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Piroplasmosis is an economically important tick-borne disease worldwide. However, little is known about the presence of Babesia spp. and Theileria spp. in ticks in Eastern and Southern Kazakhstan (ESK). During 2016 - 2019, adult ticks (at 26 sampling sites in 16 districts of 5 oblasts in ESK) were collected. Tick species were identified according to morphological and molecular characteristics. Two fragments (487 bp and 438 bp) of 18S ribosomal RNA (18S rRNA) were used to determine piroplasm species in representative 698 ticks. The genotype characteristics of Babesia caballi and Theileria equi were further analyzed by longer 18S rRNA gene fragments. A total of 6107 adult ticks (4558 parasitizing ticks and 1549 off-host ticks), including 4665 hard ticks and 1442 soft ticks, were collected from their natural hosts (cattle, horses, sheep, camels, shepherd dogs and hedgehogs) and the surrounding environment, respectively. Among the hard tick species, Dermacentor marginatus (62.59%, 2920/4665) was the most abundant, followed by Hyalomma asiaticum (19.36%, 903/4665) and Hyalomma detritum (9.95%, 464/4665). All soft ticks were identified as Argas persicus. 16S ribosomal DNA (16S rDNA) phylogenic analysis showed that several tick species in Kazakhstan, as exemplified by Haemaphysalis erinacei and D. marginatus, clustered together with conspecific ticks reported from China. Five species of piroplasms, i.e. Babesia occultans, Babesia caballi, Theileria ovis, Theileria annulata and Theileria equi, were detected in 698 representative ticks. Genotype E of T. equi in Almaty, and genotype A of B. caballi in Almaty and South Kazakhstan were identified., (Copyright © 2021. Published by Elsevier GmbH.)

Mitochondrial genomes provide new insights that help elucidating biological features, genetic evolution, and classification of protozoans. Theileria uilenbergi (T. uilenbergi), transmitted by Haemaphysalis qinghaiensis and H. longicornis, is considered as highly pathogenic to sheep and goats in China. This study reports and outlines features of its mitochondrial genome. The T. uilenbergi mitochondrial genome is a linear monomeric molecule of 6.0 kb length, which encodes three protein-coding genes named cytochrome c oxidase I (cox1), cytochrome b (cob), and cytochrome c oxidase III (cox3), as well as six large subunit (LSU) rRNA gene fragments, and ends in terminal inverted repeats (TIRs). The array structure and organization of the mitochondrial genome of T. uilenbergi is identical to that of T. parva. Phylogenetic analysis based on the amino acid sequences of cox1, cob, and cox3 genes suggests that T. uilenbergi is distantly related to the group of transforming Theileria species such as T. parva. This study contributes to a comprehensive understanding of the phylogeny and evolution of the mitochondrial genome of piroplasms and provides useful information of diagnostic marker for T. uilenbergi., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Piroplasmosis is a serious debilitating and sometimes fatal disease. Phylogenetic relationships within piroplasmida are complex and remain unclear. In the study, we assessed the relative resolution capabilities of the DNA sequences of the nuclear genes 40S ribosomal protein S5 (RPS5) and mitochondrial DNA Cytochrome c oxidase subunit III (cox3) gene in the phylogeny of Babesia and Theileria species isolates. We demonstrated that by using the cox3 gene can recover a better supported species tree for some Theileria species than when using the nuclear RPS5 gene alone, it tends to intra-specific diversity and considerable inter-specific difference. Additionally, the combined DNA sequences of the nuclear RPS5 and cox3 gene improved the inference of evolutionary relationships among Babesia and Theileria species. The mitochondrial cox3 gene outperforms nuclear RPS5 gene and yields better resolution on the intra-specific diversity of Babesia and Theileria species. However, the combined RPS5 nuclear DNA and cox3 DNA tree had more advantage in the phylogeny of Babesia and Theileria species than that of single gene alone., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Bovine theileriosis, caused by protozoan parasites of the genus Theileria, presents with various clinical symptoms. In cattle, clinical presentations and outcomes of bovine theileriosis are closely correlated with the causative Theileria spp. Thus, accurate detection and discrimination of Theileria spp. are essential for epidemiological studies and for provision of clinical management strategies. High-resolution melting (HRM) analyses of two amplicons targeting the 18S rRNA indicated that T. annulata, T. orientalis, and T. sinensis isolated from China can be accurately detected and discriminated with the lowest detection limit of 1-10 copy numbers of plasmid bearing the 18S rRNA sequence. The approach was verified with DNA samples from experimentally infected cattle and field samples. Thus, this assay is useful for diagnosis of bovine theileriosis in field samples and experimentally infected animals, and could also be applicable for the survey of parasite dynamics, epidemiological studies., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Introduction: Protozoan Theileria is transmitted by tick vectors to some animals worldwide and causes considerable economic damage. The detection of pathogens in camels is not only crucial for the preservation of species but also provides important information on the epidemiologic chain of diseases., Materials and Methods: We conducted a molecular detection in Xinjiang, China to assess the impact of Theileria infection in Bactrian camels and ticks in 2016. The 18S rRNA and MPSP gene sequences of T. sinensis obtained from Bactrian camels and ticks in Xinjiang, China have been deposited in the GenBank database., Results: PCR revealed that 6.56% of the Bactrian camel blood samples, 2.75% of Rhipicephalus sp., 3.81% of Hy. asiaticum, 4.32% of Hy. dromedarii, and 0% of D. niveus tick samples were positive for T. sinensis, respectively; no other Theileria and Babesia were found in these samples and D. niveus samples. The results showed that one Theileria parasite, Theileria sinensis, was found in Bactrian camels (Camelus bactrianus), Rhipicephalus sp., Hy. asiaticum, and Hy. dromedarii in Xinjiang, respectively., Conclusion: This is the first detection of T. sinensis DNA in Bactrian camels from China. Our results provide important data to increase the understanding of theileriosis of Bactrian camel, and will aid in taking effective measures to control theileriosis transmission to camels, cattle and other ruminants in Xinjiang, China.

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: Bovine theileriosis is a common disease transmitted by ticks, and can cause loss of beef and dairy cattle worldwide. Here, an indirect enzyme-linked immunosorbent assay (iELISA) based on Theileria luwenshuni surface protein (TlSP) was developed and used to carry out a seroepidemiological survey of bovine theileriosis in northern China., Methods: We used the BugBuster Ni-NTA His•Bind Purification Kit to purify recombinant TlSP (rTlSP), which was subsequently analyzed by Western Blotting to evaluate cross-reactivity with other pathogen-positive sera. The iELISA method based on rTlSP was successfully developed. Sera from 2005 blood samples were tested with the rTlSP-iELISA method, and blood smears from these samples were observed by microscopy., Results: The specificity of iELISA was 98.9%, the sensitivity was 98.5%, and the cut-off was selected as 24.6%. Western Blot analysis of rTlSP confirmed that there were cross-reactions with Theileria luwenshuni, Theileria uilenbergi, Theileria ovis, Theileria annulata, Theileria orientalis and Theileria sinensis. The epidemiological survey showed that the highest positive rate of bovine theileriosis was 98.3%, the lowest rate was 84.1%, and the average positive rate was 95.4% by iELISA. With microscopy, the highest positive rate was 38.9%, the lowest rate was 5.1%, and the relative average positive rate was 13.7%., Conclusions: An rTlSP-iELISA was developed to detect circulating antibodies against bovine Theileria in northern China. This is the first report on the seroprevalence of bovine theileriosis in northern China, and it also provides seroepidemiological data on bovine theileriosis in China.

Theileriosis continues to threaten the livestock industry worldwide, but comprehensive epidemiological surveys for this disease have not been conducted in the Tibet Plateau Region, China. In this study, we screened 154 cattle blood samples from the Tibet Plateau Region (Lhasa, Lhoka, and Tianzhu), China, for detection of Theileria pathogens by polymerase chain reaction (PCR) with species-specific primers. The results revealed that the prevalence was 6.9 % (2/29) for Theileria orientalis and 27.6 % (8/29) for Theileria sinensis in Lhasa, 0 % (0/30) for T. orientalis and 26.7 % (8/30) for T. sinensis in Lhoka, and 0 % (0/95) for T. orientalis and 30.5 % (29/95) for T. sinensis in Tianzhu. Interestingly, Theileria luwenshuni, which was a previously reported pathogenic Theileria sp. in sheep and goats, was detected in blood samples from cattle and yaks for the first time, with a prevalence of 10 % (3/30) in Lhoka and 1.1 % (1/95) in Tianzhu. No other Theileria sp. was detected in these samples. T. sinensis and T. orientalis infections were detected in cattle and yaks, and T. luwenshuni was discovered for the first time in cattle and yaks in the Tibet Plateau Region, China.

Theileria is a tick-borne, intracellular protozoan parasite of worldwide economic and veterinary importance in small ruminants. Here, an enzyme-linked immunosorbent assay (ELISA) was developed based on Theileria luwenshuni recombinant surface protein (rTlSP) and was used in the standardization and validation of an ELISA for the detection of circulating antibodies against ovine and caprine theileriosis. A total of 233 sera samples were used for the calculation of the cut-off value which served as a threshold between the positive and the negative sera. When the positive threshold was chosen as 19% of the specific mean antibody rate, the specificity was 97.9%, and the sensitivity was 97.1%. There was a cross-reaction with sera against Theileria uilenbergi and Theileria ovis, and no cross-reaction with sera against Babesia spp. in the ELISA and Western blotting. Two hundred forty samples collected from sheep in Gansu province were detected with blood smears and ELISA, respectively. The results showed that the positive rate of Theileria infection in Gansu province were 63.75% with rTlSP-ELISA, and 46.67% with blood smears, respectively. Our test proved that the rTlSP ELISA is suitable to diagnose Theileria infection and could be used in serological surveys to map out the prevalence of ovine and caprine theileriosis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Theileria uilenbergi and T. luwenshuni are tick-borne protozoan parasites, transmitted by Haemaphysalis qinghaiensis and H. longicornis, respectively. They are the main causative agents of theileriosis in small ruminants in China. The disease has resulted in severe economic losses and hindered the development of sheep and goat husbandry industry in the endemic regions., Methods: In this study, a colloidal gold-based immunochromatographic strip (ICS) was developed for the detection of T. uilenbergi and/or T. luwenshuni infections. A recombinant T. uilenbergi immunodominant protein (rTuIP) was used as antigen for the ICS. The nitrocellulose membrane was incubated with rTuIP on the test (T) line and anti-rTuIP antiserum on the control (C) line, respectively. The rTuIP conjugated to colloidal gold particles was used as the detection system for visualization of the lines. Then the sample pad, conjugate pad, nitrocellulose membrane and absorbent pad were assembled onto a backing plate in the appropriate order., Results: The ICS was able to detect antibodies in the sera of animals experimentally infected with T. uilenbergi from 14 to 85 days. It also reacted with the serum from T. luwenshuni infected sheep. However, there was no cross-reactivity with sera from animals infected with Babesia motasi and Anaplasma ovis. Comparison of the ICS with the rTuIP antigen based indirect enzyme-linked immunosorbent assays (ELISA) using test field samples showed good correlations with 93.1 % (81/87) sensitivity and 100 % (40/40) specificity, respectively, with an almost perfect agreement (Kappa = 0.895, P < 0.01)., Conclusion: An immunochromatographic strip test based on a recombinant T. uilenbergi immunodominant protein (rTuIP) was developed. This is a rapid test (approximately 15 min to completion) for the detection of T. uilenbergi and/or T. luwenshuni infection that is easy to perform and; delivers results that are visible to the naked eye.

Background: Bovine babesiosis and theileriosis is an important hemoprotozoal disease in cattles and yaks in tropical and subtropical regions leading to significant economic losses. In the field, the risk of co-infection between the bovine Babesia and Theileria species is very high. Thus, it is necessary to develop a simple, accurate, rapid and cost-effective method for large-scale epidemic investigation, in particular for the detection of co-infection in field., Methods: In this study, DNA sequences of a ribosomal protein S8 (RPS8) gene from eight species of cattle piroplasms in China were used to develop a species-specific PCR-RFLP diagnostic tool. The eight Theileria and Babesia species could be differentiated by digesting the RPS8 PCR product with Mbo I., Results: The sensitivity of the PCR assays was 0.1 pg DNA for Babesia species but 1 pg DNA for Theileria species. The clearly different size of the PCR-RFLP products allowed for a direct discrimination between eight bovine Theileria and Babesia species (T. annulata, T. sinensis, T. sergenti, B. ovata, B. bovis, B. bigemina, B. major and Babesia species Kashi isolate)., Conclusion: Our results indicated that the established method based on the RPS8 gene was a reliable molecular diagnostic tool for the simultaneous detection and identification of bovine Babesia and Theileria species in China, which could be applicable for the survey of parasite dynamics, epidemiological studies as well as prevention and control of the disease.

Theileria sp. OT3 was firstly detected and identified from clinically healthy sheep in Xinjiang Uygur Autonomous Region of China (XUAR) through comparing the complete 18S rDNA gene sequences available in GenBank database and the phylogenetic status based on the internal transcribed spacers (ITS1, ITS2) as well as the intervening 5.8S coding region of the rRNA gene by the methods of a partitioned multi-locus analysis in BEAST and Maximum likelihood analysis in PhyML. Moreover, the findings were confirmed by the species-specific PCR for Theileria sp. OT3 and the prevalence of Theileria sp. OT3 was 14.9% in the north of XUAR. This study is the first report on the occurrence of Theileria sp. OT3 in China., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Ovine babesiosis and theileriosis are important hemoprotozoal diseases of sheep and goats in tropical and subtropical regions that lead to economic losses in these animals. PCR-restriction fragment length polymorphism (PCR-RFLP) is a reliable molecular diagnostic tool for discriminating Theileria or Babesia species in the same host. In this study, the DNA sequences of a ribosomal protein S8 (RPS8) gene from four species of piroplasms in China were used to develop a species-specific PCR-RFLP diagnostic tool. The sensitivity of the PCR assays was 0.1 pg DNA for B. motasi and 1 pg DNA for T. uilenbergi and 10 pg DNA for Babesia sp. Xinjiang-2005 and T. luwenshuni. The clear size difference of the PCR products allowed for a direct discrimination for B. motasi, Babesia sp. Xinjiang-2005 and ovine Theileria species (T. uilenbergi and T. luwenshuni), except that the mixed infection between T. uilenbergi and T. luwenshuni may be difficult to distinguish, simply after the electrophoretic separation of the amplification products. Further T. uilenbergi and T. luwenshuni diagnoses were made by digesting the PCR product with SacI. The established method could be applicable for the survey of parasite dynamics, and epidemiological studies as well as prevention and control of the disease., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Several approaches have been developed for diagnosis of Theileria equi infection in horses and donkeys but all of them have limitations in practice. Due to numerous strengths including easy operation, cheapness and high sensitivity and specificity, LAMP has been already extensively used for surveillance of a number of diseases. We here set up a LAMP assay based on 18S rRNA gene for T. equi diagnosis. The approach was specific enough to differentiate T. equi from other evolutionary-related protozoa. Moreover, it was sensitive enough that LAMP was capable of detecting as much low as 10 copy target gene and 1 pg/μl blood genomic DNA. It was further demonstrated that LAMP was much more sensitive than canonical blood smear and comparable to PCR using test and field samples. The present results support an idea that LAMP developed in this study is reliable, reproducible and highly sensitive and specific, being a potential to be globally used for surveillance of T. equi infection in the field., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The experimental transmission of a recently identified new Theileria (China 1) species pathogenic for sheep and goats in northern China is described. Haemaphysalis qinghaiensis nymphs and adults developed from larvae and nymph engorged on sheep infected with Theileria sp. (China 1) were able to respectively transmit the Theileria sp. to splenectomized sheep. Meanwhile, H. longicornis nymphs and adults developed from larvae and nymphs engorged on sheep infected with Theileria sp. (China 1) were also able to respectively transmit this new Theileria sp. (China 1) to splenectomized sheep. These experiments suggested that the Theileria sp. (China 1) could be transmitted by at least two species of Haemaphysalis sp. ticks, H. longicornis and H. qinghaiensis, and the mode of transmission is stage to stage.

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.

Theileria parasites known as either T. buffeli, T. orientalis or T. sergenti share many characteristics and are referred to as the T. buffeli group. The 18S ribosomal RNA and merozoite-piroplasm surface protein-encoding genes display a wider genetic variation within the T. buffeli group than between well defined species like T. annulata and T. parva. Analysis of 18S rRNA gene sequences from the database showed that similar groups of related Theileria parasites occur in wild ruminants in Japan and the USA. Moreover, a recently discovered Theileria species pathogenic for small ruminants in China is phylogenetically related to these Japanese Theileria parasites. Phylogenetic analysis of all Theileria 18S rRNA genes reported thus far allowed a subdivision into eight clusters. Some of these clusters contain multiple different species, whereas others appear to contain parasites with similar biological properties whose true speciation remains at present unresolved. The consequences for 18S rRNA based diagnostic assays is discussed.

The great gerbil (Rhombomys opimus), widely distributed in Asia, is a natural reservoir for Yersinia pestis , Leishmania donovani and some species of helminths. In this study, 188 great gerbils were sampled in Alataw City and Manas County, northwestern China, and tested for the presence of Theileria , Hepatozoon and Taenia species by molecular methods. Theileria sp., named as "Candidatus Theileria xinjiangensis ", was detected in heart, liver, spleen, lung, and kidney of 6.9% rodents. Six genotypes of " Taenia sp. Rhombomys opimus ", which were close to Taenia laticollis (87.3–94.0% identities), were detected in cyst liquid of 5.3% rodents. " Hepatozoon ayorgbor- like" haemogregarines was detected in spleens of 1.6% rodents. To our best knowledge, Candidatus Theileria xinjiangensis , Hepatozoon ayorgbor -like and genotypes of " Taenia sp. Rhombomys opimus " were found for the first time in the great gerbil. These results extend our knowledge on the diversity and pathogenesis of Theileria , Hepatozoon and Taenia species. [Display omitted] • "Candidatus Theileria xinjiangensis ", causing splenomegaly in great gerbils, was the closest related to Candidatus Theileria senegalensis from Nile rat. • " Hepatozoon ayorgbor- like" haemogregarine was detected for the first time in great gerbils. • Six genotypes of " Taenia sp. Rhombomys opimus ", causing cyst in great gerbils, were closest related to Taenia laticollis. [ABSTRACT FROM AUTHOR]

Theileria annulata, a tick-borne apicomplexan protozoan, causes a lymphoproliferative disease of cattle with high prevalence in tropical and sub-tropical regions. Understanding the genetic diversity and structure of local populations will provide more fundamental knowledge for the population genetics and epidemics of protozoa. In this study, 78 samples of T. annulata collected from cattle/yaks representing 6 different geographic populations in China were genotyped using eight micro- and mini-satellite markers. High genetic variation within population, moderate genetic differentiation, and high level of diversity co-occurring with significant linkage disequilibrium were observed, which indicates there is gene flow between these populations in spite of the existence of reproductive and geographical barriers among populations. Furthermore, some degree of genetic differentiation was also found between samples from China and Oman. These findings provide a first glimpse of the genetic diversity of the T. annulata populations in China, and might contribute to the knowledge of distribution, dynamics, and epidemiology of T. annulata populations and optimize the management strategies for control. [ABSTRACT FROM AUTHOR]

Tropical theileriosis, caused by Theileria annulata , is distributed worldwide and causes great economic losses in dairy. The reliable diagnostic method is critical for prevention and control of the disease. In this study, a sporozoite and macroschizont gene 2 (spm2) protein from T . annulata was used to develop an indirect ELISA for tropical theileriosis. Specificity test showed that there were no cross-reactions with antibodies raised against other bovine piroplasm species using ELISA or western blotting. The specificity and sensitivity were 98.4% and 98.7%, respectively, with a threshold of 35.5% of the specific mean antibody rate (AbR). Furthermore, a total of 196 field sera samples collected from Xinjiang and Gansu provinces were detected by the spm2 ELISA and IFA. The results obtained with the spm2 ELISA and IFA in this study had the moderate agreement. The average positive rates of T . annulata sera samples detected in the present study were close to the prevalence of previous reports in these endemic areas. This indicated that the Spm2 ELISA could be used as a reliable diagnostic tool for serological survey of T . annulata infection in areas where Theileria parva is not present. [ABSTRACT FROM AUTHOR]