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Theileria parvacauses East Coast fever (ECF), one of the most important and lethal tick-borne diseases of cattle in sub-Saharan Africa. ECF is a considerable burden to the livestock industry, causing annual losses exceeding US $300 million. Currently, diagnosis of T. parva infections relies mainly on clinical signs, serology, and microscopic identification of parasites in either blood or lymph fluid samples. However, some of these tests might not indicate ongoing infection and they all lack the sensitivity to detect low-level infections. Molecular tests such as nested and quantitative PCR assays offer high sensitivity for detection of T. parva. However, these tests remain highly complex technologies that are impractical to use in resource-limited settings where economic losses due to the disease have the most significant impact. A field-deployable, point-of-care test will be of significant value in the treatment and control of ECF in endemic areas. For this purpose, we have developed a CRISPR-Cas12a-based pen-side tool that can sensitively and specifically detect T. parva based on the p104 gene. We describe a streamlined, field-applicable diagnostic tool comprising a 20 min recombinase polymerase amplification (RPA) reaction followed by a 60 min CRISPR-Cas12a reaction using a FAM/Biotin lateral flow strip readout. We tested two different RPA primer pairs and four different CRISPR-RNAs (crRNAs). The p104-based assay displayed high sensitivity, detecting as low as one infected lymphocyte per three microliters of blood and universally detecting eight different T. parva strains without detecting DNA from other Theileria spp. such as Theileria mutans and Theileria lestoquardi. This work opens the way for a field-applicable diagnostic tool for the sensitive point-of-care early diagnosis of T. parva infections in cattle., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background Objectives: Theileriosis is an important tick-bome hemoprotozoan disease of cattle which causes severe economic loss due to morbidity and mortality. A diagnostic test having high sensitivity, specificity and easy application at the field level is the need of the hour. In this regard Loop-mediated isothermal amplification (LAMP) is proven to be a sensitive, easy and time efficient method. One of the major obstacles for the application of LAMP is the difficulty in maintaining the cold chain to preserve reagents. Thus, the challenge is to develop a LAMP kit in a ready-to-use format with dried reagents useful for quick and simple application in field conditions., Methods: The optimized reaction of wet LAMP was followed for the standardization of dry LAMP with certain modifications which are needful. The major modification is vitrification technology of enzyme using trehalose., Results: LAMP assay (dry and wet LAMP) was found to be more sensitive (100%) when compared to microscopy (69.5%) and PCR (86.9%). It was observed that the dry LAMP reaction tubes at room temperature as well as refrigeration temperature provided successful amplification till 7 weeks., Interpretation Conclusion: The drying conditions of LAMP reagents were optimized, and finally managed to dry them in a single reaction tube without reducing the sensitivity. This technology enables us to transport LAMP kits to areas where the cold chain is not easily available., (Copyright © 2024 Copyright: © 2024 Journal of Vector Borne Diseases.)

Tropical theileriosis is a tick-borne disease that caused by Theileria annulata, and leads to substantial economic impact in endemic area. Distinguishes to other piroplasms, Theileria is the only eukaryotic parasite could transform mammalian leukocytes. At present, buparvaquone is the most effective drug used for treatment of Theileria infection. However, frequently reported of failure treatment with buparvaquone for some T. annulata isolates. Mutation of TaPIN1 was reported to be the direct reason for failure of buparvaquone treatment. Through in vitro culture, a T. annulata isolate with a TaPIN1 mutation that is similar to the reported strain was recently identified in China. In order to understand the distribution of Theileria with mutation of TaPIN1 in China, here we developed a TaqMan probe-based real-time PCR technology to detect the mutated TaPIN1 gene. The specificity, sensitivity and reproducibility of the established TaqMan Real-time PCR method were evaluated, and field cattle blood samples collected from Xinjiang Uyghur Autonomous Region were used to test its application. Among 1683 samples, 335 samples were confirmed positive for T. annulata by traditional PCR method and 34 samples were positive for buparvaquone-resistant. The TaPIN1 gene of those 34 samples was sequenced and analyzed with the published gene sequences from NCBI database. The results showed that the sequence obtained from the present study has good consistency with those published sequences. In conclusion, the TaqMan probe-based real-time PCR targeting T. annulata mutated TaPIN1 gene was successfully established and can be used to detect clinical samples to investigation of buparvaquone-resistant parasites in Xinjiang region quickly and accurately, which will be useful for guiding clinical medicine application., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Vector-borne diseases indulge in severe economic losses in the livestock industry by adversely affecting cattle breeding in tropical and subtropical zone countries, including Turkey, encompassing a wide land area representing diverse climatic conditions. This study aimed to investigate significant bovine tick-borne piroplasm, rickettsia, and some other bacterial agents by genus- or species-specific PCR and nested PCR techniques in Turkey. A total of 210 cattle blood samples were collected from sixteen provinces in different geographical regions of Turkey. PCR analyses were performed targeting the detection of Babesia/Theileria/Hepatozoon sp. 18S rRNA, Babesia/Theileria sp. 18S rRNA (V4), B. bigemina RAP-1a, B. bovis SBP-4, B. ovata AMA-1, B. naoaki AMA-1, T. annulata Tams-1, T. orientalis MPSP, T. mutans 18S rRNA, Anaplasma/Ehrlichia sp. 16S rRNA, A. marginale MSP4, A. bovis 16S rRNA, A. phagocytophilum 16S rRNA, A. capra 16S rRNA, E. ruminantium pSC20, Mycoplasma sp. 16S rRNA, and Coxiella burnetii 16S rRNA genes. Overall, 133 (63.3%) cattle were found to be infected with at least one of the following protozoan or bacterial pathogens; B. bovis, B. bigemina, B. occultans, T. annulata, T. orientalis, A. marginale, A. phagocytophilum, and Mycoplasma sp. The total prevalence of pathogens was determined as follows; 0.5% B. bovis, 0.5% B. bigemina, 1.4% B. occultans, 41.0% T. annulata, 1.4% T. orientalis, 10.5% A. marginale, 13.8% A. phagocytophilum, 0.5% A. bovis, 2.9% Uncultured Anaplasma sp., 0.5% E. minasensis, 0.5% Uncultured Ehrlichia sp., and 23.3% Mycoplasma sp. Moreover, large part of the total infection (n:133) was composed of single infections (63.9%); however, double (24.8%), triple (7.5%), quadruple (2.3%), and quintuple (1.5%) co-infections were also encountered. In addition to some bovine pathogens such as B. occultans, T. orientalis, A. bovis, M. wenyonii, and Candidatus Mycoplasma haemobos, which were rarely reported in Turkey, sequencing and phylogenetic analysis revealed the first detection of Uncultured Ehrlichia sp. (0.5%), and E. minasensis (0.5%) with 100% nucleotide sequence identities. The study also indicates that the spectrum of pathogens harbored by Turkish cattle is quite wide, and these pathogens cause multiple co-infections with various combinations, and T. annulata stands out as the primary bovine pathogen among them., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Tropical theileriosis (TT) is a tick-borne disease caused by Theileria annulata and commonly infects cattle in tropical and subtropical regions, including Algeria. It is a significant obstacle to cattle breeding programs established to improve production in Algeria. The present investigation aimed to estimate the current molecular prevalence, risk factors, and genetic characterisation of T. annulata in two bioclimatic areas of Algeria. In a cross-sectional study, 679 blood samples (629 from healthy cattle selected on farms and 50 from diseased cattle identified by veterinarians) were collected from the humid (n = 307+50) and semi-arid (n = 322) areas and screened by blood smear examination followed by polymerase chain reaction targeting cytochrome oxidase subunit 3 (cox III) mitochondrial and the 18S ribosomal RNA (18S rRNA) genes for Theileria spp. Seventy-six positive samples (56 clinically healthy and 20 with clinical signs) for Theileria spp. were confirmed to be T. annulata by the merozoïtes surface antigen-1 (Tams1) gene showing a rate of 8.9 % in clinically healthy and 40.0 % in suspected cattle. Among the 307 bloods samples collected from healthy cattle in the humid area, 25 cattle (8.1 %) were positive for T. annulata. Of the 322 healthy cattle from the semi-arid site, 31 (9.6 %) were carriers of T. annulata DNA. In subclinical population, demographic and environmental parameters analysis indicated that T. annulata infection was higher in adult crossbred cattle raised in the intensive and semi-intensive system (P<0.001). The multiple logistic regression analysis showed that age, breed, farming system, and bioclimatic area are potential risk factors for T. annulata infection in cattle (P<0.05). Multiple alignments of cox III sequences of T. annulata showed high heterogeneity with 25 polymorphic sites (nucleotide diversity π = 0.02402), resulting in two haplotypes with a low genetic diversity index (Hd) of 0.533. The 18S rRNA sequence alignment revealed only one T. annulata genotype with 100 % identity to the strains isolated from cattle and ticks in Mediterranean and Asian countries. Our preliminary results will serve as a basis for further studies on the genetic diversity and molecular epidemiology of T. annulata., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Purpose: Piroplasmosis is responsible for anemia, fever, loss of physical activity and even death in equines. In epidemiological studies, accurate diagnostic tests are essential for detecting asymptomatic carriers. This study aimed to investigate the prevalence of infection in asymptomatic horses from Lorestan province, western Iran by developing a multiplex PCR., Methods and Results: Blood samples were examined by microscopy and multiplex PCR targeting the SSU rRNA gene of Theileria equi and Babesia caballi. Out of the total of 165 horses, 19 (11.51%) and 31 (18.78%) cases were positive for piroplasms by microscopy and PCR, respectively. The detection rates of both genera were significantly higher in multiplex PCR compared to microscopy (p < 0.0001). Compared with multiplex PCR, the sensitivities of microscopy for the detection of Babesia were only 28.5%. The prevalence of T. equi infection was significantly higher in summer (p = 0.035). The prevalence of B. caballi was significantly higher in males (p = 0.038)., Conclusion: Findings indicate that the multiplex PCR described here is a sensitive technique for the detection of piroplasm DNA in carriers. Furthermore, asymptomatic carriers must be considered as an important source of infection for equids living in this region., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Theileria equi is 1 of the emerging and prevailing tick-borne hemoprotozoans adversely affecting the equids worldwide, including Pakistan. The current study aimed to investigate the prevalence and molecular characterization of T. equi in working horses (n = 194), the comparative efficacy of different diagnostic tests, associated risk factors, and hematobiochemical analysis. The blood samples of horses were subjected to microscopic examination, cELISA, and polymerase chain reaction (PCR) and the results revealed a prevalence of 9.79, 21.13, and 13.40%, respectively, for T. equi in working horses. The comparison of microscopy and cELISA results with PCR showed that cELISA had higher sensitivity (84.62%), but lower specificity (88.69%) and accuracy (88.14%) in comparison to microscopy (57.69, 97.62, and 92.27%). Molecular characterization of T. equi by phylogenetic analysis revealed a 61% resemblance of study isolates with each other OL662926, OL662925, and 82% similarity with isolate OL662924 while also showing homology with T. equi isolates of South Africa, South Korea, India, Pakistan, and Brazil. The risk factor analysis revealed a significant association (P < 0.05) of tick control status, previous tick history, tick infestation, house hygiene, deworming/vaccination, and the presence of other livestock species with T. equi infection in horses. The hematobiochemical profile revealed a significant (P < 0.05) decrease in red blood cells (RBCs), hemoglobin (Hb), packed cell volume (PCV), white blood cells (WBCs), platelet (PLT), phosphorus, and an increase in lymphocytes, granulocytes, aspartate aminotransferase (AST), glucose, bilirubin, blood urea nitrogen (BUN), and creatinine in T. equi-infected horses. The current study is the first comprehensive report for comparative evaluation of microscopy, cELISA, and PCR, assessment of epidemiological risk factors as well as hematobiochemical variations due to T. equi infection in Pakistan., (© American Society of Parasitologists 2024.)

Theileriosis is a hemoprotozoan illness of cattle in tropical regions that poses a severe economic loss to dairy farmers in the form of production loss and mortality. We designed and optimized a multiplex real-time PCR by using Taq-Man ® probe for detection and quantification of Theileria orientalis and Theileria annulata simultaneously by targeting 18 s rRna and MPSP (surface merozoite protein) genes, respectively. Fifty-five EDTA blood samples from clinically Theileria -suspected cows of three Theileria -endemic districts of Odisha were processed using acridine dye based fluorescent microscopy, Giemsa staining, and PCR. PCR revealed T. annulata and T. orientalis in 11/42 (26.11%) and 24/42 (57.14%) cases, respectively. Mixed infection due to both the Theileria spp. was recorded in 7/42 (16.66%). On comparison with gold standard test (PCR), the accuracy, sensitivity, and specificity were 92.72, 95.12, and 85.71% for Giemsa staining and 96.36, 97.56, and 92.85% for acridine orange dye. Multiplex real time PCR using Taq-Man probe detected two species of T. annulata and T. orientalis simultaneously. Acridine dye based fluorescent microscopy is comparatively easy and rapid method in detection of Thelieria spp.

Background: Theileria equi causes equine piroplasmosis, an economically significant disease that affects horses and other equids worldwide. Based on 18S ribosomal RNA (18S rRNA sequences), T. equi can be classified into five genotypes: A, B, C, D, and E. These genotypes have implications for disease management and control. However, no conventional polymerase chain reaction (PCR) assays are available to differentiate the genotypes of T. equi. To overcome this limitation, we developed and evaluated PCR assays specific for the detection of each T. equi genotype., Methods: A pair of forward and reverse primers, specifically targeting the 18S rRNA sequence of each genotype, was designed. The genotype-specific PCR assays were evaluated for their specificity using plasmids containing inserts of the 18S rRNA sequence of each genotype. Subsequently, the assays were tested on 270 T. equi-positive equine blood DNA samples (92 from donkeys in Sri Lanka and 178 from horses in Paraguay). 18S rRNA sequences derived from the PCR amplicons were analyzed phylogenetically., Results: Each genotype-specific PCR assay accurately targeted the intended genotype, and did not produce any amplicons when 18S rRNA from other T. equi genotypes or genomic DNA of Babesia caballi or uninfected horse blood was used as the template. Previous studies employing PCR sequencing methods identified T. equi genotypes C and D in the Sri Lankan samples, and genotypes A and C in the Paraguayan samples. In contrast, our PCR assay demonstrated exceptional sensitivity by detecting four genotypes (A, C, D, and E) in the Sri Lankan samples and all five genotypes in the Paraguayan samples. All the Sri Lankan samples and 93.3% of the Paraguayan samples tested positive for at least one genotype, further emphasizing the sensitivity of our assays. The PCR assays also had the ability to detect co-infections, where multiple genotypes in various combinations were detected in 90.2% and 22.5% of the Sri Lankan and Paraguayan samples, respectively. Furthermore, the sequences obtained from PCR amplicons clustered in the respective phylogenetic clades for each genotype, validating the specificity of our genotype-specific PCR assays., Conclusions: The genotype-specific PCR assays developed in the present study are reliable tools for the differential detection of T. equi genotypes., (© 2023. The Author(s).)

Background: Ovine and caprine theileriosis is a tick-borne hemoprotozoan disease, caused by Theileria spp., responsible for heavy economic losses in terms of high mortality and morbidity rates. Diagnosis of ovine theileriosis is primarily based on clinical symptoms, microscopic screening of stained blood smears, and lymph node biopsy smears, but the limitations of these detection methods against Theileria spp. infection limits their specificity., Aim: To overcome these limitations, the current study reports the differential diagnosis of theileriosis through a blood smear examination and polymerase chain reaction (PCR) in small ruminants from Pakistan., Methods: The study was conducted on 1,200 apparently healthy small ruminants (737 sheep and 463 goats). First, blood smears were screened for the presence of Theileria piroplasms in red blood cells. Second, PCR amplification based on 18S rRNA gene was performed by using primers specific to Theileria spp., Results: Out of the 1,200 samples of examined blood smears, 100 animals (8.33%) were found positive for Theileria species, which showed intra-erythrocytic bodies in the form of dot and comma shapes. Amplification of the isolated DNA from randomly collected blood samples of 737 sheep and 463 goats showed that an amplicon size of 1,098 bp was positive for Theileria spp. In total, 315 out of the 1,200 small ruminants examined in this study were found positive for Theileria spp. DNA through PCR amplification. Notably, out of the 885 blood samples negative by PCR amplification, only 15 blood samples were found positive by the blood smear test. Conversely, 230 blood samples that tested negative in the smear technique produced a specific band through PCR amplification. Overall, the sensitivity and specificity rates were 26.98% and 98.31% for the blood smear method and 73.01% and 100% for the PCR assay, respectively., Conclusion: Our finding suggests that PCR is the gold standard method compared to the conventional method of smear examination for the diagnosis of ovine and caprine theileriosis in Pakistan., Competing Interests: The authors declare that there is no conflict of interest.

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., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: The parasites of genera such as Babesia and Theileria are called piroplasmids due to the pear-shaped morphology of the multiplying parasite stages in the blood of the vertebrate host. Because of the enormous number of parasite species and the challenges of multiplex PCR, initial screening of samples using piroplasmid-specific PCR may be a more cost-effective and efficient technique to identify parasite species, especially during epidemiological studies. Accordingly, 18S rRNA PCR was standardized and optimized on common piroplasmids of different animals like cattle, buffaloes, sheep, goats, dogs, horses, and leopards., Methods: Bloods samples from 1250 animals were collected from different animals in Junagadh district of Gujarat, India. 18S rRNA PCR was standardized and optimized as a primary method for molecular screening of piroplasms in domestic and wild animals. The method was checked for its analytical sensitivity and specificity. Parasite species-specific PCR and sequencing was used to validate the test. Moreover, in-silico restriction enzyme (RE) analysis was also done to assess its applicability in PCR-RFLP., Results: Piroplasm infections were recorded in 63.3% of animals in Junagadh. The 18S rRNA PCR detected the piroplasmid DNA in as low as 39 picograms (pg) of whole blood genomic DNA isolated from microscopically Theileria positive blood samples and no reactivity was recorded from common but unrelated haemoparasites viz., Trypanosoma evansi, Hepatozoon spp., Anaplasma spp., and Ehrlichia canis was observed. The 18S rRNA PCR assay findings were confirmed by species-specific PCR and sequencing. Analysis of different sequences generated using 18S rRNA PCR revealed that the amplicon size of Babesia spp. is nearly 400 bp (393-408 bp) whereas Theileria spp. were more than 400 bp (418-424 bp). The percentage of sequence divergence among Babesia and Theileria spp. was 7.3-12.2% and 0.7-12.2%, respectively. In-silico restriction enzyme (RE) analysis reveals the presence of at least one site for a commercially available RE in 18S rRNA fragments of every parasite, which can differentiate it from its congeners., Conclusions: The presented universal oligonucleotide-based PCR assay provides a highly sensitive, specific, cost-effective, and rapid diagnostic tool for the initial screening of piroplasmids infecting domestic and wild animals and is potentially helpful for large-scale epidemiological studies., (© 2022. The Author(s) under exclusive licence to Witold Stefański Institute of Parasitology, Polish Academy of Sciences.)

Ticks are economically important obligatory blood feeding arthropods that have a pivotal role in transmission of infection. The present study was conducted in ixodid ticks collected from four districts of coastal Odisha, India to investigate the prevalence of Theileria annulata . Adult semi engorged Hyalomma anatolicum ticks ( n  = 178) were dissected, the salivary gland was isolated and DNA was extracted. A nested PCR targeting the Tams1 gene of T. annulata , utilizing two sets of primers (N516F, N517R, and Ta14136iF, Ta249R) was utilized for detection of the parasite. The PCR products were then sequenced and subjected to BLAST analysis, alignment, and phylogenetic study. Two sequences deposited in GenBank were assigned Accession No MH477290.1 and Accession No MH477291.1. The molecular investigation of T. annulata revealed an overall prevalence of 14.6% in tick vectors, and nested PCR was found to have significant ( p  < 0.05) higher results than primary PCR. A significant higher presence ( p  < 0.05) was recorded in female ticks compared with male ticks. This is the first report of detection of the parasite in tick vectors in the state of Odisha.

Theileriosis is one of the most frequently reported tick borne diseases in tropical and subtropical regions and leads to annual economic losses, such as the reduced dairy products and increased casualties. Tropical theileriosis is caused by Theileria annulata and the present study was designed to improve our knowledge of Theileria annulata infection in Pakistani cattle. In order to assess the prevalence of Theileria annulata on cattle from Multan district in the Punjab province (Pakistan) according to seasons and other risk factors, a total of 1020 blood samples (340 samples each from cross, Holstein Frisian and Sahiwal breed) were collected between 2020 and 2022. Based on Tams-1 partial sequence amplification, the overall T. annulata prevalence was estimated at 11.3% (115/1020). The highest prevalence was observed in autumn season (14.1%), followed by winter (12.9%), summer (11.4%) and spring (6.7%) season. Sahiwal cattle were most susceptible to T. annulata infection (13.2%) followed by Crossbred (11.8%) and Holstein Frisian (8.8%). Epidemiological factor analysis revealed that female cattle, cattle rose with other dairy animals at farm, tick infested cattle, and cattle raised with dogs infested with ticks were associated with the prevalence of T. annulata. White blood cells, lymphocyte (%), Monocyte (%) hemoglobin, mean cell hemoglobin, mean corpuscular hemoglobin concentration, and platelet count were significantly affected blood parameters in T. annulata positive cattle of all three breeds. Representative partial Tams-1 sequences of four Pakistani T. annulata isolates revealed a single genotype genetically close to those infecting cattle from neighboring countries like Iran, Turkey and Egypt. Longitudinal survey and phylogenetic positioning of T. annulata is recommended for epidemiological correlation, diagnosis and treatment of theileriosis in such an agricultural region of Pakistan., (Copyright © 2022. Published by Elsevier B.V.)

Equine Piroplasmosis (EP) is a tick-borne disease caused by three apicomplexan protozoan parasites, Theileria equi (T. equi), Babesia caballi (B. caballi) and T. haneyi, which can cause similar clinical symptoms. There are five known 18S rRNA genotypes of T. equi group (including T. haneyi) and three of B. caballi. Real-time PCR methods for detecting EP based on 18S rRNA analysis have been developed, but these methods cannot detect all genotypes of EP in China, especially genotype A of T. equi. In this study, a duplex real-time PCR detection method was developed for the simultaneous detection and differentiation of T. equi and B. caballi. The primers and probes for this duplex real-time PCR assay were designed based on the conserved 18S rRNA gene sequences of all genotypes of T. equi and B. caballi including Chinese strain. Double-quenched probes were used in this method, which provide less background and more signal to decrease the number of false positives relative to single-quenched probes. The newly developed real-time PCR assays exhibited good specificity, sensitivity, repeatability and reproducibility. The real-time PCR assays were further validated by comparison with a nested PCR assay and a previous developed real-time PCR for EP and sequencing results in the analysis of 506 clinical samples collected from 2019 to 2020 in eleven provinces and regions of China. Based on clinical performance, the agreements between the duplex real-time PCR assay and the nPCR assay or the previous developed real-time PCR assay were 92.5% (T. equi) and 99.4% (B. caballi) or 87.4% (T. equi) and 97.2% (B. caballi). The detection results showed that the positivity rate of T. equi was 43.87% (222/506) (10 genotype A, 1 genotype B, 4 genotype C, 207 genotype E), while that of B. caballi was 5.10% (26/506) (26 genotype A), and the rate of T. equi and B. caballi co-infection was 2.40% (12/506). The established method could contribute to the accurate diagnosis, pathogenic surveillance and epidemiological investigation of T. equi and B. caballi infections in horses., (© 2022 Wiley-VCH GmbH.)

Malawi has an estimated cattle population of 1,884,803 heads, the indigenous Malawi zebu breed accounts for 91.2%, while the exotic and crossbred accounts for the remaining 8.8%. Although ticks and tick-borne diseases are widespread in Malawi, no molecular study has been conducted to investigate the tick-borne Anaplasmataceae and piroplasms infecting cattle. To provide an insight into the current status of tick-borne pathogens (TBPs) of cattle, a molecular survey was conducted in the central and southern regions of Malawi. A total of 191 cattle of which 132 were Malawi zebu, 44 were Holstein Friesian and 15 were Holstein-Friesian/ Malawi zebu crosses were screened for Anaplasmataceae and piroplasms using the heat shock protein groEL gene and 18S rDNA, respectively. A new 18S rDNA multiplex PCR assay was designed for Babesia and Theileria species identification without sequencing. Overall, 92.3% (n = 177) of the examined animals were infected with at least one TBP. Anaplasmataceae-positive rate was 57.6% (n = 110) while for piroplasms it was 80.1% (n = 153). The detected Anaplasmataceae were Anaplasma bovis 2.6% (n = 5), Anaplasma marginale 24.6% (n = 47), Anaplasma platys-like 13.6% (n = 26), uncharacterized Anaplasma sp. 14.1% (n = 27), and uncharacterized Ehrlichia sp. 16.2% (n = 31). The detected piroplasms were Babesia bigemina 2.6% (n = 5), Theileria mutans 73.8% (n = 141), Theileria parva 33.0% (n = 63), Theileria taurotragi 12.6% (n = 24), and Theileria velifera 53.4% (n = 102). Mixed infection rate was found in 79.6% (n = 152) of the samples analyzed. This study has shown a high burden of TBPs among cattle in Malawi which highlights the need to conceive new methods to control ticks and TBPs in order to improve animal health and productivity. The newly developed multiplex PCR assay would be a useful tool especially in resource limited settings where sequencing is not available and when mixed infections are expected., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Study was undertaken in a theileriosis-endemic region of India during May 2018 to April 2019 among milch cows. Blood samples collected from apparently healthy ( n  = 65) and Theileria-suspect cows ( n  = 65) were screened against T. annulata and T. orientalis infection by SYBR Green‒based real time PCR using primers designed from the isolates of study area. Cows having single infection with T. annulata with/without clinical signs of inappetence, low milk yield, pale mucous membranes, fever, enlarged prescapular lymph node, soil licking, panting, coughing, salivation and lachrymation were subjected to further investigation where parasitaemia and piroplasms per 1000 erythrocytes ranged from 1.6 × 10 7 to 1.2 × 10 8 parasites/mL of blood and 3-24 piroplasms in moderate group (16/65), 4.4 × 10 8 to 6.9 × 10 9 parasites/mL of blood and >88 piroplasms in severe group (30/65) and 1.6 × 10 4 to 5.5 × 10 6 parasites/mL of blood and 0-1 piroplasms in asymptomatic or carriers (17/65), respectively. Study unfolded significant difference in T. annulata parasitaemia among apparently healthy and ill cows. Phylogenetic analysis of our T. annulata isolates (NCBI accession numbers MN098316, MN098317 and MN098318) exhibited maximum similarity with the isolates detected in other parts of India.

Equine piroplasmosis is a disease of horses, mules and donkeys, caused by the hemoprotozoans Babesia caballi and Theileria equi and transmitted by ticks of tropical and subtropical regions. Because the clinical signs are not specific, the diagnosis of equine piroplasmosis is difficult. In Mexico, where the environmental factors are conducive to the persistence of these pathogens, there is a lack of molecular studies to evaluate the occurrence of both parasites in horses. In the present study, matching serum and whole blood samples were obtained from 269 horses residing in 24 locations with tropical or subtropical climate and the presence of ticks. Testing of serum samples by ELISA demonstrated 55.7% seroprevalence of B. caballi and 68.4% prevalence of antibodies to T. equi. Blood samples analyzed with nPCR test were 7.8% positive to B. caballi and 78.8% positive to T. equi, while a duplex qPCR showed 15.24% positive samples to B. caballi and 59.11% to T. equi. From these results, 27 samples were sequenced for T. equi and 13 for B. caballi, confirming the presence of both horse parasites that cause equine piroplasmosis and suggesting that they are widespread in Mexico. This is the first study confirming the presence of B. caballi and T. equi in Mexico using both serological and molecular diagnostic methods. This study shows a high incidence of exposure to the etiological agents of equine piroplasmosis in horses in the studied areas., Competing Interests: The authors have declared that no competing interests exist.

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., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Equine piroplasmosis (EP) is a serious problem in the horse industry, and controlling EP is critical for international horse trading. EP is caused by two apicomplexan protozoan parasites, Theileria equi and Babesia caballi. Rapid and accurate methods that are suitable for detecting these parasites in the field are crucial to control the infection and spread of EP. In this study, we developed a card to detect antibodies against T. equi and B. caballi based on two colloidal gold immunochromatographic strips according to the principle of the double-antigen sandwich. The proteins equi merozoite antigen 1 (EMA1) and rhoptry protein BC48 are commonly used as diagnostic antigens against T. equi and B. caballi, respectively. On the strip, the purified EMA1 or BC48 protein labeled with colloidal gold was used as the detector, and nitrocellulose membranes were coated with EMA1 or BC48 and the corresponding MAb as the test and control lines, respectively. The protocol takes 10 to 15 min and requires no specialized equipment or chemical reagents, and one test can detect two EP pathogens in one card. Specificity tests confirmed there was no cross-reactivity with sera positive for common equine pathogens. Using a commercial competitive enzyme-linked immunosorbent assay (cELISA) kit for comparison, 476 clinical samples were tested with the card. The coincidence rates were 96.43% and 97.90% for T. equi and B. caballi, respectively. The field trial feedback was uniformly positive, suggesting that this diagnostic tool may be useful for controlling the spread of T. equi and B. caballi. IMPORTANCE Equine piroplasmosis (EP), caused by Theileria equi and Babesia caballi, is an important tick-borne disease of equines that is prevalent in most parts of the world. EP is considered a reportable disease by the World Organization for Animal Health (OIE). The accurate diagnosis and differentiation of T. equi and B. caballi are very important for the prevention, control, and treatment of EP. Therefore, we developed a double-antigen sandwich colloidal gold immunochromatography assay (GICG) to detect T. equi and B. caballi. Two GICG strips were assembled side by side on one card for the detection of T. equi and B. caballi, and the two EP pathogens could be detected in one test. This method was simple, rapid, and specific for the detection of EP; therefore, compared to the previous methods, this method is more suitable for pathogen diagnosis in the field.

Equine piroplasmosis (EP) is a tick-borne infectious disease highly prevalent in tropical and subtropical regions, such as Venezuela. EP affects wild and domestic equids leading to several clinical presentations, from asymptomatic to severely affected animals. In this study, thirty-three (33) sport horses under regular training activities and from endemic regions of north-central Venezuela were submitted to an observational survey, case-control, to describe the presence of clinical signs and natural EP infections. A conventional PCR assay targeting the SSU rRNA gene revealed EP etiologic agents in 13 out of 33 sampled horses (~ 39.4% infections). Nine (9) of these EP-positive samples were confirmed as infected with Babesia caballi (6/9 = 66.7%) or Theileria equi (3/9 = 33.3%) by DNA sequencing and BLASTN analyses. A phylogeny of SSU rRNA gene sequences revealed that these new B. caballi and T. equi sequences clustered within the worldwide distributed phylogenetic genotype A, respectively. No acute EP cases were observed in this study; however, six (6) PCR-positive animals displayed mild clinical signs compatible with EP, including a mild leukocytosis (P < 0.05). The heart rate variability frequency domain analysis in four (4) of these EP-positive infected animals revealed a significant (P < 0.05) higher low-frequency/high-frequency ratio suggesting a sympathovagal imbalance in these chronically infected animals. Other clinical and cardiovascular parameters were similar between the different groups. Sport horses are routinely submitted to intense training programs and sport-related activities that could lead to loss of the host-parasite equilibrium that characterizes enzootic regions, increasing the likelihood of infection reactivation and the risk of transmission. Heart rate variability analysis contributes to evaluate the sympathovagal balance and detecting homeostasis disturbances in sport horses. Molecular diagnostic tests for EP based on the detection of parasite DNA in equine blood samples should be included in the health programs of sport horses in endemic areas., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Equine piroplasmosis is caused by apicomplexan parasites, namely, Babesia caballi and Theileria equi , which are transmitted to equids principally through ticks. To ascertain the exposure of equines to agents of equine piroplasms, we tested serum samples collected from horses (n = 272) and donkeys (n = 170) in North-Western Nigeria for the presence of antibodies against B. caballi and T. equi using IFAT and ELISA. The seroprevalence of T. equi in the horses determined using IFAT and ELISA was 48.89% and 45.96%, respectively, while for B. caballi , it was 6.3% and 0.4%, respectively. For T. equi , the seroprevalence based on IFAT and ELISA results in donkeys was 14.1% and 2.9%, respectively, while for B. caballi , the seroprevalence was 2.4% and 0.6%, respectively, for ELISA and IFAT. Mixed infection detected in the horses using IFAT and ELISA was 5.5% and 0.4%, respectively, while no mixed infection was observed in the donkeys. The seroprevalence of T. equi was significantly ( P < .0001) higher than that of B. caballi in both horses and donkeys. Comparatively, the IFAT detected a greater number of piroplasm seropositive animals than ELISA, indicating a difference in their diagnostic accuracy. Findings from this study confirm the existence of equine piroplasms in both horses and donkeys in North-Western Nigeria and highlights the need for robust and effective control measures against the disease.

Theileria parasites commonly infect African wild artiodactyls. In rare roan ( Hippotragus equinus ) and sable ( H. niger ) antelopes, Theileria sp. (sable)-associated calf mortalities constrain breeding programs. The pathogenicity of most leukocyte-transforming Theileria spp. originates in their invasion of and multiplication in various mononuclear leukocytes, the transformation of both infected and uninfected leukocytes, and their infiltration of multiple organs. Understanding the pathogenesis of theileriosis can be improved by the use of immunohistochemistry (IHC) to identify the localization of the parasites in tissue sections. Our aim was to develop a reproducible IHC assay to detect leukocyte-associated Theileria parasites in formalin-fixed, paraffin-embedded roan and sable tissues. Polyclonal antibodies were purified from the sera of 5 roans from an area endemic for Theileria sp. (sable) and tested for IHC reactivity in 55 infected and 39 control roan and sable antelopes, and for antigen and species cross-reactivity in an additional 58 cases. The 3 strongest antibodies consistently detected intraleukocytic theilerial antigens in known positive cases in roan and sable antelopes, and also detected other Theileria spp. in non-hippotraginid wild artiodactyl tissues. The antibodies did not cross-react with other apicomplexan protozoa, with the exception of Cryptosporidium . Given that PCR on its own cannot determine the significance of theilerial infection in wild ruminants, IHC is a useful laboratory test with which to confirm the diagnosis in these species.

Tropical theileriosis is one of the major causes of newborn calves mortality. Observation of clinical manifestations is important while making the presumptive/tentative diagnosis of tropical theileriosis in newborn calves. The phenotypic and haemato-biochemical appraisals of tropical theileriosis could be of great help to make a holistic therapeutic plan for diseased newborn calves. Therefore, the present study aimed to evaluate the haemato-biochemical and phenotypic diagnostic markers of tropical theileriosis in newborn calves. A total of 43 newborn calves naturally infected with Theileria annulata and 16 age-matched healthy calves were enrolled. The percentage distribution of clinical markers was generalized lymph nodes enlargement (100%), pyrexia (97.67%), respiratory distress (95.34%), tick infestation (90.69%), anorexia (88.37%), pica (81.39%), pallor mucous membrane (67.44%), hyperlacrimation (58.13%) and exophthalmia (30.22%). Haemograms including TEC, Hb and HCT were found to be significantly (P ≤ 0.001) lowered in diseased calves. Remarkable alterations in the leukogram panels were not observed. Serum glucose, total protein, albumin and globulin concentrations of calves with theileriosis were significantly (P ≤ 0.001) lower than healthy ones, whereas triglycerides and total cholesterol levels of diseased calves were significantly (P ≤ 0.001) higher. Significantly (P ≤ 0.001) elevated activities of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) enzymes were observed in diseased calves. An evaluation of clinical phenotypes could be helpful to initiate quick treatment of diseased calves in field conditions and save the lives of sick calves of economically poor farmers. Altered haemato-biochemical panels to be appraised by veterinary clinicians while making a therapeutic plan of tropical theileriosis., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

An adult female sika deer (Cervus nippon aplodontus) inhabiting Nara Park, Nara, Japan, had broken bone injuries from a car accident. During its treatment, we found that the sika deer had severe anemia and the fracture remained unhealed throughout. Peripheral blood smear revealed piroplasms in the erythrocytes, which were identified as merozoites of undescribed Theileria species, widely found in sika deer in Japan. This is the report of a clinical case of Theileria infection, accompanied by severe anemia in a sika deer., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Theileria are tick-borne apicomplexan parasites, which mainly infect ruminants in tropical and subtropical regions of the world. The present study was directed to investigate the serological methods for the diagnosis of theileriosis in crossbred cattle. Blood samples (n = 176) were collected from the regional cattle populations of Bihar state situated at the Gangetic plains of India. Microscopic examination of blood smears from the cattle revealed the presence of tick-borne infectious organisms (Theileria and Anaplasma) in the region. PCR-based detection of Tams1 (Theileria annulata merozoite surface antigen) gene and the sequencing of 18S rRNA amplicon from the blood samples confirmed T. annulata as the primary causative agent of theileriosis in cattle of the Bihar region. Similarly, the amplification of the msp5 gene confirmed Anaplasma marginale infection. For the large-scale epidemiological investigation, sporozoite and macroschizont (spm2) partial gene from T. annulata was cloned in pET-28a (+) vector and overexpressed in E. coli BL21 cells. Overexpressed recombinant-Spm2 (43 kDa) was purified by Ni-NTA affinity chromatography and was used for immunodetection of theileriosis in cattle serum samples. Sequence analysis of the cloned partial spm2 gene in this study showed multiple SNPs (single nucleotide polymorphisms) in T. annulata. Recombinant-Spm2 antigen was explicitly recognised by the immunoglobulins (IgG) of the cattle naturally infected with Theileria. Further, an indirect enzyme-linked immunosorbent assay (ELISA) was developed using partial r-Spm2 antigen that exhibited high sensitivity (100 %) and specificity (90.9 %). Thus, this study suggests that partial r-Spm2 can be used as a diagnostic antigen for seroepidemiological studies of T. annulata infection in crossbred cattle., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Background: Theileriosis, trypanosomosis and babesiosis are the three major haemoprotozoan diseases causing huge economic losses worldwide. Difficulty in diagnosis of these diseases lies with the detection of carrier state with low parasitemia and concurrent infection., Purpose: The present study was conducted to standardize and evaluate multiplex PCR assay for specific, fast and simultaneous detection of Theileria annulata, Trypanosoma evansi and Babesia bovis in bovines., Methods: Positive parasitic DNA was obtained from microscopically positive samples. Simplex PCR assay was developed targeting repetitive nucleotide sequences for Trypanosoma evansi and gene coding enzyme carbamoyl phosphate synthetase II for Babesia bovis. For theileriosis conditions already standardized targeting cytochrome b gene was used. Gradient PCR assay was used to determine common amplification conditions and develop multiplex PCR assay. Limit of detection was determined using tenfold serial dilution of parasitic DNA. Blood samples collected from 117 bovines suspected for haemoparasite infection was tested by simplex and multiplex PCR assay., Results: Simplex PCR assay was able to detect Theileria annulata, Trypanosoma evansi and Babesia bovis at dilution 10 -9 , 10 -8 and 10 -8 which corresponds to copy number 1, 10 and 10, respectively, whereas of multiplex PCR assay was found to be 10 -7 dilution corresponding to 100 copy number. PCR products bands obtained in multiplex PCR assay at 257, 312 and 446 bp were easily distinguishable. Results of simplex PCR assay for detection of individual parasites revealed 48 (41.02%), 27 (23.07%) and 5 (4.27%) samples positive for T. annulata, T. evansi and B. bovis, respectively. Sixty-three (53.8%) samples were found positive by multiplex PCR assay with 15 samples (23.8%) showing mixed infection., Conclusion: Multiplex PCR assay was found to be highly specific and can be used for easy, early, sensitive, specific and simultaneous diagnosis of haemoprotozoan diseases in epidemiological survey as a robust tool.

Equine piroplasmosis (EP) is an infectious, tick-borne disease caused by the hemoprotozoan parasites, Theileria equi, Babesia caballi, and a recently reported new species, T. haneyi. Infections by these apicomplexan parasites limit performance and cause economic losses for the horse industry. Equine piroplasmosis is widespread in the northern regions of Nigeria, where an increasing portion of the animal population is composed of horses. This disease has remained epidemiologically challenging, especially as the movement of horses increases across Nigeria. In this study, blood samples from 300 horses were collected in three states of northwestern Nigeria. The presence of piroplasms was screened by nested PCR targeting 18S rDNA and positive samples were analyzed using species-specific-nested PCR-targeting genes including ema1 (T. equi), rap1 (B. caballi), and a gene coding a protein of unknown function (T. haneyi). Species-specific-nPCR results demonstrated that the prevalence of T. equi was 13.0% (39/300), B. caballi was 3.3% (10/300) and T. haneyi was 2.7% (8/300). Mixed infections with T. equi and B. caballi was 2.7% (8/300) while T. equi, B. caballi, and T. haneyi multiple infection prevalence was 0.6% (2/300). We used 18S rDNA sequences to determine close relationships between T. equi by phylogenetic analysis and demonstrated that among 57 sequences of Theileria parasites, 28 samples belonged to clade A (49%), 13 samples were found to be clade C (22%), and 16 were clade D (28%). These results demonstrate the genetic diversity of T. equi circulating in horses from Nigeria.

Theileria annulata the causative agent of bovine tropical theileriosis (BTT) is globally distributed. Rapid and accurate detection of the parasite is essential for the implementation and evaluation of mass drug administration and planned vaccination programs for controlling BTT. Loop mediated isothermal amplification (LAMP) amplifies targeted nucleic acid with a high efficacy, sensitivity and rapidity under isothermal conditions. In the present study, the internal transcriber space (ITS) gene of T. annulata was targeted for the development of a LAMP assay using pH-sensitive dye (phenol red) for enhanced visual detection of amplification. This method employed a set of specially designed four primers that recognized six distinct regions on the targeted gene. No amplification was detected with the DNA of other tested haemoprotozoans. Positive LAMP products were identified by a colour change from pink to yellow, and then rechecked by specific ladder pattern upon agarose gel electrophoresis. LAMP was able to detect infection in 63 out of 85 animals compared with blood microscopy, simple PCR and nested PCR which detected infection in 40, 49 and 64 animals, respectively. No difference in detection was seen in the colorimetric assay and the classical UV based LAMP assay., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Anaplasmosis and theileriosis are important diseases with great economic impact that affect the cattle industry worldwide. In this study, we describe the first molecularly confirmed clinical cases of anaplasmosis caused by Anaplasma marginale and of a concurrent infection with A. bovis and Theileria orientalis in Croatian cattle. Clinical signs of A. marginale-infected cows included fever, lethargy, dark urine, as well as icteric and reddish mucous membranes. Postmortem examination revealed icterus, urinary bladder filled with dark urine and splenomegaly. A marginale was observed within erythrocytes on Giemsa-stained spleen imprints. Three affected cows were successfully treated with oxytetracycline, after which no new deaths occurred in the herd. Three cows in a different herd died suddenly and were found to be concurrently infected with A. bovis and T. orientalis. Postmortem examination revealed generalized icterus and urinary bladder filled with dark urine. These cases of A. marginale, A. bovis and T. orientalis infection show that bovine anaplasmosis and theileriosis are present within Croatian cattle and should be included in differential diagnostic protocols., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

The aim of this study was to determine the seroprevalence of Babesia caballi and Theileria equi in horses imported into Shanghai port. Between 2018 and 2019, 344 horse sera samples were collected and tested for B. caballi and T. equi, using commercially available kits. Only one B. caballi seropositive sample was detected. To the best of our knowledge, this is the first report of a B. caballi seropositive in imported horses at Shanghai port, which reflects the importance of monitoring piroplasmosis seroprevalence in imported horses., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The increasing Yezo sika deer (Cervus nippon yesoensis) population is creating a large problem. Yezo sika deer are an important blood meal source, and these deer contribute to the maintenance of tick populations. Theileria spp. infections in Yezo sika deer and T. orientalis infections in cows occur at high frequencies, and the same tick species infests both deer and cows. Therefore, a specific detection method to identify deer Theileria spp. is important. In this study, we establish a novel molecular detection method for identifying Theileria spp. from deer and tick samples using loop-mediated isothermal amplification (LAMP). This method targets a metalloprotease/cell division cycle protein gene homologue. Our LAMP protocol was able to detect deer Theileria and did not show cross reactivity with other closely related protozoan parasites, including T. orientalis. The LAMP method showed sensitivity and specificity equivalent to those of nested PCR performed on the same field samples from deer and ticks. These results demonstrate the applicability of LAMP to field surveys in which the detection of deer Theileria spp. is required. In conclusion, due to its simplicity, specificity, and reliability, we suggest our LAMP protocol as an appropriate method for routine surveys to detect Yezo sika deer and ticks infected with deer Theileria spp. parasites. Additionally, this LAMP method offers great promise as a useful tool to distinguish Yezo sika deer Theileria from related Theileria parasites present in livestock., Competing Interests: Declaration of Competing Interest There are no conflicts of interest in this research study., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Equine piroplasmosis, caused by the protozoan pathogens Theileria equi or Babesia caballi , is endemic in many (sub-)tropical regions worldwide, including Southern Europe. In Germany, it mainly plays a role as an imported parasitosis. Nevetherless, tick species with known vector potential for equine piroplasms occur in Germany. Thus, a long-term establishment of autochthonous cycles after introduction via infected equids or ticks cannot be excluded. In the present case, 2 horses developed clinical T. equi infection after returning from a trail riding trip to southern France. During treatment, a feeding tick was discovered on one of the horses, which was identified as a Rhipicephalus bursa specimen. This tick species cannot establish in Germany under the current climatic conditions; nevertheless, this discovery illustrates the potential of introducing infected ticks to Germany. The 2 horses were treated with imidocarb (3 mg/kg i. m.; 3 treatments in 3-day intervals) and no adverse drug effects were recorded. To prevent establishment of autochthonous infection cycles in Germany as well as a chronic manifestation of piroplasmosis, treatment should aim at eliminating the parasites despite possible adverse effects of imidocarb., Competing Interests: Die Autoren bestätigen, dass kein Interessenkonflikt besteht., (© Georg Thieme Verlag KG Stuttgart · New York.)

The erythrocytic-stage surface protein equi merozoite antigen 1 (EMA-1) of Theileria equi is a major candidate for the development of a diagnostic antigen for equine piroplasmosis. In this study, BALB/c mice were immunized with purified recombinant EMA-1 to prepare monoclonal antibody (mAb) against T. equi EMA-1, and 1 mAb 5H2 was obtained that showed good reaction with infected red blood cells (RBC) in the indirect immunofluorescence assay (IFA). To develop a rapid serological detection method for T. equi infection in Xinjiang Uygur Autonomous Region, China, recombinant EMA-1 originating from the local T. equi strain and the mAb to EMA-1 were employed to develop an immunochromatographic test (ICT) to detect antibodies to T. equi in horse sera. The ICT showed high sensitivity and specificity and no cross-reaction with Babesia caballi. Ninety-two horse serum samples collected from Ili, Xinjiang, were tested by ICT and compared with the detection results of a commercial ELISA kit. The results showed that 56 of 92 (61%) serum samples were seropositive according to the ICT assay, and 50 (54%) samples were seropositive according to the ELISA kit. The ICT had a high coincidence (91.3%) but was more sensitive than the reference ELISA kit. To confirm whether the horses were infected by T. equi, 30 blood DNA samples from 92 horses were examined by PCR. The results showed that 14 of 30 (47%) horses were confirmed to be infected with T. equi by PCR, while 16 of 30 (53%) horses were seropositive by ICT. All PCR-positive horses were ICT-positive. The findings indicate that T. equi is endemic in Ili, Xinjiang, and that the ICT is reliable as a serological diagnosis method. The ICT developed in this study could be an efficient diagnostic tool to detect T. equi infection in horses in the Xinjiang area., (© American Society of Parasitologists 2020.)

Equine piroplasmosis (EP) is a severe disease of horses caused by the tick-borne protozoa Theileria equi (T. equi) and Babesia caballi (B. caballi). Infectious carriers are not always symptomatic, meaning there is a risk to non-enzootic areas. Regulatory tests for EP include sero-epidemiological methods for equine babesiosis, but these lack specificity due to cross-reactivity with other Babesia species. In this study, we present a real-time quantitative recombinase polymerase amplification (qRPA) method for fast simultaneous detection of both T. equi and B. caballi. In this method, primers and probes targeting the 18S rRNA gene of both T. equi and B. caballi, the ema-1 gene of T. equi and the bc48 gene of B. caballi were designed and evaluated. The sensitivity of qRPA was evaluated using the pUC57 plasmid DNA containing the target gene. For the pUC57-bc48 gene DNA, the R 2 value was 0.983 for the concentration range 0.2 ng (4.1 × 10 7 DNA copies) to 2.0 fg (4.1 × 10 1 DNA copies). For the pUC57-ema gene DNA, the R 2 value was 0.993 for the concentration range 0.2 ng (5.26 × 10 7 DNA copies) to 2.0 fg (5.26 × 10 2 DNA copies). For the pUC57-Bc18S gene DNA the R 2 value was 0.976 for the concentration range 2.0 ng (4.21 × 10 8 DNA copies) to 2.0 fg (4.21 × 10 2 DNA copies). For the pUC57-Te18S gene DNA, the R 2 value was 0.952 (Fig. S3b) for the concentration range 2.0 ng (4.16 × 10 8 DNA copies) to 2.0 fg (4.16 × 10 2 DNA copies). Furthermore, a duplex qRPA analysis was developed and optimized and the results showed that primers and probes targeting for the bc48 gene of B. caballi and the 18S rRNA gene of T. equi is the best combination for a duplex qRPA analysis in one reaction. The developed duplex qRPA assay has good specificity, and had negative amplification for several similar parasite. For DNA extracted from real horse blood specimens, this qRPA method has comparable sensitivity to traditional qPCR, but a simpler and more rapid operating process to obtain positive amplification. The qRPA, including the duplex strategy described here, could allow fast identification of the EP-causing T. equi and B. caballi, showing great potential for on-site EP screening of horses.

Aim: The present study was designed to check the molecular detection of Anaplasma marginale and Theileria annulata in blood samples of horses and donkeys collected from Dera Ghazi Khan District in Punjab and to document their phylogenetic origin and their association with studied epidemiological factors (sex and age) and complete blood count parameters, if any., Methods and Results: A total of 195 blood samples were collected from apparently healthy horses (N = 141) and donkeys (N = 54). A. marginale DNA was detected by PCR in 4.9% (7/141) horse and in 9.2% (5/54) of donkey blood samples. Prevalence of T. annulata was 5.6% (8/141) and 11.1% (6/54) in horse and donkey samples, respectively. While 1.4% (N = 2) horses and 3.7% (N = 2) donkeys were found co-infected with both parasites. Representative amplicon for both parasites was confirmed by DNA sequenced and partial DNA sequence of the major surface protein-1b encoding gene of A. marginale and cytochrome b gene from T. annulata were submitted to the GenBank database under the accession number MK792344-MK792348. Epidemiological data analysis revealed that female horses were more prone to A. marginale (P = 0.02) while female donkeys were more susceptible to A. marginale (P < 0.001) and T. annulata (P < 0.001) infection. It was observed that horse and donkey infected either with Anaplasma marginale or Theileria annulata had significantly disturbed red and white blood cell counts and their associated parameters., Conclusion: This is a first ever study regarding molecular detection of A. marginale and T. annulata in equine blood samples from Pakistan. We recommend that this multiplex PCR protocol should be used for the detection of Anaplasma marginale and Theileria annulata in livestock for their proper diagnosis and treatment.

East Coast fever is caused by Theileria parva, and poses serious concerns for dairy farmers owing to massive economic losses. In the current study, we compared three methods (DNA extraction kits, FTA-NaOH and FTA-TENT) of DNA extraction to identify the most economical and reliable method. A survey for T. parva prevalence was conducted in dairy cattle in Mbarara, Uganda. Cytochrome C oxidase subunit I (COI) and T. parva-p104 genes were amplified to compare the methods. FTA-NaOH-based polymerase chain reaction (PCR) yielded the best detection rate for both COI gene and p104 gene. Prevalence of T. parva was 45.0% and 83.3% at animal and farm-level, respectively. FTA-NaOH based-PCR is simple, highly sensitive and cost-effective tool for T. parva diagnosis in resource constrained settings.

Tropical theileriosis is a tick-borne lymphoproliferative disease of cattle caused by the apicomplexan parasite Theileria annulata, and leads to substantial economic losses to the livestock industry worldwide. Although various enzyme-linked immunosorbent assays (ELISAs) have been established to detect antibodies against T. annulata infection, a specific, rapid and reliable diagnostic assay is urgently needed for prevention and control of the disease. In the present study, a chemiluminescence immunoassay (CLIA) was developed based on the subtelomeric variable secreted protein (SVSP) of T. annulata as a sero-diagnostic antigen. Following optimization of the CLIA working parameters, the working time of the method was less than 4.5 h. The sensitivity and specificity of the established CLIA was 98.8% and 97.5%, respectively, when the cut-off value of the percent positive (PP) was 26.1% for detecting serum samples (n = 242 T. annulata positive sera, n = 158 T. annulata negative sera). After comparing 180 serum samples from Gansu province, China, the concordance rate between the CLIA and a published rSpm2 ELISA method was 72.8%. In addition, 565 serum samples of cattle collected between 2017 and 2018 from four provinces in China were detected by the CLIA, and the seroprevalence for T. annulata ranged from 53.3% to 67.3% in these regions. Our findings demonstrated that the CLIA has high specificity, sensitivity and reliability, and could be used as a rapid detection assay for epidemiological investigations of T. annulata infection., Competing Interests: Declaration of competing interest All authors declare that they have no competing conflicts of interest., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Tick-borne diseases caused by Theileria are of economic importance in domestic and wildlife ruminants. The majority of Theileria infects a limited number of host species, supporting the concept of host specificity. However, some Theileria seem to be generalists challenging the host specificity paradigm, such as Theileria sp. (sable) reported from various vertebrate hosts, including African buffalo, cattle, dogs and different antelope species. We tested the hypothesis that T. sp. (sable) uses Bovidae as hosts in general using a real-time polymerase chain reaction assay specific for T. sp. (sable) and a closely related genotype: T. sp. (sable-like). Various antelope species from the Tragelaphini (black wildebeest, blesbuck, blue wildebeest, gemsbuck, sable and waterbuck) tested positive for either T. sp. (sable) or T. sp. (sable-like). However, no African buffalo (n = 238) or cattle (n = 428) sampled in the current study tested positive, suggesting that these latter species are not carrier hosts. The results were confirmed using next-generation sequencing which also indicated at least 13 new genotypes or species found in various antelope and giraffes. Genotypes were found in single host species or in evolutionarily related hosts, suggesting that host specificity in Theileria may be a lineage specific phenomenon likely associated with tick-host-parasite co-evolution.

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.)

Oriental theileriosis, a tick-borne disease of bovids caused by members of the Theileria orientalis complex, has a worldwide distribution. Globally, at least 11 distinct genotypes of T. orientalis complex, including type 1 (chitose), type 2 (ikeda), type 3 (buffeli), types 4 to 8, and N1-N3, have been described based on the sequence of the major piroplasm surface protein (MPSP) gene. Of these 11 genotypes, mainly ikeda and chitose are known to be pathogenic and cause considerable morbidity (including high fever, anaemia, jaundice and abortion), production losses and/or mortality in cattle. Mixed infections with two or more genotypes of T. orientalis is common, but do not always lead to a clinical disease, posing challenges in the diagnosis of asymptomatic or subclinical forms of oriental theileriosis. The diagnosis of oriental theileriosis is usually based on clinical signs, the detection of piroplasms of T. orientalis in blood smears, and/or the use of serological or molecular techniques. This paper reviews current methods used for the diagnosis of T. orientalis infections and the genetic characterisation of members of the T. orientalis complex, and proposes that advanced genomic tools should be established for investigations of these and related haemoparasites.

Bovine tropical theileriosis caused by Theileria annulata is an overwhelming haemoprotozoan tick-borne disease in taurine and cross-bred cattle in Punjab, India. However, there seems to be no report from India of cutaneous nodules associated with the disease. This report describes a five-year-old cross-bred cow presented to a university clinic with a history of fever, inappetence and malaise for the past six to seven days. Clinical examination revealed normal vital parameters, pale mucous membranes, mild enlargement of the prescapular lymph nodes and multiple subcutaneous nodular masses (2-4 cm) on the neck and abdomen. Haematology revealed mild anaemia and leucopenia with 48% neutrophils, 48% lymphocytes and 4% eosinophils. Romanowsky-stained smears of fineneedle aspiration biopsy samples from swollen lymph nodes and subcutaneous masses showed an increased number of lymphoid cells, suggesting cutaneous lymphomatosis. However, a critical examination of the smears from subcutaneous nodules showed a large number of Koch's blue bodies in macrophages and lymphoblasts, and several piroplasms were also noticed within the red blood cells in lymph node smears. A peripheral blood smear revealed mild to moderate parasitaemia. Extracted DNA from the parasitologically positive blood sample was subjected to nested polymerase chain reaction (nPCR) using T. annulata speciesspecific primers encoding the 30-kiloDalton major sporozoite surface antigen. The desired 572-base pair amplified product of the nPCR was comparable to the positive control. This seems to be a rare case of T. annulata in an adult cross-bred cow, showing cutaneous nodular involvement.

Theileria and Babesia are intracellular protozoan parasites infecting a wide range of animals. In Palestine, there is limited information on the prevalence of Theileria and Babesia spp. in livestock. We used PCR of the 18S ribosomal RNA gene followed by DNA sequencing to detect and identify parasite DNA in blood samples from sheep (n = 49), goats (n = 48), horses (n = 40), camels (n = 34), donkeys (n = 28) and mules (n = 2) from four districts of Palestine. DNA of T. ovis and T. equi was detected in 19 and 2 ovine blood samples, respectively. None of the camels, donkeys, and goats were positive for T. ovis. Sheep had a significantly higher rate of infection than other animals (P < 0.05). Theileria ovis is highly prevalent in sheep, while T. equi DNA was detected in a small proportion of the equids in Palestine.

Sika deer (Cervus nippon) is widely distributed in Asian countries and is one of the most common wildlife animals in Hokkaido, Japan. Previous studies identified Theileria spp. in sika deer in Japan including Theileria sp. Thrivae belonging to T. cervi group and Theileria sp. sola belonging to T. capreoli group. However, the studies failed to differentiate these two species without sequencing. Therefore, epidemiological information on cervine theileriosis in Hokkaido, Japan is limited. This study differentiated the two Theileria spp. using restriction fragments length polymorphism (RFLP). Based on the PCR-RFLP, Theileria spp. were identified in 103 (88.0%) of 117 samples, and the prevalence of each parasites were 86.3% (n = 101) and 57.3% (n = 67) for Theileria sp. Thrivae and T. capreoli-like, respectively. Phylogenetic analysis based on the 18S rRNA showed a close relationship between Theileria sp. Thrivae and T. cervi in China. In addition, phylogenetic analysis of internal transcribed spacer regions also showed a close relationship between Theileria sp. Thrivae and T. cervi., (Copyright © 2019. Published by Elsevier B.V.)

Equine Piroplasmosis (EP) is a tick-borne disease caused by apicomplexan protozoan parasites, Babesia caballi and Theileria equi . The disease is responsible for serious economic losses to the equine industry. It principally affects donkeys, horses, mules, and zebra but DNA of the parasites has also been detected in dogs and camels raising doubt about their host specificity. The disease is endemic in tropical and temperate regions of the world where the competent tick vectors are prevalent. Infected equids remain carrier for life with T. equi infection, whilst, infection with B. caballi is cleared within a few years. This review focuses on all aspects of the disease from the historical overview, biology of the parasite, epidemiology of the disease (specifically highlighting other non-equine hosts, such as dogs and camels), vector, clinical manifestations, risk factors, immunology, genetic diversity, diagnosis, treatment, and prevention.

Brazilian Pantanal is the world´s largest wetland ecosystem, where cattle's ranching is the most important economic activity. The objective of this study was to compile some epidemiological features on equine piroplasmids from the Nhecolândia sub-region of Pantanal wetland through the evaluation of the patterns of T. equi and B. caballi infections in different groups of horses; identification of the tick species that infest horses; and to study phylogenetic relationships among Theileria equi 18S rRNA gene sequences. During October 2015, blood and serum samples were collected from 170 horses in four different categories. Ticks, after identification, had their hemolymph and eggs examined for the presence of piroplasmid sporokinets. Also we searched parasites in the peripheral blood smears of the investigated horses. The number of red blood cells (RBCs) and the packed cell volume (PCV) ​​were determined to test for anemia in the infected animals, and exposure to B. caballi and T. equi was evaluated by enzyme-linked immunosorbent assay. "Catch all primers" based on 18S rRNA gene were used in polymerase chain reactions (PCR) to detect equine piroplasmids, followed by three nested PCRs for the phylogenetic analysis. The serological results showed that 61.8% and 52.9% of the horses sampled were exposed to T. equi and B. caballi, respectively. Piroplasmid DNA was detected in 43.5% of the horses analyzed. Our sequencing revealed 98-100% identity with some sequences previously published in GenBank for T. equi, and microheterogeneity among others. We found that 51.2% of the animals sampled were infested with Dermacentor nitens, Amblyomma sculptum, and Rhipicephalus (Boophilus) microplus, singly or co-infested. Since positive and negative animals presented similar RBC and PCV values, and no sporokinets were found on blood smears, hemolymph and eggs of the ticks collected, we suggest that infected equines can act as asymptomatic carriers for piroplasmosis in the studied region. Our results together showed the enzootic characteristic of equine piroplasmids in Pantanal region highlighting the importance of using different methods for detection these parasites. Moreover, breeding mares and foals should be monitored since they displayed the greatest occurrences for molecular test (59.0% and 86.1% respectively) and tick infestations (87.2% and 63.9% respectively)., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Two haemoparasites, Theileria equi and Babesia caballi, cause equine piroplasmosis (EP), one of the most prevalent tick-borne diseases in horses. The main aim of the present study was to develop and evaluate a multinested PCR (mn-PCR) for simultaneous detection of the equine piroplasmids T. equi and B. caballi, by amplification of five genetic markers (18S rRNA, β-tubulin, cytB, EMA-1 and RAP-1). This novel assay detected a high prevalence of equine piroplasmids in 235 horse blood samples collected in Castilla-León and Extremadura, Spain. The overall prevalence of infection with equine piroplasmids by mn-PCR was 72.8% (171/235), with 66.0% (155/235) of the animals positive for T. equi and 29.4% (69/235) positive for B. caballi. The seroprevalence obtained by cELISA for the same set of samples was lower than the infection prevalence recorded by mn-PCR, for either of the two equine piroplasmids (62.6%) as well as for T. equi alone (61.7%) or B. caballi alone (3.8%). There was high agreement among the mn-PCR and cELISA assays for diagnosis of EP caused by T. equi (κ = 0.83) but not for B. caballi (κ = 0.06). A phylogenetic analysis based on the RAP-1 gene of B. caballi showed that the strains from Spain clustered with those from Israel., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

The polymorphic nature of Theileria annulata merozoite surface antigen (TAMS 1) attributes to limitation in PCR based detection of various T. annulata genotypes present in different geographical domains across the globe. Multiple reports of failure of detection of tropical theileriosis using classical N516/517 primer set in the studied area were noticed. Hence, three single PCR protocols using N516/517, TAMS F/R and NTA F/R primer sets encoding different portions of TAMS 1 gene and two nested protocols, using combinations of these three primers, were compared to find out the most suitable primer set for diagnosis of calf theileriosis in studied area. The studied area constitutes the semi-arid theileriosis endemic area of Northern India. The various PCR protocols were tested on 75 clinically confirmed cases of calf theileriosis. Alongside, 25 confirmed theileriosis negative blood samples and DNA of other haemoprotozoa were also tested for specificity of these primer sets. Results revealed that the primer set NTA F/R to be more suitable in detecting the circulating T. annulata genotypes in the studied area in comparison to the classical N516/517 primer set. None of the primers gave false positive amplification with negative samples and/or DNA of other haemoprotozoa., (Copyright © 2018. Published by Elsevier Ltd.)

The aim of this study was to determine the frequency and factors associated to Babesia caballi, Theileria equi and Trypanosoma evansi in naturally infected equids from the northeast Brazil. Blood samples from 569 equids (528 horses, 8 mules, and 33 donkeys) were collected and tested for the presence of DNA of each of these protozoan parasites by PCR. Generalized linear models were used to evaluate risk factors associated with the infection. The frequency of T. equi infection was 83.5% (475/569) - 84.3% in horses, and 73.2% in donkeys and mules. The results of the final model indicated that age (senior group) and animal species (mule and donkey group) were protective factors against this pathogen. The frequency of B. caballi infection was 24.3% (138/569) - 23.5% in horses and 34.1% in donkeys and mules. Age (adult and senior group) was considered a protective factor against B. caballi infection whereas animal species (donkey and mule group) were considered a risk factor for the infection. Trypanosoma evansi infection was not detected in any of animals. Our results suggest that equids from the area studied may be infected earlier in life with the etiological agents of equine piroplasmosis and become asymptomatic carriers.