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.     

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.     

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.     

Population diversity and multiplicity of infection in Theileria annulata

The tick-borne apicomplexan parasite Theileria annulata is endemic in many sub-tropical countries and causes the bovine disease tropical theileriosis. Although the parasite is known to be highly diverse, detailed information is lacking on the genetic structure of natural populations and levels of multiplicity of infection in the cattle host. With the widespread deployment of live attenuated vaccines and the emergence of drug-resistant parasites in the field, it is vital to appreciate the factors which shape genetic diversity of the parasite both within individual hosts and in the wider population. This study addresses these issues and represents an extensive genetic analysis of T. annulata populations in two endemic countries utilising a high-throughput adaptation of a micro- and mini-satellite genotyping system. Parasite material was collected from infected cattle in defined regions of Turkey and Tunisia to allow a variety of analyses to be conducted. All animals (n = 305) were found to harbour multiple parasite genotypes and only two isolates shared an identical predominant multi-locus profile. A modelling approach was used to demonstrate that host age, location and vaccination status play a measurable role in determining multiplicity of infection in an individual animal. Age was shown to positively correlate with multiplicity of infection and while positive vaccination status exerted a similar effect, it was shown to be due not simply to the presence of the immunising genotype. Importantly, no direct evidence was found for the immunising genotype spreading or recombining within the local parasite community. Genetic analysis confirmed the tentative conclusion of a previous study that the parasite population appears to be, in general, panmictic. Nevertheless, evidence supporting linkage disequilibrium and a departure from panmixia was uncovered in some localities and a number of explanations for these findings are advanced.     

Theileria annulata: attenuation of a schizont-infected cell line by prolonged in vitro culture is not caused by the preferential growth of particular host cell types

Flow cytometry and monoclonal antibodies to bovine leucocyte surface antigens were used to identify the types of host cells that the sporozoites of Theileria annulata infect in cattle, to determine whether virulent schizont-infected cell lines (lines) differed phenotypically from avirulent lines, and to establish whether attenuation in vitro was accompanied by the preferential growth of particular host cell types. The surface antigens of four pairs of T. annulata (Ta) (Hisar) lines derived ex vivo and in vitro, including the virulent ex vivo-derived Ta Hisar S45 line, were consistent with a myeloid origin for all lines, irrespective of their derivation. The profiles of lines derived from cattle inoculated with a virulent line showed that the schizonts liberated from inoculated cells had transferred to myeloid cells. A number of other lines infected with different stocks of T. annulata expressed myeloid markers; a single line expressed CD21, a B cell marker. During prolonged in vitro culture, the parasites in the ex vivo (virulent)- and in vitro (avirulent)-derived Ta Hisar S45 myeloid lines became clonal, as defined by glucose phosphate isomerase (GPI) polymorphism, and the virulent line became attenuated. The two lines retained phenotypic profiles indicative of a myeloid origin but coexpressed some lymphoid antigens (CD2, CD4, CD8), although not CD3. Cloned schizont-infected lines, representing the three parasite GPI isotypes which constituted the virulent line, expressed similar patterns of myeloid and lymphoid markers to the virulent parent line. Some schizont-infected clones failed to establish as lines during the early weeks of culture because the cells died as the parasites differentiated into merozoites at 37 degrees C, the temperature at which schizont-infected cells normally grow exponentially. These results provided no evidence that prolonged culture induces preferential growth or loss of particular host cell types. However, a number of the alterations in host cell surface antigens induced by prolonged culture were shown to be linked to permanent changes in the parasite genome.     

Cattle and small ruminant piroplasmosis in Macedonia, Greece

Macedonia is an endemic region of cattle as well as of sheep and goat piroplasmosis In cattle, Theileria orientalis (=T. buffeli?), the agent of Eurasian benign theileriosis, is the most common and widespread piroplasm species. T. annulata, the agent of tropical theileriosis, seems to be rare and limited to few foci, but causes very severe clinical cases, especially in imported pure-bred or cross-bred animals. Babesia bovis and B. bigemina are present in several localities, and often coexist. Cattle babesiosis cases are due to both piroplasms, but B. bovis is considered responsible for the severe and acute clinical cases. Ovine and caprine babesiosis is due to B. ovis and mainly affected imported animals as well as indigenous animals which have been transported from localities where the infection was absent. B. ovis is extremely widespread in both sheep and goats. During the last decades, no clinical cases of small ruminant theileriosis have been registered in this region. However, T. ovis, a non-pathogenic theileria, is common in sheep, but not in goats. Anaplasma ovis, a protobacterium of small ruminants, is also present in the region.     

Distribution and ecology of ticks (Acari: Ixodidae) infesting livestock in Tunisia: an overview of eighth years field collections

Ticks (Ixodidae) play a significant role as vectors of pathogens of domestic animals in Tunisia. The major losses caused by ticks are related to transmission of protozoan parasites. These include agents of tropical theileriosis and babesiosis in ruminants. Since 1991, we conducted research studies on tick population of livestock in Tunisia. This overview reports a synthesis on tick distribution, their biology and their role as vectors of pathogens in domestic animals, particularly cattle. During the whole period of the study about 15,000 tick specimens were collected from different zones of the country. A total of 14 species were identified. Hyalomma detritum detritum was the most abundant and important (vector of Theileria annulata) species infesting cattle. Hyalomma dromedarii and Hyalomma impeltatum were collected on domestic ruminants in the arid and desertic zones. Hyalomma marginatum marginatum and Hyalomma anatolicum excavatum were widespread and found on livestock hosts. Ixodes ricinus, vector of Babesia divergens and Borrelia burgdorferi sl, colonises mainly the humid zone. Boophilus annulatus and Rhipicephalus bursa infesting cattle, sheep and goats were found in the sub-humid and semi-arid zones. Haemaphysalis sulcata and Hae. punctata were collected in humid and sub-humid zones on cattle and sheep. Rhipicephalus turanicus were collected in different regions, on different animal species. Rhipicephalus sanguineus, tick of dogs, were often collected on livestock. Only few specimens of Hyalomma marginatum rufipes and Hyalomma franchinii were collected.     

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.     

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.     

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.     

A modified vaccinia Ankara virus (MVA) vaccine expressing African horse sickness virus (AHSV) VP2 protects against AHSV challenge in an IFNAR -/- mouse model

African horse sickness (AHS) is a lethal viral disease of equids, which is transmitted by Culicoides midges that become infected after biting a viraemic host. The use of live attenuated vaccines has been vital for the control of this disease in endemic regions. However, there are safety concerns over their use in non-endemic countries. Research efforts over the last two decades have therefore focused on developing alternative vaccines based on recombinant baculovirus or live viral vectors expressing structural components of the AHS virion. However, ethical and financial considerations, relating to the use of infected horses in high biosecurity installations, have made progress very slow. We have therefore assessed the potential of an experimental mouse-model for AHSV infection for vaccine and immunology research. We initially characterised AHSV infection in this model, then tested the protective efficacy of a recombinant vaccine based on modified vaccinia Ankara expressing AHS-4 VP2 (MVA-VP2).     

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%.     

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.     

An oral recombinant vaccine in dogs against Echinococcus granulosus, the causative agent of human hydatid disease: a pilot study

Dogs are the main source of human cystic echinococcosis. An oral vaccine would be an important contribution to control programs in endemic countries. We conducted two parallel experimental trials in Morocco and Tunisia of a new oral vaccine candidate against Echinococcus granulosus in 28 dogs. The vaccine was prepared using two recombinant proteins from adult worms, a tropomyosin (EgTrp) and a fibrillar protein similar to paramyosin (EgA31), cloned and expressed in a live attenuated strain of Salmonella enterica serovar typhimurium.In each country, five dogs were vaccinated with the associated EgA31 and EgTrp; three dogs received only the vector Salmonella; and six dogs were used as different controls. The vaccinated dogs received two oral doses of the vaccine 21 d apart, and were challenged 20 d later with 75,000 living protoscoleces. The controls were challenged under the same conditions. All dogs were sacrificed 26-29 d postchallenge, before the appearance of eggs, for safety reasons.We studied the histological responses to both the vaccine and control at the level of the duodenum, the natural localization of the cestode. Here we show a significant decrease of parasite burden in vaccinated dogs (70% to 80%) and a slower development rate in all remaining worms. The Salmonella vaccine EgA31-EgTrp demonstrated a high efficacy against E. granulosus promoting its potential role in reducing transmission to humans and animals.     

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.     

Preventive strategies for frequent outbreaks of Japanese encephalitis in Northern India

Japanese encephalitis (JE) remains the most important cause of acute viral encephalitis and continues to spread to hitherto unaffected regions like Indonesia, Pakistan and Australia. Approximately 60% of the world population inhabits JE endemic areas. Despite its restricted range mostly in the developing countries, a high annual incidence of 50,000 cases and about 10,000 deaths has been reported. Disease can be fatal in 25% cases. Magnitude of the problem is even more alarming since the survivors are left with serious long-term neuropsychiatric sequelae. Almost every two years, epidemics of JE occur in Indian subcontinent with a high mortality. JE virus infection results in different disease manifestations in host from mild subclinical febrile illness to clinical infections leading to encephalitis. No antiviral treatment is so far available for JE. The prevention of JE can be achieved by controlling the vector or by immunization regime. The vector control in the rural areas, which are the worst affected ones, is practically almost impossible. Three vaccines that have been implicated against JE include inactivated mouse brain derived, inactivated cell culture derived and cell culture derived live attenuated JE vaccine. But each has its own limitation. Currently, attempts to synthesize recombinant DNA vaccine are being made. New therapeutics are on the way of development like use of minocycline, short interfering RNA, arctigenin, rosmarinic acid, DNAzymes etc. However, the immune mechanisms that lead to JE are complex and need to be elucidated further for the development of therapeutics as well as safe and efficacious JE vaccines.     

Analysis of the composition of samples of Babesia bovis and the influence of different environmental conditions on genetically distinct subpopulations.

A recombinant DNA probe specific for a tandemly repeated sequence located within the BoVA1 gene of Babesia bovis was used to analyse 10 independent samples of B. bovis. Twelve different alleles of the BoVA1 gene and flanking regions were identified in the 18 different subpopulations analysed. Most samples of B. bovis originally derived from single animals contained more than one genetically distinct subpopulation. However, only one population of parasites was identified in samples of the Ka line used in Australia from 1979 until 1990 as the live attenuated vaccine strain. In contrast, the replacement attenuated vaccine line, Ta, contained two genetically distinct subpopulations of parasites. Changes in the ratios of subpopulations of parasites were identified during attenuation and under different culture conditions. Batch-to-batch variation in the composition of doses of the live attenuated vaccine may lead to differences in efficacy and in severity of the infection associated with vaccination.