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Background and Aim: Ovine theileriosis caused by Theileria ovis and Theileria lestoquardi is an important infectious disease affecting small ruminants in regions of the tropic and subtropic zones. There is limited studies about ovine theileriosis in Egypt; so the present study aims to assess the occurrence of ovine theileriosis in Egypt at the molecular level. Materials and Methods: Blood samples were collected from 115 randomly selected sheep, which were apparently healthy; the ages of the sampled sheep ranged from 1 to 5 years old, from a local breed (barkae and balade), and showed no symptoms indicating infection with Theileria spp. The study was conducted in three governorates representing Lower Egypt (Menoufia and Beheira) and Upper Egypt (El-Wady El-Geded). All blood samples were subjected to polymerase chain reaction (PCR) and semi-nested PCR to target Theileria spp. 18S rRNA genes. Positive samples were sequenced, and these sequences were analyzed using nucleotide basic local alignment search tool (BLAST). Results: Six animals (5.22%) were PCR-positive carriers for ovine theileriosis. Nucleotide BLAST and phylogenetic analyses of the six obtained sequences showed that T. ovis was present in five animals (4.37%) in Menoufia (n=2) and El-Wady El-Geded (n=3), whereas T. lestoquardi was detected in 1 animal (0.87%) in El-Wady El-Geded. Conclusion: This study is the first to provide molecular evidence, genetic characterization, and phylogenetic analysis of ovine Theileria spp. in Egypt. Specifically, T. lestoquardi and T. ovis carrier statuses of sheep were confirmed. These results highlight the importance of developing an effective control strategy against ovine theileriosis carriers that might develop and/or spread theileriosis.

In the current investigation, the effect of ascorbic acid on the in vitro growth of several piroplasm including Babesia bovis (Bartonella bovis), Baconia bigemina, B. caballi, and Theileria equi (T. equi), as well as against Brucella microti in mice was assessed. The antipiroplasm efficacy of ascorbic acid in vitro and in vivo was assessed using a fluorescence-based SYBR Green I test. Using atom pair fingerprint (APfp), we investigated the structural similarity between ascorbic acid and the commonly used antibabesial medicines, diminazene aceturate (DA) and imidocarb dipropionate (ID). In vitro cultures of B. bovis and T. equi were utilized to determine the ascorbic acid and DA interaction using the Chou-Talalay method. Ascorbic acid inhibited B. bovis, B. bigemina, T. equi, and B. caballi growth in vitro in a dose-dependent manner. The APfp results revealed that ascorbic acid and DA have a maximum structural similarity (MSS). On a T. equi culture in vitro, ascorbic acid showed a synergistic interaction with DA, with a combination index of 0.28. B. microti growth was decreased by 41% in vivo using ascorbic acid combined with a very low dosage of DA (6.25 mg kg

Background An innovative approach has been introduced for identifying and developing novel potent and safe anti-Babesia and anti-Theileria agents for the control of animal piroplasmosis. In the present study, we evaluated the inhibitory effects of Malaria Box (MBox) compounds (n = 8) against the growth of Babesia microti in mice and conducted bioinformatics analysis between the selected hits and the currently used antibabesial drugs, with far-reaching implications for potent combinations. Methods A fluorescence assay was used to evaluate the in vivo inhibitory effects of the selected compounds. Bioinformatics analysis was conducted using hierarchical clustering, distance matrix and molecular weight correlation, and PubChem fingerprint. The compounds with in vivo potential efficacy were selected to search for their target in the piroplasm parasites using quantitative PCR (qPCR). Results Screening the MBox against the in vivo growth of the B. microti parasite enabled the discovery of potent new antipiroplasm drugs, including MMV396693 and MMV665875. Interestingly, statistically significant (P Theileria equi in vitro culture in comparison with untreated cultures. MMV396693/clofazimine and MMV665875/atovaquone (AV) showed maximum structural similarity (MSS) with each other. The distance matrix results indicate promising antibabesial efficacy of combination therapies consisting of either MMV665875 and AV or MMV396693 and imidocarb dipropionate (ID). Conclusions Inhibitory and hematology assay results suggest that MMV396693 and MMV665875 are potent antipiroplasm monotherapies. The structural similarity results indicate that MMV665875 and MMV396693 have a similar mode of action as AV and ID, respectively. Our findings demonstrated that MBox compounds provide a promising lead for the development of new antibabesial therapeutic alternatives. Graphical Abstract

Tick-borne diseases mainly, theileriosis, babesiosis and anaplasmosis cause significant economic losses in livestock globally, including Turkey. The tick-borne pathogens of small ruminants in Turkey have been studied widely but information on molecular characterization and disease occurrence is still limited. In this study, both microscopy and molecular detection and characterization for Theileria spp. Babesia ovis, Anaplasma ovis and Anaplasma phagocytophilum was conducted. A total of 133 blood samples of tick-infested small ruminants (105 sheep and 28 goats) were collected from Turkey: half of the animals had clinical signs of tick-borne disease infections. Using PCR assays and microscopy, 90.2% and 45.1% of the samples were positive for at least one pathogen, respectively. Overall, the infection rates of A. phagocytophilum, B. ovis, A. ovis, Theileria spp. were 66.7%, 62.4%, 46.6% and 7.0%, respectively. Fifty-nine of the 133 (44.4%) samples were co-infected with two or more pathogens. Sex, season and B. ovis positivity were significant risk factors for occurrence of clinical disease. Sequence and phylogenetic analysis based on B. ovis 18S small subunit rRNA, A. ovis major surface protein 4, Theileria spp. 18S rRNA and A. phagocytophilum 16S rRNA genes showed that the isolates in this study clustered together in well-supported clades with those previously collected from Turkey and other countries. The study shows B. ovis as the most significant pathogen associated with clinical and fatal cases in small ruminants from Turkey. Female sex and summer season are associated with increased risk of the disease. This study shows high infection rates with the pathogens among small ruminants including A. phagocytophilum which has veterinary and public health importance.

Anti-piroplasm drugs currently on the market have proven toxicity to the host and parasite resistance. Plants are possible sources of novel drugs. Subsequently, a novel strategy should be used to find new anti-piroplasm agents that are both effective and safe. In the present study, we have evaluated the effect of turmeric (Curcuma longa) methanolic extract on the in vitro growth of Babesia (B.) bovis, B. divergens, B. caballi, and Theileria (T.) equi. The in vitro inhibitory effectiveness of turmeric was assessed using a fluorescence test. The enhancement in the in vitro inhibitory efficacy of turmeric when administrated in combination with diminazene aceturate (DA) was investigated using in vitro cultures of different piroplasm parasites. Turmeric reduced the in vitro growth of B. bovis, B. divergens, T. equi, and B. caballi with IC50 values of 0.830 ± 0.078, 0.375 ± 0.055, 1.405 ± 0.575, and 0.720 ± 0.090 mg/mL, respectively. An amount of 1 mg/mL turmeric for B. bovis, 0.5 mg/mL turmeric for B. divergens, 1 mg/mL turmeric for T. equi, and 0.5 mg/mL turmeric for B. caballi exhibited 73.43%, 80.065%, 73.47%, and 47.375% inhibitions in the growth of the parasites, respectively. When turmeric was combined with DA, its in vitro inhibitory impact on bovine Babesia and equine Babesia/Theileria parasites was amplified. These findings show that a methanolic extract of turmeric could be a promising medicinal plant for the treatment of babesiosis, especially when administered in conjunction with DA.

Equine piroplasmosis is a tick-transmitted disease that is considered one of the most serious infectious diseases affecting equines. Searching for novel antipiroplasm drugs remains indispensable due to the emergence and spreading of resistant piroplasm parasites against the limited currently used drugs, diminazene aceturate and imidocarb dipropionate. Therefore, novel drugs with specified targets need to be identified and exploited. The inhibitory effects of Medicines for Malaria Venture (MMV) Malaria Box compounds with potent in vitro anti-equine piroplasmosis activity were evaluated against the growth of B. microti in mice in this study. Using a nested PCR assay targeting the B. microti ss-rRNA gene, we investigated the far-reaching impacts of effective combinations to inhibit parasite recrudescence. Using real-time PCR, this study revealed potential targets for the found potent compounds. When used as monotherapy, screening the Malaria Box against the in vivo growth of the B. microti parasite resulted in the discovery of new, potent antipiroplasm medicines, such as MMV020275 and MMV020490. In MMV020275-treated Theileria equi in vitro culture, a statistically significant difference (P0.05) in the cGMP-dependent protein kinase (PKG) mRNA level was identified as a down-regulation in contrast to non-treated cultures. In conclusion, new potent antipiroplasm drugs, including MMV020275 and MMV020490 are identified. MMV020275 significantly down-regulate the mRNA levels of the PKG gene. Clofazimine enhanced the inhibitory efficacy of MMV compounds which is suggested to use in treatment of animal or human babesiosis in the future.

Diminazene aceturate (DA) and imidocarb dipropionate are commonly used in livestock as antipiroplasm agents. However, toxic side effects are common in animals treated with these two drugs. Therefore, evaluations of novel therapeutic agents with high efficacy against piroplasm parasites and low toxicity to host animals are of paramount importance. In this study, the 400 compounds in the Pathogen Box provided by the Medicines for Malaria Venture foundation were screened against Babesia bovis, Babesia bigemina, Babesia caballi, and Theileria equi. A fluorescence-based method using SYBR Green 1 stain was used for initial in vitro screening and determination of the half maximal inhibitory concentration (IC50). The initial in vitro screening performed using a 1 μM concentration as baseline revealed nine effective compounds against four tested parasites. Two “hit” compounds, namely MMV021057 and MMV675968, that showed IC50 100 were selected. The IC50s of MMV021057 and MMV675968 against B. bovis, B. bigemina, T. equi and B. caballi were 23, 39, 229, and 146 nM, and 2.9, 3, 25.7, and 2.9 nM, respectively. In addition, a combination of MMV021057 and DA showed additive or synergistic effects against four tested parasites, while combinations of MMV021057 with MMV675968 and of MMV675968 with DA showed antagonistic effects. In mice, treated with 50 mg/kg MMV021057 and 25 mg/kg MMV675968 inhibited the growth of Babesia microti by 54 and 64%, respectively, as compared to the untreated group on day 8. Interestingly, a combination treatment with 6.25 mg/kg DA and 25 mg/kg MMV021057 inhibited B. microti by 91.6%, which was a stronger inhibition than that by single treatments with 50 mg/kg MMV021057 and 25 mg/kg DA, which showed 54 and 83% inhibition, respectively. Our findings indicated that MMV021057, MMV675968, and the combination treatment with MMV021057 and DA are prospects for further development of antipiroplasm drugs., Graphical abstract Image 1, Highlights • Nine of 400 MMV compounds suppressed the in vitro growth of piroplasm parasites. • MMV021057 and MMV675968 showed effects similar to those of DA. • Combinations of MMV021057 and DA had additive or synergistic effects on parasites. • MMV021057 and MMV675968 moderately inhibited the growth of B. microti in mice. • A combination treatment with MMV021057 and DA highly inhibited B. microti in mice.

Babesia, Theileria, and Anaplasma are important causative agents of tick-borne diseases that severely affect sheep. However, there is paucity in the occurrence genetic diversity of the infections of tick-borne diseases in sheep in border regions, northwestern China. In this study, nested polymerase chain reaction (nPCR) assays and gene sequencing were used to identify tick-borne Babesia spp., Theileria spp., and Anaplasma spp. infections in border regions, northwestern China. Out of 323 samples tested in this study, 225 (69.7%) sheep were infected with Babesia spp., Theileria spp., and Anaplasma spp. Two hundred six (63.8%), 60 (18.6%), 54 (16.7%), 51 (15.8%), 32 (9.9%), 19 (5.9%), and 16 (5.0%) were positive for A. ovis, B. motasi-like, A. bovis, T. uilenbergi, A. phagocytophilum, T. luwenshuni, and B. motasi-like Xinjiang, respectively. The most common dual infection was with A. ovis and B. motasi-like while the most frequent triple coinfection was A. ovis, B. motasi-like, and T. uilenbergi with coinfection rates of 17.0% (55/323) and 5.0% (16/323), respectively. Sequencing analysis indicated that A. ovis MSP4, A. phagocytophilum epank1, A. bovis 16S rRNA, B. motasi-like rap1-b, B. motasi-like Xinjiang rap1-a, T. luwenshuni 18S rRNA, and T. uilenbergi 18S rRNA from border regions, northwestern China, showed 99-100% identity with documented isolates from other countries. To the best of the authors' knowledge, this is the first report of T. uilenbergi and T. luwenshuni infections of sheep in border regions, northwestern China. Furthermore, these findings provide important data for understanding the distribution of Babesia, Theileria, and Anaplasma in sheep between border countries and China.

In the present study, we conducted an epidemiological survey of Theileria equi, with sequencing analysis of the PCR product using blood–DNA samples collected from donkeys (n = 149) reared in different Egyptian provinces in Lower Egypt (Menoufia and Mersa Matruh) and middle Egypt (Giza). All animals were tested for the presence of T. equi parasite using species-specific PCR assay targeting the Equi merozoite antigen-1 (EMA-1). Nine- (6.04%) samples were positive for T. equi. The highest positive rate for infection was detected in Giza zoological garden (10.16%). Egyptian EMA-1 gene sequence exhibited a high identity with gene sequence from Italy, Japan, South Africa, Indian and Israel, the Palestinian Authority. In conclusion, data presented here revealed for the presence of T. equi in donkeys in two provinces of Egypt either in form of acute infection or carriers. These findings have economic significance and indicate the importance of introducing effective prevention and control strategies throughout Egypt to minimize the prevalence of equine piroplasmosis caused by T. equi.

Tick-borne diseases (TBDs) are serious constraints to livestock production in Tanzania and other tropical and subtropical countries and impact the livelihoods of resource-poor farming communities in the region. In Tanzania, detailed studies on tick-borne pathogens (TBPs) in cattle using sensitive molecular detection methods are scarce. The objective of this study was to investigate the occurrence and species composition of bovine TBPs in cattle kept in Zanzibar Island. A total of 236 blood samples were randomly collected in cattle population in June and July 2019. We used polymerase chain reaction (PCR) and gene sequencing to detect and identify pathogens. PCR screening of all 236 samples revealed that 64.5% of animals were infected by TBPs, including Theileria parva (34.3%), T. mutans (38.1%), T. taurotragi (30.9%), Anaplasma marginale (10.2%), Babesia bigemina (5.1%), T. velifera (3.4%) and B. bovis (2.1%). Overall a total of 86 animals (36.4%) were co-infected with up to five pathogens including T. parva, T. mutans, T. taurotragi, A. marginale and B. bigemina. The pathogens mostly involved in the co-infection were T. parva, T. taurotragi and T. mutans. Sequence analysis indicated that T. parva p104 and B. bigemina RAP1a genes are diverse among the sampled animals in Zanzibar Island, with 99.64%–100% and 99.51%–100% nucleotide sequence identity value respectively. In contrast, the A. marginale MSP-5, T. mutans 18S rRNA V4 region and B. bovis SBP-2 genes are conserved, with 100%, 99.05%-100% and 99.66%-100% nucleotide sequence identity values respectively. The phylogenetic analyses revealed that T. parva p104 and B. bigemina RAP1a gene sequences showed significant differences of genotypes, as they appear in different clades. Meanwhile, A. marginale MSP-5, T. mutans 18S rRNA V4 region and B. bovis SBP-2 gene sequences appear in the same clade with other sequences extracted from the NCBI GenBank. The epidemiological findings revealed in this study will provide important information on tick-borne diseases in Tanzania and will be used as scientific basis for planning future control strategies.

Here, we have evaluated the inhibitory effects of Medicines for Malaria Venture (MMV) Malaria Box compounds that exhibited potent in vitro anti-bovine Babesia efficacy against the growth of B. microti in mice and conducted follow-up investigations of the structural similarity between the identified potent MMV compounds and the commonly used antibabesial drugs was performed using atom Pair fingerprints (APfp). Screening the Malaria Box against the in vivo growth of the B. microti parasite helped with the discovery of new, effective anti-bovine Babesia drugs, including MMV667488, MMV007285, and MMV019881. Of note, MMV019881 exhibited the highest anti-B. microti efficacy in vivo among the screened MMV compounds. The APfp results revealed that the maximum structural similarity (MSS) was observed between MMV007285, diminazene aceturate, and imidocarb dipropionate (ID). In the same way, clofazimine (CF) and MMV667488 showed the MSS with either each other based on the analysis. The distance matrix and molecular weight correlation findings highlight the possible potential antibabesial efficacy of MMV667488, ID, and CF when administrated as a combination therapy. In conclusion, in the current study new potent antibabesial drug, MMV019881 was identified. CF and MMV667488 showed the MSS with either each other based on the hierarchical clustering analysis (HCA) and such relation is confirmed by the distance matrix and molecular weight correlation findings. Such combination therapy might have a potential as a novel regime for treating animal or human babesiosis.

The effect of Zingiber officinale rhizome methanolic extract (ZOR) on the in vitro growth of bovine Babesia (B. bovis, B. bigemina, and B. divergens) and equine piroplasm (B. caballi, and Theileria equi) parasites and on the growth of B. microti in mice was evaluated in this study. The possible in vitro synergistic interaction between ZOR and either diminazene aceturate (DA) or potent Medicines for Malaria Venture (MMV) hits from the malaria box was also investigated. In vitro, ZOR reduced the growth of B. bovis, B. bigemina, T. equi, and B. caballi in a dose-dependent manner. B. divergens was the most susceptible parasite to the in vitro inhibitory effect of ZOR. DA and MMV compounds enhanced the in vitro inhibitory antibabesial activity of ZOR. 12.5 mg/kg DA when administrated in combination with ZOR in mice exhibited a significant inhibition (P 0.05) in B. microti growth better than those observed after treatment with 25 mg/kg DA monotherapy. These findings suggest that ZOR could be a viable medicinal plant for babesiosis treatment, particularly when combined with a modest dose of either DA or powerful anti-B. bigemina MMV hits.

In Uganda, ticks and tick-borne diseases (TBDs) pose a big challenge to farmers. They reduce cattle productivity and cause severe economic damage. Several studies have documented the prevalence of tick-borne pathogens in cattle; however, their genetic characteristics and the role of wildlife-livestock interaction in the epidemiology of the TBDs are not well documented. This study assessed the prevalence and genetic diversity of various tick-borne pathogens (TBPs) as well as the risk factors associated with the occurrence of TBPs in blood samples of 208 randomly selected cattle from 16 farms located around Queen Elizabeth National Park (QENP) in Kasese District in western Uganda. Farming practices, disease challenges, and the level of wildlife-livestock interactions were assessed by a questionnaire survey amongst farm owners. Polymerase chain reaction (PCR) assays revealed that 62.9% (131/208) cattle samples were positive for one or more pathogens. Using specific PCR assays, we detected Theileria spp., Theileria parva, Anaplasma marginale, Anaplasma platys-like, and Babesia bigemina at 50.5%, 27.9%, 19.2%, 11.5% and 8.7%, respectively. We also confirmed the infection of samples by Theileria velifera and Theileria mutans after sequencing the Theileria spp. 18S rRNA gene. The risk factors associated with the occurrence of TBPs included communal grazing, herd size, age, and proximity to QENP. Phylogenetic analysis of the T. parva p104 gene showed a high identity to the previous isolates from Uganda and other East African countries and clustered closer to the buffalo (Syncerus caffer) isolates, suggesting a possible cross-species transmission. The sequences of A. marginale groEL and B. bigemina RAP-1a formed well-supported clades with high identities to the previous isolates identified from central and eastern Uganda. The isolates obtained from A. phagocytophilum 16S rRNA gene sequences showed relationship with A. platys-like, Anaplasma sp., uncultured Anaplasma species and A. phagocytophilum isolates from Africa, Asia, Europe, and the USA. The findings of the present study showed that TBDs are still a burden to farmers and that management practices in this area may increase the transmission of pathogens between livestock and wildlife.

application/pdf, N-acetyl-L-cysteine is known to have antibacterial, antiviral, antimalarial, and antioxidant activities. Therefore, the in vitro inhibitory effect of this hit was evaluated in the present study on the growth of Babesia and Theileria parasites. The in vitro growth of Babesia bovis, Babesia bigemina, Babesia divergens, Theileria equi, and Babesia caballi that were tested was significantly inhibited (P < 0.05) by micromolar concentrations of N-acetyl-L-cysteine. The inhibitory effect of N-acetyl-L-cysteine was synergistically potentiated when used in combination with diminazene aceturate on B. bovis and B. caballi cultures. These results indicate that N-acetyl-L-cysteine might be used as a drug for the treatment of babesiosis, especially when used in combination with diminazene aceturate. (C) 2017 Elsevier Inc. All rights reserved.

There is an innovative approach to discovering and developing novel potent and safe anti- Babesia and anti- Theileria agents for the control of animal piroplasmosis. Large-scale screening of 400 compounds from a Malaria Box (a treasure trove of 400 diverse compounds with antimalarial activity has been established by Medicines for Malaria Venture) against the in vitro growth of bovine Babesia and equine Babesia and Theileria parasites was performed, and the data were published in a brief with complete dataset from 236 screens of the Malaria Box compounds. Therefore, in this review, we explored and discussed in detail the in vitro inhibitory effects of 400 antimalarial compounds (200 drug-like and 200 probe-like) from the Malaria Box against Babesia ( B. ) bovis , B. bigemina , B. caballi , and Theileria ( T. ) equi . Seventeen hits were the most interesting with regard to bovine Babesia parasites, with mean selectivity indices (SIs) greater than 300 and half maximal inhibitory concentration (IC 50 s) ranging from 50 to 410 nM. The most interesting compounds with regard to equine Babesia and Theileria parasites were MMV020490 and MMV020275, with mean SIs > 258.68 and > 251.55, respectively, and IC 50 s ranging from 76 to 480 nM. Ten novel anti– B. bovis , anti– B. bigemina , anti– T. equi , and anti– B. caballi hits, MMV666093, MMV396794, MMV006706, MMV665941, MMV085203, MMV396693, MMV006787, MMV073843, MMV007092, and MMV665875, with nanomole levels of IC 50 were identified. The most interesting hits were MMV396693, MMV073843, MMV666093, and MMV665875, with mean SIs greater than 307.8 and IC 50 s ranging from 43 to 630 nM for both bovine Babesia and equine Babesia and Theileria parasites. Screening the Malaria Box against the in vitro growth of Babesia and Theileria parasites helped with the discovery of new drugs than those traditionally used, diminazene aceturate and imidocarb dipropionate, and indicated the potential of the Malaria Box in finding new, potent antibabesial drugs.

Background Developing new antibabesial drugs with a low toxic effect to the animal and with no resistance from Babesia parasites is in urgent demand. In this concern, the antimalarial, anticancer and antioxidant effect of thymoquinone (TQ), a phytochemical compound found in the plant Nigella sativa, has been reported. Therefore, in the present study, the antibabesial effect of this compound was evaluated on the growth of piroplasm parasites. Results Significant inhibition (P < 0.05) of the in vitro growth of piroplasm parasites were observed after treatment by TQ with IC50 values of 35.41 ± 3.60, 7.35 ± 0.17, 0.28 ± 0.016, 74.05 ± 4.55 and 67.33 ± 0.94 μM for Babesia bovis, Babesia bigemina, Babesia divergens, Theileria equi and Babesia caballi, respectively. The in vitro inhibitory effect of TQ was significantly enhanced (P < 0.05) when used in combination with either diminazene aceturate on bovine Babesia and equine Babesia and Theileria cultures. In B. microti-infected mice, oral and intraperitoneal administrations of TQ showed significant (P < 0.05) inhibition of parasite growth at a dose of 70 mg/kg and 50 mg/kg, respectively, compared to the control group. Conclusions The obtained results indicate that thymoquinone might be a promising medicinal compound for use in the treatment of animal piroplasmosis. Electronic supplementary material The online version of this article (10.1186/s13071-019-3296-z) contains supplementary material, which is available to authorized users.

Enoxacin is a broad-spectrum 6-fluoronaphthyridinone antibacterial agent (fluoroquinolones) structurally related to nalidixic acid used mainly in the treatment of urinary tract infections and gonorrhea. Also it has been shown recently that it may have cancer inhibiting effect. The primary antibabesial effect of Enoxacin is due to inhibition of DNA gyrase subunit A, and DNA topoisomerase. In the present study, enoxacin was tested as a potent inhibitor against the in vitro growth of bovine and equine Piroplasms. The in vitro growth of five Babesia species that were tested was significantly inhibited (P

Objective To use two diagnostic antigens belonging to the frequently associated in Theileria domain, Theileria equi ( T. equi ) protein 82 (Te 82) and T. equi 104 kDa microneme-rhoptry antigen precursor (Te 43), to diagnose T. equi infection in horses as compared with equi merozoite antigen-2 (EMA-2). Methods In the current study, we applied a cocktail-ELISA containing two antigens (EMA-2 + Te 82) to diagnose T. equi infection either in experimentally infected horses or in field infection. Results Our findings have revealed that a cocktail formula of EMA-2 + Te 82 provided a more practical and sensitive diagnostic candidate for diagnosing T. equi infection in horses as compared with Te 82 or Te 43 alone. Conclusions The ELISA technique using a cocktail formula of EMA-2 + Te 82 offers a practical and sensitive diagnostic tool for diagnosing T. equi infection in horses and using of this promising cocktail formula will be applicable for epidemiological surveys and will help control the infection in horses.

Ticks carry and transmit a wide range of pathogens (bacteria, viruses, and protozoa) that are of importance to humans and animals globally. However, information about the tick-borne pathogens harbored by ticks in the Xinjiang Uygur Autonomous Region (XUAR), northwestern China, is scarce. This study investigated the occurrence of tick species of domestic animals and tick-borne pathogens by using morphological molecular identification and sequence analysis in Turpan, Qitai, Altay, Hejing, Nileke, and Zhaosu counties (XUAR). A total of 5822 adult ticks (females and males) from 12 tick species were identified from 5 animal species (cattle, goats, sheep, camels, and horses) in 6 counties in the XUAR. Collected tick species included Dermacentor marginatus (24.7 %), Dermacentor nuttalli (20.8 %), Hyalomma anatolicum (13.7 %), Dermacentor niveus (13.1 %), Haemaphysalis punctata (10.7 %), Dermacentor silvarum (7.1 %), Dermacentor pavlovskyi (3.9 %), Hyalomma asiaticum (2.2 %), Rhipicephalus pumilio (1.9 %), Rhipicephalus sanguineus sensu lato (0.7 %), Rhipicephalus turanicus (0.6 %), and Hyalomma asiaticum kozlovi (0.6 %). Furthermore, 750 partially engorged adult ticks (females and males), including H. anatolicum (250), D. nuttalli (250), and D. marginatus (250), were individually separated according to species and sampling site, used for DNA extraction, and then screened for tick-borne pathogens. The most common pathogen was Rickettsia raoultii (36.80 %), followed by Brucella sp. (26.2 %), Anaplasma ovis (22.4 %), Babesia caballi (14.8 %), Theileria equi (8.7 %), and Theileria ovis (8.5 %). The sequencing of 6 genes showed a 96-100 % nucleotide identity between the sequences in this study and those deposited in GenBank. This study provides a scientific reference for the prevention and control of tick-borne diseases in the XUAR.

The treatment of bovine and equine piroplasmosis is limited to diminazene aceturate (DA) and imidocarb dipropionate. To address this challenge, we need to explore novel drug compounds and targets. Topoisomerases are potential drug targets because they play a vital role in solving topological errors of DNA strands during replication. This study documented the effectiveness of topoisomerase inhibitors, nitidine chloride (NC) and camptothecin (Cpt), on the growth of Babesia and Theileria parasites. The half maximal inhibitory concentrations (IC50s) against B. bovis, B. bigemina, B. caballi, and T. equi were 1.01 ± 0.2, 5.34 ± 1.0, 0.11 ± 0.03, and 2.05 ± 0.4 μM for NC and 11.67 ± 1.6, 4.00 ± 1.0, 2.07 ± 0.6, and 0.33 ± 0.02 μM for Cpt, respectively. The viability experiment revealed that 4, 10, and 4 μM treatments of NC or 48, 8, and 8 μM treatments of Cpt were sufficient to stop the in vitro regrowth of B. bovis, B. bigemina, and B. caballi, respectively. However, T. equi regrew in all of the concentrations used. Moreover, increasing the concentration of NC and Cpt to 16 μM and 1.2 μM (8 × IC50) did not eliminate T. equi. The micrographs of B. bigemina and B. caballi taken at 24 h and 72 h showed deformed merozoites and remnants of parasites within the red blood cell (RBC), respectively. The treatments of 25 mg/kg DA and 20 mg/kg NC administered intraperitoneally and 20 mg/kg NC given orally showed 93.7, 90.7, and 83.6% inhibition against Babesia microti (B. microti), respectively, compared to the untreated group on day 8. In summary, NC and Cpt were effective against Babesia and Theileria parasites in vitro. Moreover, 20 mg/kg NC administered intraperitoneally was as effective as 25 mg/kg DA against B. microti in mice and showed no toxic symptoms in mice. The results indicate that NC may, after further evaluations, prove to be an alternative drug against bovine and equine piroplasmoses.

Heat shock protein 90 (Hsp90) is a chaperone protein that stabilizes cells during stress or non-stress responses. Previous reports have shown that Hsp90 is a potential drug target to suppress the multiplication of several protozoan parasites. In this study, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), an Hsp90 inhibitor, was evaluated for its inhibitory effect on five in vitro cultures of Babesia and Theileria species, including B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi, and on the multiplication of a B. microti–infected mouse model. 17-DMAG showed the inhibitory effect in all of the species tested. The half maximum inhibition concentration (IC50) of 17-DMAG on B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi was 77.6 ± 2.9, 62.4 ± 1.9, 183.8 ± 3.2, 88.5 ± 9.6, and 307.7 ± 7.2 nM, respectively. The toxicity assay on MDBK and NIH/3T3 cell lines showed that 17-DMAG affected the viability of cells with an IC50 of 15.5 ± 4 and 8.8 ± 2 μM, respectively. Since the IC50s were much lower on the parasites than on the host cell lines, the selectivity index were high for all tested species. Furthermore, the two-drug combination of 17-DMAG with diminazene aceturate (DA) and atovaquone (AV) showed synergism or addition on in vitro cultures of Babesia and Theileria parasites. In the mouse model, 17-DMAG at a concentration of 30 mg/kg BW effectively inhibited the multiplication of B. microti. Moreover, if combined with DA or AV, 17-DMAG showed a comparable inhibition at the half dose. Taken together, these results indicate that 17-DMAG is a potent drug for treating piroplamosis. The data warrant further evaluation of 17-DMAG as an antibabesial drug and as an option in combination with atovaquone for the treatment of human babesiosis., Graphical abstract Image 1, Highlights • 17-DMAG inhibits the in vitro multiplication of Babesia and Theileria parasites. • Combination of 17-DMAG with diminazene aceturate or atovaquone were also effective. • 17-DMAG also inhibits the multiplication of B. microti in mice. • 17-DMAG is a new treatment option for babesiosis in animal and human.

Cattle, buffaloes, and sheep are the main sources of meat and milk in Egypt, but their productivity is thought to be greatly reduced by hemoprotozoan parasitic diseases. In this study, we analyzed the infection rates of Babesia bovis, Babesia bigemina, Theileria annulata, and Theileria orientalis, using parasite-specific PCR assays in blood-DNA samples sourced from cattle (n=439), buffaloes (n=50), and sheep (n=105) reared in Menoufia, Behera, Giza, and Sohag provinces of Egypt. In cattle, the positive rates of B. bovis, B. bigemina, T. annulata, and T. orientalis were 3.18%, 7.97%, 9.56%, and 0.68%, respectively. On the other hand, B. bovis and T. orientalis were the only parasites detected in buffaloes and each of these parasites was only found in two individual DNA samples (both 2%), while one (0.95%) and two (1.90%) of the sheep samples were positive for B. bovis and B. bigemina, respectively. Sequence analysis showed that the B. bovis Rhoptry Associated Protein-1 and the B. bigemina Apical Membrane Antigen-1 genes were highly conserved among the samples, with 99.3-100% and 95.3-100% sequence identity values, respectively. In contrast, the Egyptian T. annulata merozoite surface antigen-1 gene sequences were relatively diverse (87.8-100% identity values), dispersing themselves across several clades in the phylogenetic tree containing sequences from other countries. Additionally, the T. orientalis Major Piroplasm Surface Protein (MPSP) gene sequences were classified as types 1 and 2. This is the first report of T. orientalis in Egypt, and of type 2 MPSP in buffaloes. Detection of MPSP type 2, which is considered a relatively virulent genotype, suggests that T. orientalis infection may have veterinary and economic significance in Egypt. In conclusion, the present study, which analyzed multiple species of Babesia and Theileria parasites in different livestock animals, may shed an additional light on the epidemiology of hemoprotozoan parasites in Egypt.

In present study, blood samples were collected randomly from 439 cows at three main regions of Egypt (northern, central and southern). Molecular diagnosis of Babesia and Theileria infections by PCR amplification of DNA (gene) fragments, then cloning and sequencing of the positive samples were conducted. A questionnaire was created to imply the assumed risk factors and logistic regression statistical analysis was carried out to appraise the potential factors on the animal level. The results revealed that 49 (11.16%) and 45 (10.25%) cattle were infected with Babesia and Theileria parasites, respectively. B. bigemina (7.97%) and T. annulata (9.56%) were the most prevalent parasites. For Babesia sp., final multivariate logistic regression analysis showed a significant association between the infection and irregular use of antiprotozoal drugs (P = 0.003; OR: 0.28; 95% CI: 0.12-0.65), management practice (P = 0.029; OR: 6.66; 95% CI: 1.21-36.59) and ecology area (P = 0.006; OR: 5.62; 95% CI: 1.63-19.31). However, for Theileria sp. infection, animal breed (P = 0.003; OR: 0.44; 95% CI: .45-1.00) and irregular use of antiprotozoal drugs (P

A rapid and accurate assay for evaluating antibabesial drugs on a large scale is required for the discovery of novel chemotherapeutic agents against Babesia parasites. In the current study, we evaluated the usefulness of a fluorescence-based assay for determining the efficacies of antibabesial compounds against bovine and equine hemoparasites in in vitro cultures. Three different hematocrits (HCTs; 2.5%, 5%, and 10%) were used without daily replacement of the medium. The results of a high-throughput screening assay revealed that the best HCT was 2.5% for bovine Babesia parasites and 5% for equine Babesia and Theileria parasites. The IC50 values of diminazene aceturate obtained by fluorescence and microscopy did not differ significantly. Likewise, the IC50 values of luteolin, pyronaridine tetraphosphate, nimbolide, gedunin, and enoxacin did not differ between the two methods. In conclusion, our fluorescence-based assay uses low HCT and does not require daily replacement of culture medium, making it highly suitable for in vitro large-scale drug screening against Babesia and Theileria parasites that infect cattle and horses.