Control of Theileria annulata in Iran

Tropical theileriosis or Mediterranean Coast Fever - caused by Theileria annulata - is a disease of cattle widely distributed across southern Europe, north Africa and central Asia. Its distribution broadly corresponds with that of its main ixodid tick vectors Hyalomma excavatum and H. detritum (Fig. 1). 'Exotic' cattle (Bos taurus) are particularly susceptible with mortalities up to 40-80% in some areas, whereas indigenous cattle (B. indicus) generally suffer much lower mortalities (about 10%) confined mainly to calves. But because imported non-immune cattle are so susceptible, T. annulata represents a major constraint to livestock improvement programmes in many parts of the middle east and Asia. Cattle that recover from T. annulata infection generally show a solid, long-lasting immunity. For many years there have been programmes to protect cattle by inoculation with blood from sick animals, and more recently using live attenuated T. annulata vaccines prepared from cultured schizont-infected lymphoid cells. This article reviews a 14 year immunization programme against T. annulata in Iran.     

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.     

Loop-mediated isothermal amplification (LAMP) assays for the detection of Theileria annulata infection in China targeting the 18S rRNA and ITS sequences

We have developed two loop-mediated isothermal amplification (LAMP) assays for the detection of Theileria annulata, an economically important cattle disease in China that occurs in subtropical and tropical areas. These assays target the ribosomal RNA (18S rRNA) and ITS LAMP sequences. The primer set for each gene target consists of four primers, and each set recognizes six distinct regions on the target gene to allow for the highly specific detection of T. annulata. The specific ladder bands were amplified from the autologous genomic DNA of four Chinese-laboratory-preserved standard T. annulata stocks, and there were no cross-reactions with the genomic DNA of normal bovine blood and other protozoan species. The LAMP assays were sufficiently sensitive to detect 0.1 pg/μl of genomic DNA. Furthermore, DNA extracted from blood collected from cattle experimentally infected with T. annulata (18-105 days post-infection) was amplified, demonstrating the high sensitivity of these primers. Of the 351 field samples collected from China, 24.5% were positively detected by two LAMP primers, and 18.2% were found to be positive for T. annulata infection by PCR. These results indicate that the LAMP assay could be a potential diagnostic tool for epidemiological studies of T. annulata infection in China.     

Detection of Theileria annulata by the PCR in ticks (Acari: Ixodidae) collected from cattle in Mauritania

We report on the detection of Theileria annulata in infected Hyalomma ticks by the PCR using primers derived from the gene encoding the 30 kDa major merozoite surface antigen (Tams1–1). No inhibition of the PCR was observed and as little as 0.1 pg of parasite DNA, corresponding to 12 sporozoites, could be detected in non-infected tick DNA samples, spiked with T. annulata genomic DNA. Hyalomma dromedarii ticks, fed on a calf experimentally infected with T. annulata, were used to validate the PCR further. The infection rate in the adult ticks, fed as nymphs during the febrile reaction, was high (62%), dropped to zero for 1 day in tick batches that engorged after treatment with ButalexTM and increased to 30% 2 days later and 38% of the ticks acquired the infection after feeding as nymphs during a carrier state piroplasm parasitaemia of less than 0.1%. As an internal control, 16S tick rDNA sequences could be amplified from T. annulata-negative tick samples. Finally, 202 adult ticks from Mauritania, collected from zebu cattle carrying low levels of Theileria piroplasms, were tested by the PCR. Thirty-eight out of 52 (73%) and 17 out of 30 (57%) H. dromedarii from the Gorgol and Trarza regions, respectively and two out of 30 (7%) Hyalomma marginatum rufipes from the Gorgol region were positive. Hyalomma marginatum rufipes, Rhipicephalus evertsi evertsi and Rhipicephalus guilhoni from the Trarza region were negative. These findings confirm that H. dromedarii is the main vector of T. annulata in Mauritania and that the PCR is a useful method of determining the infection rates in ticks collected from cattle carrying low levels of T. annulata piroplasms.     

Prevalence of blood parasites in cattle from wilayates of Annaba and El Tarf east Algeria

Between June and September 2002, a preliminary study was conducted to assess the prevalence of blood parasites of cattle in eastern Algeria. Fifty-four bovines of different genotypes were submitted to clinical examination. From each animal, blood smears were made and stained by Giemsa. Four species of parasites, namely Theileria annulata, T. orientalis, Babesia bovis and Anaplasma marginale were encountered. Fifty animals carried single or multiple infections with blood parasites and four were found negative. The rate of single infections (72.3%, n = 39) was almost three times higher than multiple infections (20.3%, n = 11). The high percentage of single infections was recorded with T. annulata (53.7%). However single infection with Anaplasma marginale (7.4 %), B. bovis (5.6%) and T orientalis (5.6%) were very low compared to T. annulata infection. The rates of mixed infection were as follows: T. annulata/A. marginale 9.3%, T. annulata/T. orientalis 5.6%, A. marginale/T. orientalis 3.7% and B. bovis/A. marginale 1.9%.     

The protozoan parasite, Theileria annulata, induces a distinct acute phase protein response in cattle that is associated with pathology

Acute phase proteins (APP) are synthesised in the liver in response to the systemic presence of high levels of pro-inflammatory cytokines. Bacteria are considered to be strong inducers of APP whereas viruses are weak or non-inducers of APP. Very few reports have been published on APP induction by parasites. Here, we report that the tick-borne protozoan parasite of cattle, Theileria annulata, induced an atypical acute phase response in cattle. Following experimental infection, serum amyloid A (SAA) appeared first, followed by a rise in alpha(1) acid glycoprotein (alpha(1)AGP) in all animals, whereas haptoglobin, which is a major APP in cattle, only appeared in some of the animals, and generally at a low level. All three APP only became elevated around or after the appearance of schizonts in draining lymph nodes and after the first observed temperature rise. Increased alpha(1)AGP levels coincided with the appearance of piroplasms. The production of SAA and alpha(1)AGP correlated strongly with each other, and also with some clinical measures of disease severity including the time to fever, development of leucopaenia, parasitaemia and mortality. These results are consistent with the hypothesis that T. annulata causes severe pathology in susceptible cattle by inducing high levels of pro-inflammatory cytokines.     

Development of recombinant antigen vaccines for the control of theileriosis

Immunization against Theileria parva involves infection with sporozoites and simultaneous treatment with a long-acting tetracycline. For T. annulata, immunization is achieved by inoculation of attenuated schizont-infected lymphocytes. The two methods are inadequate because of the use of live organisms and the methods are also bedevilled by the multiplicity of strains, particularly of T. parva. For these reasons, alternative methods of control are being sought. In this review an attempt is made to highlight advances towards subunit vaccines against T. parva and T. annulata. Several candidate antigens which are thought to induce protective responses have been identified and recombinant DNA technology is being employed to produce these antigens in bulk. Relevant antigens may be delivered as subunit vaccines by using recombinant vaccinia virus or attenuated Salmonella spp. as carriers of the genes expressing these antigens. It is likely that effective vaccines against T. parva and T. annulata will have to elaborate immune responses against both the sporozoite and schizont stages of the parasite.     

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.     

Control of tropical theileriosis (Theileria annulata infection) of cattle

Tropical bovine theileriosis caused by Theileria annulata and transmitted by ticks of the genus Hyalomma may be controlled by one or more of the following methods: i) management, with particular emphasis on movement control; ii) vector control by application of acaricides, preventing transmission of disease; iii) treatment of clinical disease using specific chemotherapeutics; iv) immunization with live vaccines; and v) the use of cattle resistant to ticks or the disease. Of these the most important and effective control method is the use of a live cell culture vaccine attenuated by prolonged culture in vitro of mononuclear cells persistently infected with macroschizonts of T. annulata. This vaccine, used chiefly in susceptible taurine dairy cattle, can now be complemented by using novel chemotherapeutic naphthoquinones--parvaquone and buparvaquone--which are very effective in treatment of the clinical disease in these valuable cattle.     

Tropical theileriosis in Tunisia: epidemiology and control

In Tunisia, tropical theileriosis (Theileria annulata infection of cattle) is one of the major diseases affecting cattle in the summer. Each year about 2,500 clinical cases are recorded in the country, mainly in pure-bred animals. An attempt was made in the North of Tunisia to estimate the direct cost related to clinical cases of TT. In the endemic regions, three states of endemicity were described according to cattle age categories at highest disease risk: (i) endemic stability, (ii) low endemic instability, (iii) high endemic instability. The characterisation of these endemic situations, which are conditioned by the levels of the vector tick population and the quantitative aspects of the infection in ticks, are highly relevant for the development of a live attenuated cell line vaccine against TT targeting the cattle population at disease risk in Tunisia. A research programme was set up to develop the attenuation of four local parasite stocks. Two cell lines infected with two distinct T. annulata stocks were tested in the field on pure-bred animals of different age groups from regions with high disease incidence. The vaccination with each of the two cell lines showed a high efficacy. However significant differences in vaccine reactions rates were observed between the two attenuated cell lines emphasising the importance of achieving an optimal balance between protection and vaccine tolerance particularly in pure-bred lactating cows.     

Characterization of Theileria species by PCR using specific target sequences

Theileriosis is an infectious disease in tropical countries and in the Mediterranean area. It is caused by Theileria, a haemoprotozoan, transmitted by vectors belonging to the Ixodidae. In Southern Italy and in Sicily the infection is due mainly to T. annulata, but in some cases other species are involved in the disease. The authors describe a method to identify theileriosis in cattle blood samples, using PCR and hybridization techniques. Different primer sets were used to amplify different DNA target sequences, both genus and species specific. Blood samples from cattle were collected in Sicily. The DNA extracted from blood samples was employed first to detect the presence of the 18S ribosomal subunit gene specific for Theileria genus. Successively the positive samples were analysed to identify the species, T. annulata or T. buffeli/orientalis, using as target sequences for amplification respectively a fragment of the TAMS-1 and p33/34 antigens gene. Here the authors describe for the first time the presence of T. buffeli/orientalis infection in Sicilian herds. In fact 66% of positive blood samples were T. buffeli/orientalis infected.     

Immunity in theileriosis

The theilerioses can be separated on the basis of their principal pathogenic features, into a lymphoproliferative group caused by Theileria parva and T. annulata in cattle, and T. hirci in goats and sheep, and a haemoproliferative group caused by T. sergenti and T. mutans both in cattle. In the former group, proliferation of parasites within lymphoid cells followed by lymphodestruction are the main pathogenic features; whereas in the latter group, invasion and destruction of erythrocytes, causing anaemia, are more important. In addition, a number of other theilerial parasites which cause mild or inapparent infections, are found in domestic livestock. This review focuses on T. parva, the causative agent of East Coast fever (ECF) in cattle in East and Central Africa, because it is the most pathogenic species and the immunology of ECF has been more intensively studied than that of the other theilerioses.     

Detection of Theileria annulata carrier cattle by PCR

A simple method for treating bovine blood samples for direct detection of T. annulata in carriers, after polymerase chain reaction (PCR) amplification of small subunit ribosomal RNA (SSU rRNA) gene is described. The threshold of detection of the PCR-assay was an erythrocytic parasitaemia of 0.00008% corresponding to 16 infected bovine erythrocytes. In 50 known carriers, 42 were positive in PCR, in which 8 cattle revealed presence of T. annulata in stained blood smear under microscope.     

Bos taurus and Bos indicus (Sahiwal) calves respond differently to infection with Theileria annulata and produce markedly different levels of acute phase proteins

Disease-resistant livestock could provide a potentially sustainable and environmentally sound method of controlling tick and tick-borne diseases of livestock in the developing world. Advances in the knowledge and science of genomics open up opportunities to identify selectable genes controlling disease resistance but first, breeds and individuals with distinguishable phenotypes need to be identified. The Bos indicus breed, Sahiwal, has been exploited in dairy breeding programmes, because it is resistant to ticks and has relatively good performance characteristics compared to other indigenous cattle breeds of tropical regions. The analyses reported here show that Sahiwal calves were also more resistant than European Bos taurus (Holstein) dairy breed calves to tick-borne tropical theileriosis (Theileria annulata infection). Following experimental infection with T. annulata sporozoites, a group of Sahiwal calves all survived without treatment, with significantly lower maximum temperatures (P<0.01) and lower rates of parasite multiplication (P<0.05) than a group of Holstein calves, which all had severe responses. Although the Sahiwals became as anaemic as the Holsteins, other measures of pathology, including enlargement of the draining lymph node and the acute phase proteins, alpha1 acid glycoprotein and haptoglobin, were significantly less in the Sahiwals than in the Holsteins (P<0.05). Additionally, the Sahiwals had significantly lower resting levels of alpha1 acid glycoprotein than the Holsteins (P<0.05). Production of a third acute phase proteins, serum amyloid A, had very similar kinetics in both breeds. Acute phase proteins are produced in response to systemic release of the kinds of pro-inflammatory cytokines that are thought to be responsible for the pyrexic, cachectic and anorexic responses characteristic of tropical theileriosis. The prolonged production of alpha1 acid glycoprotein in the Holsteins is indicative of chronic production of circulating pro-inflammatory cytokines. In contrast, Sahiwals appear able to overcome infection with T. annulata as well as limit pathology by preventing the over-stimulation of pathways involving these cytokines.     

Studies on theileriosis in Southern Spain

Mediterranean theileriosis is widely distributed throughout Southern Spain, where it causes economic losses of considerable importance. In recent studies on 221 fighting bulls from 29 locations of different provinces, mainly in Southern Spain, the seroprevalence was 75.1% by IFAT. On the other hand, we are carrying out studies on beef and dairy breeds from eight provinces in the southern half of Spain where the positivity by IFAT ranges from 50 to more than 90% of the sampled cattle. Studies on the isolation of T. annulata strains, the demonstration of the vectorial role of some tick species, and cattle immunisation, have been carried out as well. In order to isolate the causative agent of the disease, infected blood or biopsies of lymph nodes from infected animals were used to establish in vitro cultures of T. annulata-infected lymphoblastoid cells, in six different cell lines. Attenuation of the parasites was achieved by growth in successive passages of a cell line by in vitro culture. Nowadays, a tissue culture vaccine has been developed from a local strain which is tried in experimental calves and under field condition with successful results. Besides, some studies on the transmission of T. annulata to cattle by the tick Hyalomma lusitanicum have been done.     

Detection of natural infections with Theileria annulata on calves at first theileriosis season: comparison of the Indirect Fluorescent Antibody Test (IFAT) and blood smears

The Indirect Fluorescent Antibody Test (IFAT) remains so far the most commonly used test for sero-epidemiological investigations on tropical theileriosis (infection of cattle with Theileria annulata). The present studies evaluated the ability of both IFAT with schizont antigen (schizont IFAT) and blood smears to detect infected animals just after the theileriosis season. This evaluation was performed on a group of 89 calves of known infection status for T. annulata at first disease season, from farms with endemic stability for tropical theileriosis. An additional retrospective group of 84 cattle free of infection was also used for the estimation of the specificity of the schizont IFAT. The sensitivity and the specificity of schizont IFAT were 88.9% (64/72) and 97% (98/101), respectively. Blood smears showed a lower sensitivity of 63.9% (46/72). The agreement between the two detection techniques and the infection status of the animals, evaluated by the Kappa coefficient, was 0.85 and 0.64 for IFAT and blood smears, respectively.     

ApiAP2 Factors as Candidate Regulators of Stochastic Commitment to Merozoite Production in Theileria annulata

Background

Differentiation of one life-cycle stage to the next is critical for survival and transmission of apicomplexan parasites. A number of studies have shown that stage differentiation is a stochastic process and is associated with a point that commits the cell to a change over in the pattern of gene expression. Studies on differentiation to merozoite production (merogony) in T. annulata postulated that commitment involves a concentration threshold of DNA binding proteins and an auto-regulatory loop.

Principal Findings

In this study ApiAP2 DNA binding proteins that show changes in expression level during merogony of T. annulata have been identified. DNA motifs bound by orthologous domains in Plasmodium were found to be enriched in upstream regions of stage-regulated T. annulata genes and validated as targets for the T. annulata AP2 domains by electrophoretic mobility shift assay (EMSA). Two findings were of particular note: the gene in T. annulata encoding the orthologue of the ApiAP2 domain in the AP2-G factor that commits Plasmodium to gametocyte production, has an expression profile indicating involvement in transmission of T. annulata to the tick vector; genes encoding related domains that bind, or are predicted to bind, sequence motifs of the type 5''-(A)CACAC(A) are implicated in differential regulation of gene expression, with one gene (TA11145) likely to be preferentially up-regulated via auto-regulation as the cell progresses to merogony.

Conclusions

We postulate that the Theileria factor possessing the AP2 domain orthologous to that of Plasmodium AP2-G may regulate gametocytogenesis in a similar manner to AP2-G. In addition, paralogous ApiAP2 factors that recognise 5''-(A)CACAC(A) type motifs could operate in a competitive manner to promote reversible progression towards the point that commits the cell to undergo merogony. Factors possessing AP2 domains that bind (or are predicted to bind) this motif are present in the vector-borne genera Theileria, Babesia and Plasmodium, and other Apicomplexa; leading to the proposal that the mechanisms that control stage differentiation will show a degree of conservation.     

Characterization of Anamnestic T-cell Responses Induced by Conventional Vaccines against Contagious Bovine Pleuropneumonia

A better understanding of how T1 vaccination confers immunity would facilitate the rational design of improved vaccines against contagious bovine pleuropneumonia (CBPP). We show here that mycoplasmas-induced recall proliferation and IFN-γ responses are detected in cattle that received multiple shots of T1 vaccines. These anamnestic responses were under the strict control of CD4⁺ T lymphocytes. Moreover, CD62L expression indicated that both CD4⁺ effector memory (Tem) and central memory (Tcm) T lymphocytes are elicited in these animals. Comparative analysis with data from cattle that completely recovered from CBPP infection revealed similar anamnestic T-cell responses albeit at a lower magnitude for T1-vaccinated animals, particularly in the Tcm compartment. In conclusion, we discuss how our current understanding of T-cell responses will contribute to ongoing efforts for the improvement of future CBPP vaccines.     

Alteration of host cell phenotype by Theileria annulata and Theileria parva: mining for manipulators in the parasite genomes

The apicomplexan parasites Theileria annulata and Theileria parva cause severe lymphoproliferative disorders in cattle. Disease pathogenesis is linked to the ability of the parasite to transform the infected host cell (leukocyte) and induce uncontrolled proliferation. It is known that transformation involves parasite dependent perturbation of leukocyte signal transduction pathways that regulate apoptosis, division and gene expression, and there is evidence for the translocation of Theileria DNA binding proteins to the host cell nucleus. However, the parasite factors responsible for the inhibition of host cell apoptosis, or induction of host cell proliferation are unknown. The recent derivation of the complete genome sequence for both T. annulata and T. parva has provided a wealth of information that can be searched to identify molecules with the potential to subvert host cell regulatory pathways. This review summarizes current knowledge of the mechanisms used by Theileria parasites to transform the host cell, and highlights recent work that has mined the Theileria genomes to identify candidate manipulators of host cell phenotype.