection rate was calculated as 2.44% (CI 1.88-3.17). The MLE of the infection varied among tick species, ranging from 0.85% (CI 0.23-2.34) in Haemaphysalis spp. nymph to 17.93% (CI 6.94-37.91) in D. marginatus. Pathogens identified in ticks included Theileria annulata, Babesia ovis, Babesia crassa, Anaplasma/Ehrlichia spp., Anaplasma ovis, Ehrlichia canis, Anaplasma phagocytophilum, Hepatozoon canis and Hepatozoon felis. Most tick pools were infected with a single pathogen. However, four pools infected with H. canis displayed infections with B. crassa, A. phagocytophilum and E. canis. The sequencing indicated that Anaplasma/Ehrlichia spp. was 100% identical to the sequence of Ehrlichia sp. Firat 2 and 3 previously identified from Hyalomma anatolicum.

Infection of small ruminants and their red blood cells with Theileria annulata schizonts.

Theileria annulata, the causative agent of tropical theileriosis, is a protozoan parasite that also causes lymphoproliferative diseases in cattle. In vivo, parasitized cells undergo clonal expansion and infiltrate both the lymphoid and non-lymphoid tissues of the infected host. To determine whether the small ruminants and their red blood cells (RBCs) were invaded by T. annulata schizonts or not, T. annulata schizonts were used to infect bovine, ovine and caprine RBCs in vitro, and sheep and goats in vivo. The results showed that the schizonts infected bovine, ovine and caprine RBCs in vitro, but not sheep and goats, which showed only an increase in body temperature and no development of piroplasms. To our knowledge, this is the first report of infection of small ruminants and their RBCs by T. annulata schizonts.

Lymphocytes and macrophages are infected by Theileria equi, but T cells and B cells are not required to establish infection in vivo.

Theileria equi has a biphasic life cycle in horses, with a period of intraleukocyte development followed by patent erythrocytic parasitemia that causes acute and sometimes fatal hemolytic disease. Unlike Theileria spp. that infect cattle (Theileria parva and Theileria annulata), the intraleukocyte stage (schizont) of Theileria equi does not cause uncontrolled host cell proliferation or other significant pathology. Nevertheless, schizont-infected leukocytes are of interest because of their potential to alter host cell function and because immune responses directed against this stage could halt infection and prevent disease. Based on cellular morphology, Theileria equi has been reported to infect lymphocytes in vivo and in vitro, but the specific phenotype of schizont-infected cells has yet to be defined. To resolve this knowledge gap in Theileria equi pathogenesis, peripheral blood mononuclear cells were infected in vitro and the phenotype of infected cells determined using flow cytometry and immunofluorescence microscopy. These experiments demonstrated that the host cell range of Theileria equi was broader than initially reported and included B lymphocytes, T lymphocytes and monocyte/macrophages. To determine if B and T lymphocytes were required to establish infection in vivo, horses affected with severe combined immunodeficiency (SCID), which lack functional B and T lymphocytes, were inoculated with Theileria equi sporozoites. SCID horses developed patent erythrocytic parasitemia, indicating that B and T lymphocytes are not necessary to complete the Theileria equi life cycle in vivo. These findings suggest that the factors mediating Theileria equi leukocyte invasion and intracytoplasmic differentiation are common to several leukocyte subsets and are less restricted than for Theileria annulata and Theileria parva. These data will greatly facilitate future investigation into the relationships between Theileria equi leukocyte tropism and pathogenesis, breed susceptibility, and strain virulence.

A Bovine Lymphosarcoma Cell Line Infected with Theileria annulata Exhibits an Irreversible Reconfiguration of Host Cell Gene Expression.

Theileria annulata, an intracellular parasite of bovine lymphoid cells, induces substantial phenotypic alterations to its host cell including continuous proliferation, cytoskeletal changes and resistance to apoptosis. While parasite induced modulation of host cell signal transduction pathways and NFκB activation are established, there remains considerable speculation on the complexities of the parasite directed control mechanisms that govern these radical changes to the host cell. Our objectives in this study were to provide a comprehensive analysis of the global changes to host cell gene expression with emphasis on those that result from direct intervention by the parasite. By using comparative microarray analysis of an uninfected bovine cell line and its Theileria infected counterpart, in conjunction with use of the specific parasitacidal agent, buparvaquone, we have identified a large number of host cell gene expression changes that result from parasite infection. Our results indicate that the viable parasite can irreversibly modify the transformed phenotype of a bovine cell line. Fifty percent of genes with altered expression failed to show a reversible response to parasite death, a possible contributing factor to initiation of host cell apoptosis. The genes that did show an early predicted response to loss of parasite viability highlighted a sub-group of genes that are likely to be under direct control by parasite infection. Network and pathway analysis demonstrated that this sub-group is significantly enriched for genes involved in regulation of chromatin modification and gene expression. The results provide evidence that the Theileria parasite has the regulatory capacity to generate widespread change to host cell gene expression in a complex and largely irreversible manner.

Recruitment of EB1, a master regulator of microtubule dynamics, to the surface of the Theileria annulata schizont.

The apicomplexan parasite Theileria annulata transforms infected host cells, inducing uncontrolled proliferation and clonal expansion of the parasitized cell population. Shortly after sporozoite entry into the target cell, the surrounding host cell membrane is dissolved and an array of host cell microtubules (MTs) surrounds the parasite, which develops into the transforming schizont. The latter does not egress to invade and transform other cells. Instead, it remains tethered to host cell MTs and, during mitosis and cytokinesis, engages the cell's astral and central spindle MTs to secure its distribution between the two daughter cells. The molecular mechanism by which the schizont recruits and stabilizes host cell MTs is not known. MT minus ends are mostly anchored in the MT organizing center, while the plus ends explore the cellular space, switching constantly between phases of growth and shrinkage (called dynamic instability). Assuming the plus ends of growing MTs provide the first point of contact with the parasite, we focused on the complex protein machinery associated with these structures. We now report how the schizont recruits end-binding protein 1 (EB1), a central component of the MT plus end protein interaction network and key regulator of host cell MT dynamics. Using a range of in vitro experiments, we demonstrate that T. annulata p104, a polymorphic antigen expressed on the schizont surface, functions as a genuine EB1-binding protein and can recruit EB1 in the absence of any other parasite proteins. Binding strictly depends on a consensus SxIP motif located in a highly disordered C-terminal region of p104. We further show that parasite interaction with host cell EB1 is cell cycle regulated. This is the first description of a pathogen-encoded protein to interact with EB1 via a bona-fide SxIP motif. Our findings provide important new insight into the mode of interaction between Theileria and the host cell cytoskeleton.

Naturally occurring infections of cattle with Theileria lestoquardi and sheep with Theileria annulata in the Sudan.

Theileria annulata is endemic in northern Sudan, hindering all efforts at upgrading cattle for milk production. T. lestoquardi clinical cases occur throughout the year and causes annual outbreaks that result in substantial losses in sheep. In the northern Sudan both cattle and small ruminants are frequently raised together and/or share common grazing grounds at river banks. In an attempt to evaluate field cross infectivity of Theileria lestoquardi and T. annulata in cattle and sheep respectively, a PCR analysis was carried out on samples collected from closely reared sheep and cattle using both T. annulata and T. lestoquardi specific primers. A total of 19 sheep out of 51 (37.3%) were positive for T. lestoquardi while four sheep (7.8%) showed T. annulata specific amplicons. A total of 38 out of 52 (73.1%) surveyed cattle were PCR positive for T. annulata and only two (3.8%) showed T. lestoquardi specific bands. These findings indicate complex epidemiology of both infections in areas where both parasites are transmitted by the same vector and call for further investigations of this phenomenon.

Serum acute phase proteins in control and Theileria annulata infected water buffaloes (Bubalus bubalis).

This study was carried out to ascertain the changes in acute phase proteins (APPs) and pro-inflammatory cytokines in Theileria annulata infected water buffalo. Thirty infected water buffaloes and 20 parasitologically free were used. In the present study there was significant (P ≤ 0.05) increase in haptoglobin (Hp), serum amyloid A (SAA), ceruloplasmin, α1-acid glycoprotein (AGP) and fibrinogen levels (2.18 ± 0.29 g/l, 156.58 ± 3.48 mg/l, 31.23 ± 1.25mg/dl, 370.23 ± 33.21 mg/l and 16.17 ± 1.18 g/l, respectively) in T. annulata infected water buffaloes when compared to healthy ones (0.13 ± 0.01 g/l, 23.9 ± 0.56 mg/l, 21.23 ± 1.21 mg/dl, 240.53 ± 22.45 mg/l and 4.2 ± 0.1 6g/l, respectively). Moreover, there was significant (P ≤ 0.05) increase in the levels of TNF-α, IL-1α, IL-6, IL-12, IL-1ß and IFN-γ (2.55 ± 0.12 ng/ml, 98.32 ± 4.21 pg/ml, 152.32 ± 5.62 pg/ml, 26.44 ± 1.43 ng/ml, 240.33 ± 20.45 pg/ml and 123.65 ± 5.67 pg/ml, respectively) in T. annulata infected water buffaloes when compared to healthy ones (0.42 ± 0.04 ng/ml, 55.32 ± 3.21 pg/ml, 88.23 ± 3.21 pg/ml, 7.45 ± 0.67 ng/ml, 98.33 ± 3.45 pg/ml and 34.76 ± 1.56 pg/ml, respectively). There was also significant decrease (P ≤ 0.05) in the Hb content, PCV%, RBCs and WBCs counts in the diseased water buffaloes compared to the control ones. Neutropenia, eosinopenia, lymphopenia, monocytopenia and thrombocytopenia were also recorded. The biochemical changes revealed significant (P ≤ 0.05) elevation in the levels of AST, ALT, ALP, LDL-c, VLDL-c, BHBA and NEFA, with significant (P ≤ 0.05) decrease in the levels of total proteins, albumin, globulins, cholesterol, triglyceride, glucose, G6PD, calcium and phosphorus in T. annulata infected water buffaloes when compared to healthy ones. It could be concluded that APPs and pro-inflammatory cytokines could be used as a valuable biomarkers in T. annulata infected water buffaloes (Bubalus bubalis).

Modelling the transmission dynamics of Theileria annulata: model structure and validation for the Turkish context.

A mathematical model that describes the transmission dynamics of Theileria annulata is proposed that consists of 2 host components: the Hyalomma tick population and a compartmental model of T. annulata infection in the cattle population. The model was parameterized using data describing tick infestation and the infection status of cattle in Turkey from 2006 to 2008. The tick attachment rates are highly seasonal and because of the temporal separation of infectious and susceptible ticks virtually all ticks are infected by carrier cattle, so that annual peaks of disease in cattle do not impact on infection in the Hyalomma tick population. The impact of intervention measures that target the tick population both on the host and in the environment and their impact on the transmission of T. annulata were investigated. Interventions that have a limited 'one-off' impact and interventions that have a more permanent impact were both considered. The results from the model show the importance of targeting ticks during the period when they have left their first host as nymphs but have yet to feed on their second host.

Clinical and haematological study on water buffaloes (Bubalus bubalis) and crossbred cattle naturally infected with Theileria annulata in Sharkia province, Egypt.

This study was conducted to investigate the clinical and haematological findings in water buffaloes and crossbred cattle naturally infected with Theileria annulata with special reference to the clinical picture of tropical theileriosis in Egyptian buffaloes. A total 50 field cases of buffaloes and cattle was clinically and laboratory investigated from March to June 2008. Forty-four buffaloes and cattle out of 50 were naturally infected with T. annulata and showed typical signs of infection. Six animals showed no clinical signs and were free from external, internal, and blood parasites. The clinical findings of examined cattle and buffaloes showed typical signs of tropical theileriosis: fever, enlargement of the superficial lymph nodes, severe lacrimation, bilateral conjunctivitis, photophobia, and corneal opacity. It was clear that the severity of clinical signs in infected buffaloes was more prominent than in infected cattle with persistence of some lesions after recovery as corneal opacity and pulmonary lesions. Haematological analysis revealed a significant decrease in RBCS count, PCV%, haemoglobin amount, and WBCs in the infected animals when compared to the control group. It was concluded from our study that T. annulata infection is associated with impairment and alteration of blood parameters in both cattle and water buffaloes. Theileriosis in water buffaloes might cause irreversible ocular changes that could lead to complete blindness. Data obtained in this study might be the basis for subsequent studies under natural and experimental field conditions.