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

East Coast fever, a tick-borne cattle disease caused by the Theileria parva parasite, is among the biggest natural killers of cattle in East Africa, leading to over 1 million deaths annually. Here we report on the genetic analysis of a cohort of Bos indicus (Boran) cattle demonstrating heritable tolerance to infection with T . parva ( h 2 = 0.65, s.e. 0.57). Through a linkage analysis we identify a 6 Mb genomic region on bovine chromosome 15 that is significantly associated with survival outcome following T . parva exposure. Testing this locus in an independent cohort of animals replicates this association with survival following T . parva infection. A stop gained variant in a paralogue of the FAF1 gene in this region was found to be highly associated with survival across both related and unrelated animals, with only one of the 20 homozygote carriers (T/T) of this change succumbing to the disease in contrast to 44 out of 97 animals homozygote for the reference allele (C/C). Consequently, we present a genetic locus linked to tolerance of one of Africa’s most important cattle diseases, raising the promise of marker-assisted selection for cattle that are less susceptible to infection by T . parva .

Piroplasmosis is an important tick-borne disease in several regions, and can lead to significant economic animal production losses. The current study aimed to systematically examine the incidence of bovine piroplasmosis in Kashgar, Xinjiang, to provide baseline data for the effective prevention and control of this disease among bovines in the region. A total of 1403 bovine blood samples from 12 sampling points were screened via PCR with universal Piroplasma primers targeting the 18S rRNA locus and specific Theileria annulata primers targeting the cytochrome b ( COB ) gene. The overall prevalence of bovine Piroplasma was 65.9% (925/1403). Three species of pathogenic Theileria , including T. annulata , T. orientalis , and T. sinensis , were detected, and the infection rates for these species were 65.1% (913/1403), 0.5% (7/1403), and 0.1% (1/1403), respectively. The mixed infection rate for T. orientalis and T. annulata was 0.3% (4/1403). No Babesia was detected in this study. In conclusion, bovine piroplasmosis was still common in Kashgar and T. annulata was the dominant species, and a mixed infection of T. annulata and T. orientalis was detected. Notably, T. sinensis was reported for the first time in this region. Therefore, strategies for the prevention and control of bovine piroplasmosis should be enhanced.

Multigene families often play an important role in host-parasite interactions. One of the largest multigene families in Theileria parva, the causative agent of East Coast fever, is the T. parva repeat (Tpr) gene family. The function of the putative Tpr proteins remains unknown. The initial publication of the T. parva reference genome identified 39 Tpr family open reading frames (ORFs) sharing a conserved C-terminal domain. Twenty-eight of these are clustered in a central region of chromosome 3, termed the “Tpr locus”, while others are dispersed throughout all four nuclear chromosomes. The Tpr locus contains three of the four assembly gaps remaining in the genome, suggesting the presence of additional, as yet uncharacterized, Tpr gene copies. Here, we describe the use of long-read sequencing to attempt to close the gaps in the reference assembly of T. parva (located among multigene families clusters), characterize the full complement of Tpr family ORFs in the T. parva reference genome, and evaluate their evolutionary relationship with Tpr homologs in other Theileria species. We identify three new Tpr family genes in the T. parva reference genome and show that sequence similarity among paralogs in the Tpr locus is significantly higher than between genes outside the Tpr locus. We also identify sequences homologous to the conserved C-terminal domain in five additional Theileria species. Using these sequences, we show that the evolution of this gene family involves conservation of a few orthologs across species, combined with gene gains/losses, and species-specific expansions.

Bovine theileriosis caused by several Theileria species including Theileria annulata, Theileria parva, Theileria orientalis, Theileria mutans, and Theileria sinensis is a significant hemoprotozoan tick-borne disease. Among these, Theileria species, T. annulata, which causes tropical theileriosis (TT), is regarded as one of the most pathogenic and is responsible for high mortality. At present, most conventional diagnostic methods for tropical theileriosis are time-consuming and laborious and cannot distinguish newfound T. sinensis in China. Therefore, a high sensitivity and specificity real-time quantitative PCR method based on the TA19140 target molecule was developed, and the method was found to be specific for T. annulata. No cross-reaction was observed with T. sinensis, T. orientalis, Babesia bovis, Babesia bigemina, or Hyalomma anatolicum which is negative for T. annulata. A total of 809 field samples from different regions of China were analyzed by using the developed qPCR and conventional PCR. The positive samples for T. annulata detected by real-time qPCR and conventional PCR were 66/809 (8.16%) and 20/809 (2.47%), respectively, and all positive amplicons by qPCR were confirmed by Sanger sequencing. The results showed that the developed qPCR for the T. annulata 19,140 gene was more sensitive than conventional PCR. In addition, we first discovered that TA19140 was mainly expressed at the schizont and merozoite stages of T. annulata by relative quantification. The protein encoded by the TA19140 gene may be used as a potential diagnostic antigen for tropical theileriosis. In conclusion, a real-time quantitative PCR diagnostic method targeting the TA19140 gene was successfully established and could be used for both the quantitative and qualitative analysis of T. annulata infection from cattle and vector ticks, which will greatly help to control and diagnosis of tropical theileriosis.

Theileriosis is a tick-borne disease that causes enormous losses in the dairy industry. There are several species of Theileria that can infect bovines. Generally, more than one species are prevalent in any geographical area; thus, chances of co-infections are high. Differentiation of these species may not be possible by microscopic examination or serological tests. Therefore, in this study, a multiplex PCR assay was standardized and evaluated for rapid and simultaneous differential detection of two species of Theileria viz . , Theileria annulata and Theileria orientalis . Species-specific primers were designed to target the merozoite piroplasm surface antigen gene (TAMS1) of T. annulata and the major piroplasm surface protein gene of T. orientalis , yielding specific amplicon of 229 bp and 466 bp, respectively. The sensitivity of multiplex PCR was 10 2 and 10 3 copies for T. annulata and T. orientalis , respectively. The simplex and multiplex PCRs were specific and showed no cross-reactivity with other hemoprotozoa for either primer. For comparative evaluation, blood samples from 216 cattle were tested by simplex and multiplex PCR for both species. Using multiplex PCR, 131 animals were found infected for theileriosis, of which 112 were infected with T. annulata , five were infected with T. orientalis , and 14 had mixed infections. This is the first report of T. orientalis from Haryana, India. Representative sequences of T. annulata (ON248941) and T. orientalis (ON248942) were submitted in GenBank. The standardized multiplex PCR assay used in this study was specific, sensitive, for the screening of field samples.

Aim to construct a simple and efficient diagnostic assay for Theileria annulata and Theileria sergenti, a multiplex polymerase chain reaction (PCR) method was developed in this study. Following the alignment of the related sequences, two primer sets were designed specific targeting on T. annulata cytochrome b (COB) gene and T. sergenti internal transcribed spacer (ITS) sequences. It was found that the designed primers could react in one PCR system and generating amplifications of 818 and 393 base pair for T. sergenti and T. annulata, respectively. The standard genomic DNA of both species Theileria was serial tenfold diluted for testing the sensitivity, while specificity test confirmed both primer sets have no cross-reaction with other Theileria and Babesia species. In addition, 378 field samples were used for evaluation of the utility of the multiplex PCR assay for detection of the pathogens infection. The detection results were compared with the other two published PCR methods which targeting on T. annulata COB gene and T. sergenti major piroplasm surface protein (MPSP) gene, respectively. The developed multiplex PCR assay has similar efficient detection with COB and MPSP PCR, which indicates this multiplex PCR may be a valuable assay for the epidemiological studies for T. annulata and T. sergenti.

Piroplasmosis, a disease of domestic and wild animals, is caused by tick-borne protozoa in the genera of Theileria and Babesia. There is limited information available about the prevalence of piroplasmosis in ticks in China, and to assess the potential threat of piroplasmosis in China, we investigated the infections of ovine and bovine Babesia and Theileria species in ticks collected from cattle, yaks, sheep, horses, and camels in several regions of China where tick-borne diseases have been reported. In total, 652 ticks were collected from the animals in 6 provinces of China. Babesia spp. and Theileria spp. were detected with a PCR-RLB method and identified by sequencing. Overall, 157 ticks (24.1%) were infected with 5 Babesia and 4 Theileria species. Among tested tick samples, 134 (20.6%) were single infections with 1 of 7 piroplasm species, with Theileria annulata (118/652, 18.1%) being dominant. Only 23 (3.5%) tick samples were double or triple infected, Theileria luwenshuni and Theileria sinensis (18/652, 2.8%) were frequently observed in co-infections. Some piroplasm species were carried by ticks that were not previously reported to be vectors.

Background There had been isolated reports of the presence of novel Theileria annulata genotypes based on the 18S rRNA gene sequence data from India, Pakistan and Saudi Arabia; but, these studies were restricted to limited field samples. Additionally, no comparative study has been conducted on all the isolates of this parasite from different countries whose sequences are available in the nucleotide databases. Therefore, we aimed to study the genetic diversity of T. annulata based on all available nearly complete 18S rRNA gene sequences in the GenBank™. Out of a total of 312 gene sequences of T. annulata available in the NCBI database, only 70 nearly complete sequences (> 1527 bp) were used for multiple sequence alignment. Results The maximum likelihood tree obtained using TN93 + G + I model manifested two major clades. All the valid host-cell transforming Theileria species clustered in one clade. The T. annulata designated sequences occupying this clade clustered together, excluding two isolates (DQ287944 and EU083799), and represented the true T. annulata sequences ( n  = 54). DQ287944 and EU083799 exhibited close association with Theileria lestoquardi . In addition, 14 Indian sequences formed a large monophyletic group with published Theileria orientalis sequences. The broad range of sequence identity (95.8–100%) of T. annulata designated sequences indicated the presence of different Theileria spp. A closer analysis revealed the presence of three Theileria spp., namely, T. annulata , T. orientalis, and two isolates (DQ287944 and EU083799) closely related to T. lestoquardi . The true T. annulata sequences manifested 98.8–100% nucleotide identity within them. EU083799 and 14 misidentified Indian T. annulata sequences exhibited the highest similarity with T. lestoquardi (98.6–98.8%) and T. orientalis (98.0-99.9%) in comparison with the other Theileria spp. of domestic and wild ruminants. Conclusion In the course of analyzing the genetic diversity of T. annulata , we identified the nearly complete 18S rRNA gene sequences of other Theileria spp. that have not only been misidentified as T. annulata in the GenBank™, but are also published as T. annulata . Moreover, a high level of sequence conservation was noticed in the 18S rRNA gene of true T. annulata and T. orientalis sequences.

Theileria annulata and T. parva are closely related protozoan parasites that cause lymphoproliferative diseases of cattle. We sequenced the genome of T. annulata and compared it with that of T. parva to understand the mechanisms underlying transformation and tropism. Despite high conservation of gene sequences and synteny, the analysis reveals unequally expanded gene families and species-specific genes. We also identify divergent families of putative secreted polypeptides that may reduce immune recognition, candidate regulators of host-cell transformation, and a Theileria-specific protein domain [frequently associated in Theileria (FAINT)] present in a large number of secreted proteins.