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.

Dynamics and within-host interaction of Theileria lestoquardi and T. ovis among naive sheep in Oman.

Mixed species infections of Theileria spp. are common in nature. Experimental and epidemiological data suggest that mixed species infections elicit cross-immunity that can modulate pathogenicity and disease burden at the population level. The present study examined within-host interactions, over a period of 13 months during natural infections with two Theileria spp., pathogenic (T. lestoquardi) and non-pathogenic (T. ovis), amongst a cohort of naive sheep in Oman. In the first two months after exposure to infection, a high rate of mortality was seen among sheep infected with T. lestoquardi alone. However, subsequently mixed-infections of T. lestoquardi and T. ovis prevailed, and no further death occurred. The overall densities of both parasite species were significantly higher as single infection vs mixed infection and the higher relative density of pathogenic T. lestoquardi indicated a competitive advantage over T. ovis in mixed infection. The density of both species fluctuated significantly over time, with no difference in density between the very hot (May to August) and warm season (September to April). A high degree of genotype multiplicity was seen among T. lestoquardi infections, which increased with rising parasite density. Our results illustrate a potential competitive interaction between the two ovine Theileria spp., and a substantial reduction in the risk of mortality in mixed parasite infections, indicating that T. ovis confers heterologous protection against lethal T. lestoquardi infection.

A longitudinal study of the effect of Theileria orientalis Ikeda type infection on three New Zealand dairy farms naturally infected at pasture.

The aims of this study were to monitor the change in Theileria orientalis Ikeda type infection intensity, haematocrit, milk production and reproduction on three New Zealand spring calving dairy herds, over the 2014-2015 milking season. Three spring calving dairy farms, A, B and C, from high risk (endemically stable), low risk (endemically unstable), and zero risk (disease-free) tick areas respectively were followed through the 2014-2015 milking season. On Farms, A and B, 100 cows were randomly selected at the first visit, and the same cows blood sampled every month thereafter, whilst on Farm C, the whole herd was blood sampled bimonthly (140 cows). Blood samples were tested for haematocrit, by centrifugation, and Ikeda infection intensity, using qPCR. Animals that were Ikeda type PCR positive at the first sampling were described as prevalence cases and cows that were negative at the first sampling and became PCR positive during the sampling period were described as incidence cases. Production and reproduction data were accessed through LIC MINDA® and milk production data was standardised to energy corrected milk (ECM). In addition, the effect of buparvaquone (BPQ) treatment on milk production was estimated on Farm B. The prevalence of infection at the first sampling was 100 % on Farm A, 57 % on Farm B and 26 % on Farm C. The incidence risk of infection over the sampling period on Farms B and C was 25 % and 2 % and the incident rate was 0.026 and 0.002 cases per cow-month respectively. The average infection intensity for prevalence cases on all farms was low throughout the milking season, <7000 Ikeda organisms/µL however, cases of anaemia still occurred. There was no direct effect of infection intensity on milk production or from being a prevalence case compared to an uninfected cow on milk production, across all farms. However, on Farm B there was a loss of 266 kg (95 % CI 82 ̶ 450) ECM (∼20 kg milk solids) for incidence cases and a loss of 458 kg (95 % CI 211 ̶ 710) of ECM for buparvaquone treated cows, compared to uninfected cows. No significant effect of Ikeda infection on reproduction could be shown for Farms B and C, reproductive data for Farm A was not available. The effect of T. orientalis Ikeda type infection on production and reproduction appears to be minimal once animals have passed through the acute phase of infection and reached the chronic, asymptomatic carrier phase of infection.

Economic Significance of Tropical Theileriosis on a Holstein Friesian Dairy Farm in Pakistan.

The dairy industry in Pakistan is booming, and investors are anxious to fund dairy farms that are using high-milk-producing (exotic) cattle breeds such as Holstein Friesians that are not native to the country. Unfortunately, the benefits of increased milk production do not provide resistance to pathogens present in regions where the exotic breeds are introduced. Therefore, the current study was conducted to evaluate the economic impact of Theileria annulata on a commercial Holstein Friesian dairy farm in the District of Ranjanpur, in the Province of Punjab, Pakistan. The economic impact of T. annulata infection was calculated for cattle with subclinical and clinical theileriosis. Losses were estimated based on milk production, morbidity, mortality, and tick control costs (organophosphate sprays). Animals were classified into groups after screening for mastitis, teat abnormality, abnormal parturition, intestinal parasites, and hemoparasites ( T. annulata, Babesia spp., and Anaplasma spp.). Microscopy was done for hemoparasites and intestinal parasites. PCR was used to confirm microscopic identification of T. annulata. Animals were classified into 3 groups: group A (normal), group B (subclinical theileriosis), and group C (acute theileriosis). Hemoparasites were observed microscopically in 28.7% of cows. Theileria annulata was found in 8%, and the herd incidence (new cases) of T. annulata was 2.8%. Milk production, animal rectal temperature, and body condition scores between group A and groups B and C were significantly different ( P < 0.05). But the enlargement of sub-scapular lymph node and interval of body condition score of the 3 groups were not significant ( P > 0.05). The total expenditure incurred due to theileriosis was US $74.98 per animal and 13.83% of total farm costs. Hence theileriosis caused significant economic loss of US $18,743.76 (0.02 million) on this Holstein Friesian dairy.

Variation in cardiac markers and electrocardiographic alterations in young calves naturally infected with bovine tropical theileriosis.

The present study was designed to assess the deleterious effects of bovine tropical theileriosis on the cardiovascular system and the consequent myocardial involvement in young calves. Myocardial effects in parasitic diseases are often neglected. Hemolytic anemia, associated secondary hypoxia, and vasculitis are cardinal features of bovine theileriosis. In the present study, electrocardiogram (ECG) alongside serum cardiac troponin I (cTnI) and creatinine phosphokinase-myocardial band (CPK-MB) concentrations were analyzed in infected, treated, and control groups of young calves. Non-significant alterations were noticed in ECG. However, certain signs like sinus tachycardia, first-degree AV block, atrial premature complex, left atrial hypertrophy, and right atrial hypertrophy were found on consistent basis in infected calves. A significant increase in the serum concentration levels of cTnI and CPK-MB was noticed in infected calves followed by significant fall in both these biomarkers post treatment. cTnI and CPK-MB can definitely be used as myocardial markers in theileriosis-affected animals.

Application of system dynamics and participatory spatial group model building in animal health: A case study of East Coast Fever interventions in Lundazi and Monze districts of Zambia.

East Coast Fever (ECF) is the most economically important production disease among traditional beef cattle farmers in Zambia. Despite the disease control efforts by the government, donors, and farmers, ECF cases are increasing. Why does ECF oscillate over time? Can alternative approaches such as systems thinking contribute solutions to the complex ECF problem, avoid unintended consequences, and achieve sustainable results? To answer these research questions and inform the design and implementation of ECF interventions, we qualitatively investigated the influence of dynamic socio-economic, cultural, and ecological factors. We used system dynamics modelling to specify these dynamics qualitatively, and an innovative participatory framework called spatial group model building (SGMB). SGMB uses participatory geographical information system (GIS) concepts and techniques to capture the role of spatial phenomenon in the context of complex systems, allowing stakeholders to identify spatial phenomenon directly on physical maps and integrate such information in model development. Our SGMB process convened focus groups of beef value chain stakeholders in two distinct production systems. The focus groups helped to jointly construct a series of interrelated system dynamics models that described ECF in a broader systems context. Thus, a complementary objective of this study was to demonstrate the applicability of system dynamics modelling and SGMB in animal health. The SGMB process revealed policy leverage points in the beef cattle value chain that could be targeted to improve ECF control. For example, policies that develop sustainable and stable cattle markets and improve household income availability may have positive feedback effects on investment in animal health. The results obtained from a SGMB process also demonstrated that a "one-size-fits-all" approach may not be equally effective in policing ECF in different agro-ecological zones due to the complex interactions of socio-ecological context with important, and often ignored, spatial patterns.

Detection of Theileria parva in tissues of cattle undergoing severe East Coast fever disease show significant parasite DNA accumulation in the spleen.

Infiltration and proliferation of Theileria parva infected lymphocytes in bovine host lymphoid organs is one of the hallmarks of T. parva infection. The relative abundance of parasites within infected host tissues, both lymphoid and non-lymphoid is however unknown. Using quantitative PCR, we have shown that significantly higher concentrations of T. parva DNA are detected in the spleens of cattle undergoing severe disease compared to other organs.

An experimental ovine Theileriosis: The effect of Theileria lestoquardi infection on cardiovascular system in sheep.

The malignant ovine theileriosis is caused by Theileria lestoquardi, which is highly pathogenic in sheep. Theileriosis involves different organs in ruminants, but the effect of the disease on the cardiovascular system is unclear. To understand the pathogenesis of T. lestoquardi on the cardiovascular system, Baluchi breed sheep were infected with the mentioned parasite by releasing unfed adults of Hyalomma anatolicum anatolicum, which were infected with T. lestoquardi. The infected sheep were clinically examined on days 0, 2, 5, 7, 10, 12, 14, 17, and 21, and the blood samples were collected for biochemical parameters measurement. At termination of the experiment, the infected sheep were euthanized and pathological examinations of heart tissue were conducted. During experimental infection of sheep with T. lestoquardi, activities of cardiac troponin I (cTnI), lactate dehydrogenase, and aspartate aminotransferase, were significantly increased (P˂0.05), while a conspicuous decrease (P˂0.05) was observed in creatine phosphokinase activities. Alterations made in biochemical factors almost coincided with the presence of piroplasm in the blood and schizont in lymph nodes. Maximum and minimum of parasitemia in the sheep stood between 3.3% and 0.28%, respectively. In addition, electrocardiography revealed sinus tachycardia, sinus arrhythmia, sino-atrial block and ST-elevation, atrial premature beat, and alteration in QRS and in T waves' amplitude. Heart histopathological examination showed hyperemia, infiltration of mononuclear inflammatory cells into interstitial tissue, endocarditis, and focal necrosis of cardiac muscle cells. In addition, in one of the sheep, definite occurrence of infarction was observed. The results indicate that T. lestoquardi infection has devastating pathological impacts on the cardiovascular system of sheep. Furthermore, measurement of the cTnI amount is a useful biochemical factor for diagnosis and for better understanding of the severity and progression of the disease and its effects on cardiac tissue.

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.

Theileria-transformed bovine leukocytes have cancer hallmarks.

The genus Theileria includes tick-transmitted apicomplexan parasites of ruminants with substantial economic impact in endemic countries. Some species, including Theileria parva and Theileria annulata, infect leukocytes where they induce phenotypes that are shared with some cancers, most notably immortalization, hyperproliferation, and dissemination. Despite considerable research into the affected host signaling pathways, the parasite proteins directly responsible for these host phenotypes remain unknown. In this review we outline current knowledge on the manipulation of host cells by transformation-inducing Theileria, and we propose that comparisons between cancer biology and host-Theileria interactions can reveal chemotherapeutic targets against Theileria-induced pathogenesis based on cancer treatment approaches.

Detection of Theileria equi in spleen and blood of asymptomatic piroplasm carrier horses.

This study aimed to determine whether asymptomatic horses naturally infected with Theileria equi retain infected erythrocytes in the spleen and whether the presence of the hemoparasite in this organ is associated with parasitemia. We collected samples from 25 adult horses without clinical signs of any disease. From each animal, we collected whole blood samples from the jugular vein and a splenic puncture blood sample. All samples were submited to blood cell counts and detection of Theileria or Babesia. DNA extraction and PCR were performed in all samples for identification of piroplasm infection (T. equi and B. caballi). From the 25 horses evaluated for piroplasm detection by PCR, seven horses (28%) were positive in jugular vein blood but negative in splenic blood samples, five horses (20%) were positive in splenic blood samples but negative in jugular vein blood samples, and 13 horses (52%) were positive in both jugular vein and splenic blood samples. The hematological evaluation revealed anemia in 13 of 25 (52%) infected horses, lymphopenia in five (20%), neutrophilia in two (8%), neutropenia in one (4%), and thrombocytopenia in one (4%) infected horse. The present study demonstrated that several (20%) of the asymptomatic piroplasm carrier horses did not show parasitemia, but show infected erythrocytes in the spleen.

Innate immunity to tropical theileriosis.

The intracellular protozoan parasite Theileria annulata causes a severe, and often fatal, disease of pure and cross-bred cattle in tropical and subtropical countries. The present review refers to the importance of innate immunity as far as it is known to date in this infectious disease. Specifically, macrophages and the mediators produced by these cells are outlined. In addition, the latest findings concerning cattle breed differences in susceptibility to T. annulata infection in relation to macrophage activation are discussed.

The host responses in sheep artificially infected with Theileria sp. (China).

Studies on host responses in sheep artificially infected with Theileria sp. (China) were discussed and summarized mainly on typical high fever periods, merozoits and schizoon observation, antibody response.

A PKA survival pathway inhibited by DPT-PKI, a new specific cell permeable PKA inhibitor, is induced by T. annulata in parasitized B-lymphocytes.

T. annulata, an intracellular pathogenic parasite of the Aplicomplexa protozoan family infects bovine B-lymphocytes and macrophages. Parasitized cells that become transformed survive and proliferate independently of exogenous growth factors. In the present study, we used the isogenic non parasitized BL3 and parasitized TBL3 B cell lines, as a model to evaluate the contribution of two-major PI3-K- and PKA-dependent anti-apoptotic pathways in the survival of T. annulata parasitized B lymphocytes. We found that T. annulata increases PKA activity, induces over-expression of the catalytic subunit and down-regulates the pro-survival phosphorylation state of Akt/PKB. Consistent with a role of PKA activation in survival, two pharmacological inhibitors H89 and KT5720 ablate PKA-dependent survival of parasitized cells. To specifically inhibit PKA pro-survival pathways we linked the DPTsh1 peptide shuttle sequence to PKI(5-24) and we generated DPT-PKI, a cell permeable PKI. DPT-PKI specifically inhibited PKA activity in bovine cell extracts and, as expected, also inhibited the PKA-dependent survival of T. annulata parasitized TBL3 cells. Thus, parasite-dependent constitutive activation of PKA in TBL3 cells generates an anti-apoptotic pathway that can protect T. annulata-infected B cells from apoptosis. These results also indicate that DPT-PKI could be a powerful tool to inhibit PKA pathways in other cell types.

Accelerated binding of autoantibody to red blood cells with increasing anaemia in cattle experimentally infected with Theileria sergenti.

Anaemia is the most important clinical manifestation in cattle infected with Theileria sergenti. In order to determine the mechanism of red blood cells (RBC) destruction in anaemic cattle, we studied the binding of autoantibody (IgG) to RBC during the development of anaemia in T. sergenti infection. The low levels of IgG-bound RBC before the development of anaemia were triggered in proportion with the progression of anaemia and parasitaemia. Our results suggest an accelerated destruction of RBC in anaemic cattle by IgG-dependent phagocytosis.

Theileria-induced leukocyte transformation.

The intracellular protozoan parasites Theileria parva and T. annulata transform the cells they infect, inducing uncontrolled proliferation. This is not a trivial event as, in addition to permanently switching on the complex pathways that govern all steps of the cell cycle, the built-in apoptotic safety mechanisms that prevent 'illegitimate' cell replication also need to be inactivated. Recent experiments show that the NF-kappa B and phosphoinositide 3-kinase (PtdIns-3K) pathways are important participants in the transformation process. I kappa B kinase (IKK), a pivotal kinase complex in the NF-kappa B pathway, is recruited to the parasite surface where it becomes activated. The PtdIns-3K/Akt/PKB pathway is also constitutively activated in a parasite-dependent manner, but contrary to IKK, activation is probably not triggered by direct association with the parasite.

Observation on the schizont stage of an unidentified Theileria sp. in experimentally infected sheep.

The schizont stage of an unidentified Theileria sp. infective for small ruminants was observed. Intact sheep were infected with adult Haemaphysalis qinghaiensis collected from fields where ovine theileriosis was prevalent. When the infested sheep developed theileriosis, tissue and organ smears were prepared. Theileria schizonts were demonstrated in liver, spleen, lung, kidney, lymph node and peripheral blood. Most of the schizonts were found outside the host cells, which was probably a smear artifact. This study adds our knowledge that lung and kidney can be parasitized by schizonts of Theileria sp.

The role of cytokines in immunity and immunopathogenesis of pirolasmoses.

Cells involved in innate and adapted immunity produce cytokines capable of orchestrating the immune response to Babesia and Theileria infections. Thus, CD4-postive T cells recognise peptide fragments of the parasites in the context of the major histocompatibility complex (MHC) class II antigen and produce gamma interferon (IFN-gamma) to activate macrophages for enhanced phagocytosis and intracellular killing of the parasites. In addition, CD4-positive T cells produce interleukin 2 (IL-2) which is essential for the clonal expansion of CD8-positive T cells. The latter cells kill Theileria-infected host leucocytes in an MHC class I-dependent manner. On the other hand, the overproduction of proinflammatory cytokines contributes to disease progress.

A survey of Theileria sergenti infection, daily weight gain and conception proportions in 85 herds of grazing heifers in Japan.

A survey of Theileria sergenti infections, daily weight gain and conception proportion was conducted in 85 herds of grazing heifers in Japan. Basic information and epidemiological data from participating herds were obtained by mailed questionnaires, which were completed by field veterinarians. The average cumulative incidence and proportion of treated animals in the participating herds were 25.7 and 21.1%, respectively. The average daily weight gain and conception proportion were 0.51 kg per day and 56.9%, respectively. The basic information and epidemiological data had a large range and standard deviation, which reflect the wide diversity of the grazing herds in Japan. Herds with heavy tick infestation had significantly higher cumulative incidence and proportion of treated animals, therefore, this factor can be a good estimator to predict the occurrence and loss by theileriosis of the herds. The present questionnaire survey was useful for obtaining information about herds in different regions, and this survey method can be applied to the research of other animal diseases in Japan.

Theileriosis in a Missouri beef herd caused by Theileria buffeli: case report, herd investigation, ultrastructure, phylogenetic analysis, and experimental transmission.

A 6-year-old Simmental cow infected with Theileria buffeli had a clinical disease characterized by theilerial parasitemia, macrocytic normochromic anemia with acanthocytosis and spherocytosis, lymphoid hyperplasia (lymphocytosis, edematous lymphadenomegaly), dysproteinemia, evidence of liver disease, and a low serum antibody titer against T. buffeli. The cow was in a herd in which all cattle originated in Missouri; 22/75 (29%) of cattle had a theilerial parasitemia and 26/75 (35%) had titers to T. buffeli of > or =1:160. Classification of the Missouri bovine organism as T. buffeli was based on DNA sequencing and comparison to sequences for T. buffeli and Theileria sp. type A obtained from GenBank. Intraerythrocytic veils and piroplasms were seen during transmission electron microscopy. The organism was successfully transmitted to two splenectomized calves, which developed mild anemias while parasitemic. Blood from the second calf was used as the source of T. buffeli antigen for an indirect immunofluorescence antibody test. Theilerial isolates from a Missouri white-tailed deer were also sequenced and resembled Theileria sp. types F and G and were not consistent with the bovine organism.