A Theileria annulata parasite with a single mutation, methionine 128 to isoleucine (M128I), in cytochrome B is resistant to buparvaquone.

Tropical theileriosis is a fatal leukemic-like disease of cattle caused by the tick-transmitted protozoan parasite Theileria annulata. The economics of cattle meat and milk production is severely affected by theileriosis in endemic areas. The hydroxynaphtoquinone buparvaquone (BPQ) is the only available drug currently used to treat clinical theileriosis, whilst BPQ resistance is emerging and spreading in endemic areas. Here, we chronically exposed T. annulata-transformed macrophages in vitro to BPQ and monitored the emergence of drug-resistant parasites. Surviving parasites revealed a significant increase in BPQ IC50 compared to the wild type parasites. Drug resistant parasites from two independent cloned lines had an identical single mutation, M128I, in the gene coding for T. annulata cytochrome B (Tacytb). This in vitro generated mutation has not been reported in resistant field isolates previously, but is reminiscent of the methionine to isoleucine mutation in atovaquone-resistant Plasmodium and Babesia. The M128I mutation did not appear to exert any deleterious effect on parasite fitness (proliferation and differentiation to merozoites). To gain insight into whether drug-resistance could have resulted from altered drug binding to TaCytB we generated in silico a 3D-model of wild type TaCytB and docked BPQ to the predicted 3D-structure. Potential binding sites cluster in four areas of the protein structure including the Q01 site. The bound drug in the Q01 site is expected to pack against an alpha helix, which included M128, suggesting that the change in amino acid in this position may alter drug-binding. The in vitro generated BPQ resistant T. annulata is a useful tool to determine the contribution of the various predicted docking sites to BPQ resistance and will also allow testing novel drugs against theileriosis for their potential to overcome BPQ resistance.

Epidrugs: alternative chemotherapy targeting Theileria annulata schizont stage parasites.

The growing emergence of resistance to current anti-theilerial agents necessitates the exploration of alternative approaches to drug discovery. This study evaluated the antiparasitic efficacy of 148 compounds derived from an epigenetic inhibitor library against the schizont stage of a Theileria annulata-infected cell line. Initial screening at a concentration of 10 µM identified 27 compounds exhibiting promising anti-theilerial activity. Further investigation, including determination of the 50% inhibitory concentration (IC50) and host cell cytotoxicity assay, highlighted seven highly effective compounds (SAHA, BVT-948, Trichostatin A, Methylstat, Plumbagin, Ryuvidine, and TCE-5003) against T. annulata-infected cells. Analysis of the active compounds revealed their inhibitory action against various human targets, such as HDAC (SAHA and Trichostatin A), SET domain (Ryuvidine), PRMT (BVT-948 and TCE-5003), histone demethylase (Methylstat), and ROS/apoptosis inducer (Plumbagin). We identified gene orthologs of these targets in Theileria and conducted molecular docking studies, demonstrating effective binding of the compounds with their respective targets in the parasite, supported by in vitro data. Additionally, we performed in silico ADME/T predictions, which indicated potential mutagenic and hepatotoxic effects of Plumbagin, Methylstat, and TCE-5003, rendering them unsuitable for drug development. Conversely, SAHA, Trichostatin A, and BVT-948 showed promising characteristics and may represent potential candidates for future development as chemotherapeutic agents against tropical theileriosis. These findings provide valuable insights into the search for novel anti-theilerial drugs and offer a basis for further research in this area.IMPORTANCETheileria annulata is a protozoan parasite responsible for tropical theileriosis, a devastating disease affecting cattle. Traditional chemotherapy has limitations, and the study explores the potential of epidrugs as an alternative treatment approach. Epidrugs are compounds that modify gene expression without altering the underlying DNA sequence, offering a novel way to combat parasitic infections. This research is pivotal as it addresses the urgent need for innovative therapies against T. annulata, contributing to the development of more effective and targeted treatments for infected livestock. Successful implementation of epidrugs could not only enhance the well-being of cattle but also have broader implications for the control of parasitic diseases, showcasing the paper's significance in advancing veterinary science and improving livestock health globally.

Anti-parasitic benzoxaboroles are ineffective against Theileria parva in vitro.

East Coast Fever (ECF) is a disease affecting cattle in sub-Saharan Africa, caused by the tick-borne Apicomplexan pathogen Theileria parva. The disease is a major problem for cattle farmers in affected regions and there are few methods of control, including a complex infection and treatment vaccine, expensive chemotherapy, and the more widespread tick control through acaricides. New intervention strategies are, therefore, sorely needed. Benzoxaboroles are a versatile class of boron-heterocyclic compounds with demonstrable pharmacological activity against a diverse group of pathogens, including those related to T. parva. In this study, the in vitro efficacy of three benzoxaboroles against the intracellular schizont stage of T. parva was investigated using a flow cytometry approach. Of the benzoxaboroles tested, only one showed any potency, albeit only at high concentrations, even though there is high protein sequence similarity in the CPSF3 protein target compared to other protozoan pathogen species. This finding suggests that benzoxaboroles currently of interest for the treatment of African animal trypanosomiasis, toxoplasmosis, cryptosporidiosis and malaria may not be suitable for the treatment of ECF. We conclude that testing of further benzoxaborole compounds is needed to fully determine whether any lead compounds can be identified to target T. parva.

Selection of genotypes harbouring mutations in the cytochrome b gene of Theileria annulata is associated with resistance to buparvaquone.

Buparvaquone remains the only effective therapeutic agent for the treatment of tropical theileriosis caused by Theileria annulata. However, an increase in the rate of buparvaquone treatment failures has been observed in recent years, raising the possibility that resistance to this drug is associated with the selection of T. annulata genotypes bearing mutation(s) in the cytochrome b gene (Cyto b). The aim of the present study was: (1) to demonstrate whether there is an association between mutations in the T. annulata Cyto b gene and selection of parasite-infected cells resistant to buparvaquone and (2) to determine the frequency of these mutations in parasites derived from infected cattle in the Aydin region of Türkiye. Susceptibility to buparvaquone was assessed by comparing the proliferative index of schizont-infected cells obtained from cattle with theileriosis before and/or after treatment with various doses of buparvaquone, using the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colourimetric assay. The DNA sequence of the parasite Cyto b gene from cell lines identified as resistant or susceptible was determined. A total of six nonsynonymous and six synonymous mutations were identified. Two of the nonsynonymous mutations resulted in the substitutions V135A and P253S which are located at the putative buparvaquone binding regions of cytochrome b. Allele-specific PCR (AS-PCR) analyses detected the V135A and P253S mutations at a frequency of 3.90% and 3.57% respectively in a regional study population and revealed an increase in the frequency of both mutations over the years. The A53P mutation of TaPIN1 of T. annulata, previously suggested as being involved in buparvaquone resistance, was not detected in any of the clonal cell lines examined in the present study. The observed data strongly suggested that the genetic mutations resulting in V135A and P253S detected at the putative binding sites of buparvaquone in cytochrome b play a significant role in conferring, and promoting selection of, T. annulata genotypes resistant to buparvaquone, whereas the role of mutations in TaPIN1 is more equivocal.

In vivo activity and atom pair fingerprint analysis of MMV665941 against the apicomplexan parasite Babesia microti, the causative agent of babesiosis in humans and rodents.

The effect of MMV665941 on the growth of Babesia microti (B. microti) in mice, was investigated in this study using a fluorescence-based SYBR Green I test. Using atom Pair signatures, we investigated the structural similarity between MMV665941 and the commonly used antibabesial medicines diminazene aceturate (DA), imidocarb dipropionate (ID), or atovaquone (AV). In vitro cultures of Babesia bovis (B. bovis) and, Theileria equi (T. equi) were utilized to determine the MMV665941 and AV interaction using combination ratios ranged from 0.75 IC50 MMV665941:0.75 IC50 AV to 0.50 IC50 MMV665941:0.50 IC50 AV. The used combinations were prepared depending on the IC50 of each drug against the in vitro growth of the tested parasite. Every 96 h, the hemolytic anemia in the treated mice was monitored using a Celltac MEK-6450 computerized hematology analyzer. A single dose of 5 mg/kg MMV665941 exhibited inhibition in the B. microti growth from day 4 post-inoculation (p.i.) till day 12 p.i. MMV665941 caused 62.10%, 49.88%, and 74.23% inhibitions in parasite growth at days 4, 6 and 8 p.i., respectively. Of note, 5 mg/kg MMV665941 resulted in quick recovery of hemolytic anemia caused by babesiosis. The atom pair fingerprint (APfp) analysis revealed that MMV665941 and atovaquone (AV) showed maximum structural similarity. Of note, high concentrations (0.75 IC50) of MMV665941 and AV caused synergistic inhibition on B. bovis growth. These findings suggest that MMV665941 might be a promising drug for babesiosis treatment, particularly when combined with the commonly used antibabesial drug, AV.

Metabolomic profiling of bovine leucocytes transformed by Theileria annulata under BW720c treatment.

BACKGROUND: When Theileria annulata infects host cells, it undertakes unlimited proliferation as tumor cells. Although the transformed cells will recover their limited reproductive characteristics and enter the apoptosis process after treatment with buparvaquone (BW720c), the metabolites and metabolic pathways involved are not clear. METHODS: The transformed cells of T. annulata were used as experimental materials, and the buparvaquone treatment group and DMSO control group were used. Qualitative and quantitative analysis was undertaken of 36 cell samples based on the LC-QTOF platform in positive and negative ion modes. The metabolites of the cell samples after 72 h of drug treatment were analyzed, as were the different metabolites and metabolic pathways involved in the BW720c treatment. Finally, the differential metabolites and metabolic pathways in the transformed cells were found. RESULTS: A total of 1425 metabolites were detected in the negative ion mode and 1298 metabolites were detected in the positive ion mode. After drug treatment for 24 h, 48 h, and 72 h, there were 56, 162, and 243 differential metabolites in negative ion mode, and 35, 121, and 177 differential metabolites in positive ion mode, respectively. These differential metabolites are mainly concentrated on various essential amino acids. CONCLUSION: BW720c treatment induces metabolic disturbances in T. annulata-infected cells by regulating the metabolism of leucine, arginine, and L-carnitine, and induces host cell apoptosis.

Genetic characterisation of the Theileria annulata cytochrome b locus and its impact on buparvaquone resistance in bovine.

Control of tropical theileriosis, caused by the apicomplexan Theileria annulata, depends on the use of a single drug, buparvaquone, the efficacy of which is compromised by the emergence of resistance. The present study was undertaken to improve understanding of the role of mutations conferring buparvaquone resistance in T. annulata, and the effects of selection pressures on their emergence and spread. First, we investigated genetic characteristics of the cytochrome b locus associated with buparvaquone resistance in 10 susceptible and 7 resistant T. annulata isolates. The 129G (GGC) mutation was found in the Q01 binding pocket and 253S (TCT) and 262S (TCA) mutations were identified within the Q02 binding pocket. Next, we examined field isolates and identified cytochrome b mutations 129G (GGC), 253S (TCT) and 262S (TCA) in 21/75 buffalo-derived and 19/119 cattle-derived T. annulata isolates, providing evidence of positive selection pressure. Both hard and soft selective sweeps were identified, with striking differences between isolates. For example, 19 buffalo-derived and 7 cattle-derived isolates contained 129G (GGC) and 253S (TCT) resistance haplotypes at a high frequency, implying the emergence of resistance by a single mutation. Two buffalo-derived and 12 cattle-derived isolates contained equally high frequencies of 129G (GGC), 253S (TCT), 129G (GGC)/253S (TCT) and 262S (TCA) resistance haplotypes, implying the emergence of resistance by pre-existing or recurrent mutations. Phylogenetic analysis further revealed that 9 and 21 unique haplotypes in buffalo and cattle-derived isolates were present in a single lineage, suggesting a single origin. We propose that animal migration between farms is an important factor in the spread of buparvaquone resistance in endemic regions of Pakistan. The overall outcomes will be useful in understanding how drug resistance emerges and spreads, and this information will help design strategies to optimise the use and lifespan of the single most drug use to control tropical theileriosis.

Detection and genetic characterization indicates circulation of a possible new Theileria species (Theileria sp. Yokoyama) in India.

Bovine tropical theileriosis, a tick-borne disease, causes huge economic loss to the Indian dairy industry. Theileriosis in India is mainly caused by Theileria annulata, although the presence of T. orientalis has also been reported. The present study was undertaken to investigate the deaths of cross-bred Holstein Friesen (CBHF) cows on a farm in the state of Telangana, India. Deceased animals had recently calved and prior to death had developed high fever (107 °F) and anaemia. Infected cows were infested with ticks (Hyalomma species). Theileria piroplasms were noticed in the Giemsa stained blood smears. PCR assays further confirmed the presence of Theileria in the blood samples of the infected cows. Partial Tams1 gene sequences from the infected animals shared 99.87% to 100% identity scores with the sequences of Sri Lankan isolates recently proposed as a novel Theileria species (provisionally designated as Theileria sp. Yokoyama). To the best of our knowledge, this is the first report of the novel species of Theileria from India. Infected animals were effectively treated with buparvaquone and oxytetracycline. The introduction of new animals into the farm without risk assessment was found to be a major cause of the outbreak.

Ascorbic acid co-administration with a low dose of diminazene aceturate inhibits the in vitro growth of Theileria equi, and the in vivo growth of Babesia microti.

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-1). The results imply that ascorbic acid /DA could be a viable combination therapy for the treatment of T. equi and that it could be utilized to overcome the resistance of Babesia parasites to full doses of the regularly used antibabesial medication, DA.

In vitro growth inhibitory effect of selected 18ß-glycyrrhetinic acid esters on Theileriaannulata.

Glycyrrhetinic acid (GA) is one of the important Pentacyclic Triterpenoids (PT) found in the roots of licorice. This study aimed to evaluate the in vitro growth inhibitory effect of 18ß-GA (18ß-Glycyrrhetinic acid) and C-30 esters against Theileria annulata, the causative agent of Tropical Bovine Theileriosis. C-30 esters of 18ß-GA were synthesized and their structures were elucidated using spectroscopy. The pharmacodynamic properties of 18ß-GA and its C-30 esters were predicted using DataWarrior and Swiss ADME tools. Cattle isolates of T. annulata schizont-infected bovine lymphoblastoid cells were cultured using standard conditions and the growth inhibitory effect of GA and its esters were evaluated using MTT assay. The isopropyl ester of 18ß-GA (GI50- 1.638 µM; R2- 0.818) showed improved anti-theileriosis efficacy than other 18ß-GA derivatives. The propyl (GI50 - 5.549 µM), ethyl (GI50 - 5.638 µM), and benzyl (GI50 - 7.431 µM) esters also showed considerable inhibitory effect. The GI50 value for 18ß-GA was recorded as 6.829 µM. This study throws light on the usefulness of 18ß-GA and its esters for the treatment of Tropical Bovine Theileriosis.

MMV560185 from pathogen box induces extrinsic pathway of apoptosis in Theileria annulata infected bovine leucocytes.

Tropical theileriosis is a lymphoproliferative disease caused by the intracellular schizonts of Theileria annulata, an apicomplexan parasite. It causes severe infection in cattle and the untreated cattle would possibly die within 3-4 weeks of infection. The chemotherapy for this disease is largely dependent on the use of hydroxynaphthoquinone, namely buparvaquone. There have been reports recently of the development of resistance against this drug in T. annulata. Hence, identification of new drug molecule(s) or repurposing of existing drug molecule(s) against T. annulata is quite important. Here, we present the screening of 400 compounds included in the open-access Pathogen box from Medicine for Malaria Venture (MMV) to discover the novel compounds with potential inhibitory activity against T. annulata infected bovine leucocytes. We identified two compounds, MMV000062 and MMV560185, with IC50 values of 2.97 µM and 3.07 µM, respectively. MMV000062 and MMV560185 were found non-toxic to BoMac cells with CC50 values 34 µM and > 100 µM, respectively. The therapeutic indices of these compounds, MMV000062 and MMV560185, were calculated as more than 33 and 11, respectively. Further, it was observed that the parasite-infected cells under long-term culture were unable to recover with these compounds. We further deciphered that MMV560185 kills the infected cell by activation of TNFR-1 mediated extrinsic pathway of the apoptosis. The phenotypic characteristics of apoptosis were confirmed by Transmission Electron Microscopy. Our results suggest that it may be possible to develop MMV560185 further for chemotherapeutics of tropical theilerosis.

Successful treatment of severe form of bovine tropical theileriosis in dairy cattle and genotyping of Theileria annulata isolates of Tamil Nadu, India.

Bovine tropical theileriosis (BTT) is a tick-borne protozoan disease of cattle and responsible for major economic losses to the dairy farmers in India. This report describes diagnosis, genotyping and successful treatment of heavy infection of Theileria annulata in an organized dairy farm at Kattupakkam, Chennai. Four cross bred cows of 2 to 5 years of age showed clinical signs i.e., anorexia, salivation and panting. Clinical examination revealed pyrexia (40.0 °C to 40.1 °C), pale mucus membranes, enlarged prescapular lymph nodes and haemoglobinuria. The peripheral blood smear examination of infected cows revealed presence of piroplasm within the RBCs indicating high parasitemia. Haematology results suggested that decreased levels of Hb, RBC, WBC and PCV in the infected cows when compared with normal reference values. There were increased serum ALT and AST values and reduced serum total protein, albumin, calcium and phosphorous values in the infected cows. Semi-nested PCR using T. annulata specific oligonucleotide primers amplified 199 bp of the partial T. annulata 18S rRNA gene. Presence of four satellite markers TS6, TS8, TS9, and TS12 in the Theileria annulata isolates 1 and 2 indicating that the isolates were the same haplotype and suggested the infection in the farm was due to a single haplotype of T. annulata parasite. Based on the clinical signs, microscopic examination of blood smear and molecular diagnosis, the condition was diagnosed as tropical theileriosis. Infected cows were successfully treated with a single deep intramuscular injection of buparvaquone (Zubion®, INTAS pharmaceuticals LTD, Ahmedabad, India) along with supportive medication.

Dynamic methylation of histone H3K18 in differentiating Theileria parasites.

Lysine methylation on histone tails impacts genome regulation and cell fate determination in many developmental processes. Apicomplexa intracellular parasites cause major diseases and they have developed complex life cycles with fine-tuned differentiation events. Yet, apicomplexa genomes have few transcription factors and little is known about their epigenetic control systems. Tick-borne Theileria apicomplexa species have relatively small, compact genomes and a remarkable ability to transform leucocytes in their bovine hosts. Here we report enriched H3 lysine 18 monomethylation (H3K18me1) on the gene bodies of repressed genes in Theileria macroschizonts. Differentiation to merozoites (merogony) leads to decreased H3K18me1 in parasite nuclei. Pharmacological manipulation of H3K18 acetylation or methylation impacted parasite differentiation and expression of stage-specific genes. Finally, we identify a parasite SET-domain methyltransferase (TaSETup1) that can methylate H3K18 and represses gene expression. Thus, H3K18me1 emerges as an important epigenetic mark which controls gene expression and stage differentiation in Theileria parasites.

Endochin-like quinolone-300 and ELQ-316 inhibit Babesia bovis, B. bigemina, B. caballi and Theileria equi.

BACKGROUND: The most common apicomplexan parasites causing bovine babesiosis are Babesia bovis and B. bigemina, while B. caballi and Theileria equi are responsible for equine piroplasmosis. Treatment and control of these diseases are usually achieved using potentially toxic chemotherapeutics, such as imidocarb diproprionate, but drug-resistant parasites are emerging, and alternative effective and safer drugs are needed. The endochin-like quinolones (ELQ)-300 and ELQ-316 have been proven to be safe and efficacious against related apicomplexans, such as Plasmodium spp., with ELQ-316 also being effective against Babesia microti, without showing toxicity in mammals. METHODS: The inhibitory effects of ELQ-300 and ELQ-316 were assessed on the growth of cultured B. bovis, B. bigemina, B. caballi and T. equi. The percentage of parasitized erythrocytes was measured by flow cytometry, and the effect of the ELQ compounds on the viability of horse and bovine peripheral blood mononuclear cells (PBMC) was assessed by monitoring cell metabolic activity using a colorimetric assay. RESULTS: We calculated the half maximal inhibitory concentration (IC50) at 72 h, which ranged from 0.04 to 0.37 nM for ELQ-300, and from 0.002 to 0.1 nM for ELQ-316 among all cultured parasites tested at 72 h. None of the parasites tested were able to replicate in cultures in the presence of ELQ-300 and ELQ-316 at the maximal inhibitory concentration (IC100), which ranged from 1.3 to 5.7 nM for ELQ-300 and from 1.0 to 6.0 nM for ELQ-316 at 72 h. Neither ELQ-300 nor ELQ-316 altered the viability of equine and bovine PBMC at their IC100 in in vitro testing. CONCLUSIONS: The compounds ELQ-300 and ELQ-316 showed significant inhibitory activity on the main parasites responsible for bovine babesiosis and equine piroplasmosis at doses that are tolerable to host cells. These ELQ drugs may be viable candidates for developing alternative protocols for the treatment of bovine babesiosis and equine piroplasmosis.

Protein Kinase Inhibitors Arrested the In-Vitro Growth of Theileria equi.

INTRODUCTION: Theileria equi is an intra-erythrocytic apicomplexean protozoa that infect equines. Protein kinases (PK), key molecules of the apicomplexean life cycle, have been implicated as significant drug targets. The growth inhibitory efficacy of PK inhibitors against Theileria/Babesia animal parasites have not been documented so far. METHODS: The present study aimed to carry out in-vitro growth inhibitory efficacy studies of four novel drug molecules-SB239063, PD0332991 isethionate, FR180204 and apigenin, targeting different protein kinases of T. equi. A continuous microaerophilic stationary-phase culture (MASP) system was established for propagation of T. equi parasites. This in-vitro culture technique was used to assess the growth inhibitory effect of protein kinase targeted drug molecules, whereas diminazene aceturate was taken as control drug against T. equi. The inhibitory concentration (IC50) was determined for comparative analysis. The potential cytotoxicity of the drug molecule was also assessed on horse's peripheral blood mononuclear cells (PBMCs) cell line. RESULTS: SB239063 and diminazene aceturate drugs significantly inhibited (p < 0.05) the in-vitro growth of T. equi parasite at 0.1 µM, 1 µM, 10 µM, 50 µM and 100 µM concentration at ≥ 48 h of incubation period and respective IC50 values were 4.25 µM and 1.23 µM. Furthermore, SB239063 was not cytotoxic to the horse PBMCs and found safer than diminazine aceturate drug. PD0332991 isethionate and FR180204 are extracellular signal-regulated kinase (ERK) inhibitors and significantly (p < 0.05) inhibited T. equi in-vitro growth at higher concentrations (≥ 48 h of incubation period) with respective IC50 value of 10.41 µM and 21.0 µM. Lower concentrations of these two drugs were not effective (p > 0.05) even after 96 h of treatment period. Apigenin (protein kinase-C inhibitor) drug molecule was unsuccessful in inhibiting the T. equi parasite growth completely. After 96 h of in-vitro treatment period, a parasite viability study was performed on drug-treated T. equi parasitized RBCs. These drugs-treated parasitized RBCs were collected and transferred to wells containing fresh culture media (without drug) and naïve host RBCs. Drug-treated RBCs collected from SB239063, PD0332991, diminazene aceturate treatment (1 µM to 100 µM concentration) were unsuccessful in growing/multiplying further. Apigenin drug-treated T. equi parasites were live after 96 h of treatment. CONCLUSION: It may be concluded that SB239063 was the most effective drug molecule (being lowest in IC50 value) out of the four different protein kinase inhibitors tested in this study. This drug molecule has insignificant cytotoxic activity against horse's PBMCs.

A longitudinal study of the effect of Theileria orientalis Ikeda type infection on three New Zealand dairy farms naturally infected at pasture.

The aims of this study were to monitor the change in Theileria orientalis Ikeda type infection intensity, haematocrit, milk production and reproduction on three New Zealand spring calving dairy herds, over the 2014-2015 milking season. Three spring calving dairy farms, A, B and C, from high risk (endemically stable), low risk (endemically unstable), and zero risk (disease-free) tick areas respectively were followed through the 2014-2015 milking season. On Farms, A and B, 100 cows were randomly selected at the first visit, and the same cows blood sampled every month thereafter, whilst on Farm C, the whole herd was blood sampled bimonthly (140 cows). Blood samples were tested for haematocrit, by centrifugation, and Ikeda infection intensity, using qPCR. Animals that were Ikeda type PCR positive at the first sampling were described as prevalence cases and cows that were negative at the first sampling and became PCR positive during the sampling period were described as incidence cases. Production and reproduction data were accessed through LIC MINDA® and milk production data was standardised to energy corrected milk (ECM). In addition, the effect of buparvaquone (BPQ) treatment on milk production was estimated on Farm B. The prevalence of infection at the first sampling was 100 % on Farm A, 57 % on Farm B and 26 % on Farm C. The incidence risk of infection over the sampling period on Farms B and C was 25 % and 2 % and the incident rate was 0.026 and 0.002 cases per cow-month respectively. The average infection intensity for prevalence cases on all farms was low throughout the milking season, <7000 Ikeda organisms/µL however, cases of anaemia still occurred. There was no direct effect of infection intensity on milk production or from being a prevalence case compared to an uninfected cow on milk production, across all farms. However, on Farm B there was a loss of 266 kg (95 % CI 82 ̶ 450) ECM (∼20 kg milk solids) for incidence cases and a loss of 458 kg (95 % CI 211 ̶ 710) of ECM for buparvaquone treated cows, compared to uninfected cows. No significant effect of Ikeda infection on reproduction could be shown for Farms B and C, reproductive data for Farm A was not available. The effect of T. orientalis Ikeda type infection on production and reproduction appears to be minimal once animals have passed through the acute phase of infection and reached the chronic, asymptomatic carrier phase of infection.

Evaluation of the efficacy of ivermectin against Theileria orientalis infection in grazing cattle.

BACKGROUND: Raising cattle on pastures is known to be beneficial for animal welfare and cost reduction. However, grazing is associated with the risk of contracting tick-borne diseases, such as theileriosis. Here, the efficacy of ivermectin against these diseases and associated clinical symptoms were evaluated. RESULTS: A total of 68 cattle from a grazing cattle farm were selected and divided into two groups: the control group (17 cattle) with no preventive treatment and the ivermectin-treated group (51 cattle) in which cattle were treated with pour-on ivermectin prior to grazing. The infection rates of Theileria orientalis and the red blood cell (RBC) profile (e.g., RBC count, hematocrit value, and hemoglobin concentration) were compared in the spring (before grazing) and summer (during grazing) between the two groups. Based on PCR amplification of the major piroplasm surface protein (MPSP) gene, 12 cattle were positive for T. orientalis infection. Phylogenetic analysis revealed that the isolates identified in this study consisted of three MPSP types (1, 2, and 7). The T. orientalis infection rate in the control group during grazing was 3-fold higher than that in the ivermectin-treated group. Moreover, differences in RBC parameters during grazing were greater in the control group than in the ivermectin-treated group. In particular, the hematocrit value was significantly reduced in the control group. CONCLUSIONS: The results of this study demonstrated that ivermectin had protective effects against T. orientalis infection and RBC hemolysis in grazing cattle.

THEILERIOSIS IN MOUNTAIN BONGO REPATRIATED TO KENYA: A CLINICAL AND MOLECULAR INVESTIGATION.

Mountain bongo (Tragelaphus euryceros isaaci) from Kenya were exported to zoological institutions in North America and Europe in the 1970s and 1980s. In the following 20-30 years bongo numbers declined in Kenya and the Mountain Bongo Repatriation Project was launched. This resulted in 18 adult bongo, descendants of the original translocated bongo, being repatriated from the United States to Kenya in 2004. These newly arrived bongo were inadvertently exposed to heavy tick infestation on release in a conservancy on the slopes of Mount Kenya. Mortality and morbidity occurred during the third week after arrival. Theileria sp. infection was apparent from the history, clinical signs, and necropsy findings, and Theileria-like parasites were detected microscopically in samples from sick and dead animals. Four bongo died before the outbreak was controlled. In order to identify the Theileria parasite conclusively, molecular amplification techniques were used. A combination of reverse line blotting, with small subunit ribosomal RNA (SSU rRNA) polymerase chain reaction (PCR) amplification and nucleotide sequencing, identified the protozoan parasite Theileria taurotragi, suggesting this as the most probable cause of mortality and morbidity in the repatriated bongo.

Screening the Medicines for Malaria Venture Pathogen Box against piroplasm parasites.

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 < 0.3 µM and a selectivity index (SI)> 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.

Ellagic acid microspheres restrict the growth of Babesia and Theileria in vitro and Babesia microti in vivo.

BACKGROUND: There are no effective vaccines against Babesia and Theileria parasites; therefore, therapy depends heavily on antiprotozoal drugs. Treatment options for piroplasmosis are limited; thus, the need for new antiprotozoal agents is becoming increasingly urgent. Ellagic acid (EA) is a polyphenol found in various plant products and has antioxidant, antibacterial and effective antimalarial activity in vitro and in vivo without toxicity. The present study documents the efficacy of EA and EA-loaded nanoparticles (EA-NPs) on the growth of Babesia and Theileria. METHODS: In this study, the inhibitory effect of EA, ß-cyclodextrin ellagic acid (ß-CD EA) and antisolvent precipitation with a syringe pump prepared ellagic acid (APSP EA) was evaluated on four Babesia species and Theileria equi in vitro, and on the multiplication of B. microti in mice. The cytotoxicity assay was tested on Madin-Darby bovine kidney (MDBK), mouse embryonic fibroblast (NIH/3T3) and human foreskin fibroblast (HFF) cell lines. RESULTS: The half-maximal inhibitory concentration (IC50) values of EA and ß-CD EA on B. bovis, B. bigemina, B. divergens, B. caballi and T. equi were 9.58 ± 1.47, 7.87 ± 5.8, 5.41 ± 2.8, 3.29 ± 0.42 and 7.46 ± 0.6 µM and 8.8 ± 0.53, 18.9 ± 0.025, 11 ± 0.37, 4.4 ± 0.6 and 9.1 ± 1.72 µM, respectively. The IC50 values of APSP EA on B. bovis, B. bigemina, B. divergens, B. caballi and T. equi were 4.2 ± 0.42, 9.6 ± 0.6, 2.6 ± 1.47, 0.92 ± 5.8 and 7.3 ± 0.54 µM, respectively. A toxicity assay showed that EA, ß-CD EA and APSP EA affected the viability of cells with a half-maximal effective concentration (EC50) higher than 800 µM. In the experiments on mice, APSP EA at a concentration of 70 mg/kg reduced the peak parasitemia of B. microti by 68.1%. Furthermore, the APSP EA-atovaquone (AQ) combination showed a higher chemotherapeutic effect than that of APSP EA monotherapy. CONCLUSIONS: To our knowledge, this is the first study to demonstrate the in vitro and in vivo antibabesial action of EA-NPs and thus supports the use of nanoparticles as an alternative antiparasitic agent.