Inadequate Differentiation of Theileria orientalis Genotypes buffeli and ikeda in a Multiplexed Tandem PCR (MT-PCR) Assay Using the p23 Gene as a Marker.
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ABSTRACT:
SUBMITTER: Gebrekidan H
PROVIDER: S-EPMC5277536 | biostudies-literature | 2017 Feb
REPOSITORIES: biostudies-literature
Publications
Inadequate Differentiation of Theileria orientalis Genotypes buffeli and ikeda in a Multiplexed Tandem PCR (MT-PCR) Assay Using the p23 Gene as a Marker.
Gebrekidan Hagos H Gasser Robin B RB Jabbar Abdul A
Journal of clinical microbiology 20161207 2
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Project description:BackgroundTheileria orientalis (Apicomplexa: Piroplasmida) has caused clinical disease in cattle of Eastern Asia for many years and its recent rapid spread throughout Australian and New Zealand herds has caused substantial economic losses to production through cattle deaths, late term abortion and morbidity. Disease outbreaks have been linked to the detection of a pathogenic genotype of T. orientalis, genotype Ikeda, which is also responsible for disease outbreaks in Asia. Here, we sequenced and compared the draft genomes of one pathogenic (Ikeda) and two apathogenic (Chitose, Buffeli) isolates of T. orientalis sourced from Australian herds.ResultsUsing de novo assembled sequences and a single nucleotide variant (SNV) analysis pipeline, we found extensive genetic divergence between the T. orientalis genotypes. A genome-wide phylogeny reconstructed to address continued confusion over nomenclature of this species displayed concordance with prior phylogenetic studies based on the major piroplasm surface protein (MPSP) gene. However, average nucleotide identity (ANI) values revealed that the divergence between isolates is comparable to that observed between other theilerias which represent distinct species. Analysis of SNVs revealed putative recombination between the Chitose and Buffeli genotypes and also between Australian and Japanese Ikeda isolates. Finally, to inform future vaccine studies, dN/dS ratios and surface location predictions were analysed. Six predicted surface protein targets were confirmed to be expressed during the piroplasm phase of the parasite by mass spectrometry.ConclusionsWe used whole genome sequencing to demonstrate that the T. orientalis Ikeda, Chitose and Buffeli variants show substantial genetic divergence. Our data indicates that future researchers could potentially consider disease-associated Ikeda and closely related genotypes as a separate species from non-pathogenic Chitose and Buffeli.
| S-EPMC5921998 | biostudies-literature
Project description:Oriental theileriosis is an emerging, tick-borne disease of bovines in the Asia-Pacific region and is caused by one or more genotypes of the Theileria orientalis complex. This study aimed to establish and validate a multiplexed tandem PCR (MT-PCR) assay using three distinct markers (major piroplasm surface protein, 23-kDa piroplasm membrane protein, and the first internal transcribed spacer of nuclear DNA), for the simultaneous detection and semiquantification of four genotypes (Buffeli, Chitose, Ikeda, and type 5) of the T. orientalis complex. Analytical specificity, analytical sensitivity, and repeatability of the established MT-PCR assay were assessed in a series of experiments. Subsequently, the assay was evaluated using 200 genomic DNA samples collected from cattle from farms on which oriental theileriosis outbreaks had occurred, and 110 samples from a region where no outbreaks had been reported. The results showed the MT-PCR assay specifically and reproducibly detected the expected genotypes (i.e., genotypes Buffeli, Chitose, Ikeda, and type 5) of the T. orientalis complex, reliably differentiated them, and was able to detect as little as 1 fg of genomic DNA from each genotype. The diagnostic specificity and sensitivity of the MT-PCR were estimated at 94.0% and 98.8%, respectively. The MT-PCR assay established here is a practical and effective diagnostic tool for the four main genotypes of T. orientalis complex in Australia and should assist studies of the epidemiology and pathophysiology of oriental theileriosis in the Asia-Pacific region.
| S-EPMC4290960 | biostudies-literature
Project description:Theileria orientalis Ikeda genotype is a parasite that causes a disease in cattle that results in major economic issues in Asia, New Zealand, and Australia. The parasite is transmitted by Haemaphysalis longicornis ticks, which have recently been reported in numerous states throughout the eastern United States. Concurrently, cattle in Virginia showed clinical signs consistent with a hemoprotozoan infection. We used amplicons specific for the major piroplasm surface protein and small subunit rDNA of piroplasms to test blood samples from the cattle by PCR. Bidirectional Sanger sequencing showed sequences with 100% identity with T. orientalis Ikeda genotype 2 sequences. We detected the parasite in 3 unrelated herds and from various animals sampled at 2 time points. Although other benign T. orientalis genotypes are endemic to the United States, detection of T. orientalis Ikeda genotype might represent a risk for the cattle industry in Virginia.
| S-EPMC6711211 | biostudies-literature
Project description:Theileria orientalis Ikeda genotype, a parasite causing a disease in cattle that leads to significant economic challenges in Asia, New Zealand, and Australia, has been identified in seven U.S. States since 2017. Two previously validated PCR tests for Theileria followed by DNA sequencing were performed to test blood samples collected from 219 cattle in Alabama, USA, during the period of 2022-2023. Bidirectional Sanger sequencing revealed that the MPSP gene sequences (639-660 bp) from two cattle in Lee and Mobile Counties of Alabama exhibited a 100% match with those of recognized T. orientalis Ikeda strains, and showed similarities ranging from 76% to 88% with ten other T. orientalis genotypes. A high copy number of T. orientalis Ikeda was detected in the blood of infected cattle (ALP-1: 1.7 × 105 and 1.3 × 106/mL whole blood, six months apart; ALP-2: 7.1 × 106/mL whole blood). Although the confirmed competent vector for T. orientalis Ikeda, Haemaphysalis longicornis tick, has not yet been identified in Alabama, the persistent nature of T. orientalis Ikeda infection and the detection of a high pathogen burden in seemingly healthy cattle in this study suggest that other tick species, as well as shared needles and dehorning procedures, could facilitate pathogen transmission within the herd. Continued investigations are necessary for the surveillance of T. orientalis Ikeda and Haemaphysalis longicornis ticks in Alabama and other U.S. states, along with assessing the pathogenicity of T. orientalis Ikeda infections in cattle.
| S-EPMC10674541 | biostudies-literature
Project description:Theileria orientalis causes losses to cattle producers in Eastern Asia, Oceania and, more recently, North America. One pathogenic genotype (Ikeda) has been sequenced to the chromosomal level, while only draft genomes exist for globally distributed Chitose and Buffeli genotypes. To provide an accurate comparative gene-level analysis and help further understand their pathogenicity, we sequenced isolates of the Chitose and Buffeli genotypes of T. orientalis using long-read sequencing technology. A combination of several long-read assembly methods and short reads produced chromosomal-level assemblies for both Fish Creek (Chitose) and Goon Nure (Buffeli) isolates, including the first complete and circular apicoplast genomes generated for T. orientalis. Comparison with the Shintoku (Ikeda) reference sequence showed both large and small translocations in T. orientalis Buffeli, between chromosomes 2 and 3 and chromosomes 1 and 4, respectively. Ortholog clustering showed expansion of ABC transporter genes in Chitose and Buffeli. However, differences in several genes of unknown function, including DUF529/FAINT-domain-containing proteins, were also identified and these genes were more prevalent in Ikeda and Chitose genotypes. Phylogenetics and similarity measures were consistent with previous short-read genomic analysis. The generation of chromosomal sequences for these highly prevalent T. orientalis genotypes will also support future studies of population genetics and mixed genotype infections.
| S-EPMC9323827 | biostudies-literature
Project description:Protozoan parasites of the genus Theileria are tick-borne parasites that have been found in many species of mammals. More than a dozen species of Theileria have been found in cattle, water buffalo, sheep, and goats. Theileria orientalis is a non-pathogenic blood protozoan parasite that was detected and identified during a regular investigation of piroplasmida infection in indigenous cattle in the spring of 2019 in Northern Provinces of Iran. In total, 92 blood samples were collected from different areas of Guilan and Mazandaran Provinces, Iran during the spring. The Giemsa stained blood smears did not show any parasitic infection; however, T. orientalis was identified by 18S rRNA gene polymerase chain reaction (PCR) and DNA sequencing. The specific sequenced DNA for T. orientalis was registered in GenBank under the accession number MN453385. The partial 18S rRNA gene sequence of the obtained DNA showed 100% nucleotide identity with reference sequences for the T. orientalis that have been registered from Europe, Africa, and Asia. Additionally, molecular phylogenetic studies have shown that T. orientalis Iran GC98-01 isolate belongs to nonpathogenic T. orientalis type 3 (buffeli). In this study, the indigenous Bos indicus cattle were detected as asymptomatic carriers of Theileria spp. infection. Here, we identified and genotyped T. orientalis for the first time as T. orientalis type 3 (buffeli) in Iran using molecular phylogenetic analysis and registered the 18S rRNA gene sequence of the T. orientalis GC98-01 isolate in GenBank. Moreover, rare T. annulata infection was detected in cattle using semi-nested PCR in Mazandaran (Miankaleh peninsula). The T. orientalis can be differentiated from other Theileria and Babesia haemoprotozoan parasites by specific molecular assays.
| S-EPMC8410183 | biostudies-literature
Project description:The ikeda and chitose genotypes of Theileria orientalis, which for many years were thought to be benign, cause a disease that results in significant economic losses in the cattle industry. This study was carried out in order to determine the genotypes of T. orientalis in cattle in Kyrgyzstan, and 149 archived DNA samples known to be T. orientalis were analyzed by the PCR amplification of the major piroplasm surface protein (MPSP) gene region. Single-Strand Conformation Polymorphism (SSCP) analysis was performed to uncover the nucleotide changes in the archived DNA samples, and 15 samples showing different band profiles were subjected to sequence analysis. As a result of the sequence analysis, it was seen that the samples belonged to the buffeli and chitose A genotypes. In order to identify mixed genotypes, PCR was performed using primers specific for these genotypes, and buffeli (type 3), chitose (type 1) and buffeli+chitose were found to be positive in 26.2%, 2% and 71.8% of samples, respectively. As a result of this study, we showed the presence of buffeli (type 3) and chitose (type 1) genotypes of T. orientalis in cattle in Kyrgyzstan. Comprehensive epidemiological studies are needed to understand the clinical infections caused by the pathogenic chitose A and to determine the geographical distribution and different genotypes of T. orientalis.
| S-EPMC9606894 | biostudies-literature
Project description:IntroductionTheileria orientalis, an economically significant tick-borne hemoparasite, infects cattle globally. The T. orientalis Ikeda genotype, transmitted by Haemaphysalis longicornis ticks, is associated with clinical manifestations characterized by anemia, abortions, and mortality, although subclinical infections prevail. Despite the common occurrence of subclinical infections, therapeutic interventions targeting T. orientalis Ikeda in such cases are currently lacking, impeding effective parasite control measures. To address this critical knowledge gap, we assessed the efficacy of buparvaquone (BPQ) in eliminating the T. orientalis Ikeda, US isolate, in sub-clinically infected cattle.MethodsTwelve sub-clinically infected calves, identified by the presence of T. orientalis in peripheral blood alongside the absence of fever and anemia, were enrolled in the study. Six calves received two treatments of the BPQ label dose (2.5 mg/kg) at a 48-h interval, while additional three calves received the drug at a dosage of 6 mg/kg following the same regimen. Three untreated calves served as controls.Results and discussionEndpoint and quantitative PCR analyses revealed that BPQ exerted a transient effect on T. orientalis parasitemia. Parasites remained undetectable in peripheral blood until weeks 4 and 11 post-treatment in animals administered 2.5 mg/kg and 6 mg/kg of BPQ, respectively. Intriguingly, following recrudescence, administering 6 mg/kg to animals previously treated with 2.5 mg/kg did not result in a reduction in parasite load. Pharmacokinetic analysis data suggested that escalating the dosage led to a less than proportional increase in serum concentrations of BPQ. Moreover, a significant yet reversible decrease (p < 0.05) in blood urea nitrogen was observed in animals treated with the drug, irrespective of the dosage. Despite parasitemia relapse, animals treated with 6 mg/kg BPQ exhibited a noteworthy decrease (p < 0.05) in IgG levels specific to the T. orientalis major piroplasm surface protein compared to controls and animals treated with 2.5 mg/kg of the drug.ConclusionBPQ did not demonstrate efficacy in clearing subclinical T. orientalis Ikeda infection. Future investigations are warranted to explore innovative therapeutic modalities that, in synergy with vaccines and diagnostic assays, can facilitate the development of comprehensive programs aimed at controlling and eradicating this parasite.
| S-EPMC11310158 | biostudies-literature
Project description:BackgroundTheileria orientalis is a tick-borne hemoparasite that causes anemia, ill thrift, and death in cattle globally. The Ikeda strain of T. orientalis is more virulent than other strains, leading to severe clinical signs and death of up to 5% of affected animals. Within the Asia-Pacific region, where it affects 25% of Australian cattle, T. orientalis Ikeda has a significant economic impact on the cattle industry. In 2017, T. orientalis Ikeda was detected in a cattle herd in Albermarle County, Virginia, United States. Months earlier, the U.S. was alerted to the invasion of the Asian longhorned tick, Haemaphysalis longicornis, throughout the eastern U.S. Abundant H. longicornis ticks were identified on cattle in the T. orientalis-affected herd in VA, and a subset of ticks from the environment were PCR-positive for T. orientalis Ikeda. A strain of T. orientalis from a previous U.S. outbreak was not transmissible by H. longicornis; however, H. longicornis is the primary tick vector of T. orientalis Ikeda in other regions of the world. Thus, the objective of this study was to determine whether invasive H. longicornis ticks in the U.S. are competent vectors of T. orientalis Ikeda.MethodsNymphal H. longicornis ticks were fed on a splenectomized calf infected with the VA-U.S.-T. orientalis Ikeda strain. After molting, a subset of adult ticks from this cohort were dissected, and salivary glands assayed for T. orientalis Ikeda via qPCR. The remaining adult ticks from the group were allowed to feed on three calves. Calves were subsequently monitored for T. orientalis Ikeda infection via blood smear cytology and PCR.ResultsAfter acquisition feeding on a VA-U.S.-T. orientalis Ikeda-infected calf as nymphs, a subset of molted adult tick salivary glands tested positive by qPCR for T. orientalis Ikeda. Adult ticks from the same cohort successfully transmitted T. orientalis Ikeda to 3/3 naïve calves, each of which developed parasitemia reaching 0.4-0.9%.ConclusionsOur findings demonstrate that U.S. H. longicornis ticks are competent vectors of the VA-U.S.-T. orientalis Ikeda strain. This data provides important information for the U.S. cattle industry regarding the potential spread of this parasite and the necessity of enhanced surveillance and control measures.
| S-EPMC7962341 | biostudies-literature
Project description:Theileria orientalis is an emerging apicomplexan pathogen of cattle occurring in areas populated by the principal vector tick, Haemaphysalis longicornis. Unlike transforming Theileria spp. that induce cancer-like proliferation of lymphocytes via their schizont stage, T. orientalis destroys host erythrocytes during its piroplasm phase resulting in anaemia. The underlying pathogenic processes of T. orientalis infection are poorly understood; consequently, there are no vaccines for prevention of T. orientalis infection and chemotherapeutic options are limited. To identify antigens expressed during the piroplasm phase of T. orientalis, including those which may be useful targets for future therapeutic development, we examined the proteome across three common genotypes of the parasite (Ikeda, Chitose and Buffeli) using preparations of piroplasms purified from bovine blood. A combination of Triton X-114 extraction, one-dimensional electrophoresis and LC-MS/MS identified a total of 1113 proteins across all genotypes, with less than 3% of these representing host-derived proteins. Just over three quarters of T. orientalis proteins (78%) identified were from the aqueous phase of the TX-114 extraction representing cytosolic proteins, with the remaining 22% from the detergent phase, representing membrane-associated proteins. All enzymes involved in glycolysis were expressed, suggesting that this is the major metabolic pathway used during the T. orientalis piroplasm phase. Proteins involved in binding and breakdown of haemoglobin were also identified, suggesting that T. orientalis uses haemoglobin as a source of amino acids. A number of proteins involved in host cell interaction were also identified which may be suitable targets for the development of chemotherapeutics or vaccines.
| S-EPMC9610513 | biostudies-literature