Antitheilerial Activity of the Anticancer Histone Deacetylase Inhibitors.

The apicomplexan parasite, Theileria annulata, is the most prevalent hemoprotozoan in livestock, causing significant economic losses worldwide. It is essential to develop new and improved therapeutics, as current control measures are compromised by the development of resistance against the only available antitheilerial drug, buparvaquone (BPQ). Histone deacetylase inhibitors (HDACi) were shown to treat cancer effectively and revealed in vitro antiparasitic activity against apicomplexan parasites such as Plasmodium and Toxoplasma. In this study, we investigated the antitheilerial activity of the four anti-cancer HDACi (vorinostat, romidepsin, belinostat, and panobinostat) against the schizont stage of T. annulata parasites. All four HDACi showed potent activity and increased hyperacetylation of the histone-4 protein. However, based on the low host cell cytotoxicity and IC50 values, vorinostat (0.103 µM) and belinostat (0.069 µM) were the most effective showing antiparasitic activity. The parasite-specific activities of the HDACi (vorinostat and belinostat) were evaluated by western blotting using parasite-specific antibodies and in silico analysis. Both vorinostat and belinostat reduced the Theileria infected cell viability by downregulating anti-apoptotic proteins and mitochondrial dysfunction, leading to caspase-dependent cell apoptosis. The HDACi caused irreversible and antiproliferative effects on the Theileria infected cell lines. Our results collectively showed that vorinostat and belinostat could be used as an alternative therapy for treating Theileria parasites.

Population Genetic Analysis of the Theileria annulata Parasites Identified Limited Diversity and Multiplicity of Infection in the Vaccine From India.

Background: Apicomplexan parasite Theileria annulata causes significant economic loss to the livestock industry in India and other tropical countries. In India, parasite control is mainly dependent on the live attenuated schizont vaccine and the drug buparvaquone. For effective disease control, it is essential to study the population structure and genetic diversity of the Theileria annulata field isolates and vaccine currently used in India. Methodology/Results: A total of 125 T. annulata isolates were genotyped using 10 microsatellite markers from four states belonging to different geographical locations of India. Limited genetic diversity was observed in the vaccine isolates when compared to the parasites in the field; a level of geographical substructuring was evident in India. The number of genotypes observed per infection was highest in India when compared to other endemic countries, suggesting high transmission intensity and abundance of ticks in the country. A reduced panel of four markers can be used for future studies in these for surveillance of the T. annulata parasites in India. Conclusion: High genetic variation between the parasite populations in the country suggests their successful spread in the field and could hamper the disease control programs. Our findings provide the baseline data for the diversity and population structure of T. annulata parasites from India. The low diversity in the vaccine advocates improving the current vaccine, possibly by increasing its heterozygosity. The reduced panel of the markers identified in this study will be helpful in monitoring parasite and its reintroduction after Theileria eradication.

Genetic Profiling Reveals High Allelic Diversity, Heterozygosity and Antigenic Diversity in the Clinical Isolates of the Theileria annulata From India.

Tropical theileriosis caused by Theileria annulata infection is a significant livestock disease affecting cattle health and productivity resulting in substantial monetary losses in several countries. Despite the use of an effective vaccine for disease control still, a high incidence of infection is reported from India. One of the many reasons behind the ineffective disease control can be the existence of genetically diverse T. annulata parasite population in India. Therefore, studies focusing on understanding the genotypes are warranted. In this study, we have performed a genetic analysis of the Indian T. annulata field cell lines and the vaccine line using microsatellite markers, Genotyping based sequencing (GBS) and tams1 gene polymorphism. The degree of allelic diversity and multiplicity of the infection was determined to be high in the Indian population. No geographical sub-structuring and linkage disequilibrium were observed in the population. High population diversity was found which were similar with countries like Oman, Tunisia, and Turkey in contrast to Portugal and China. The presence of multiple genotypes as determined by microsatellite marker genotyping, GBS analysis and tams1 gene polymorphism point toward a panmictic parasite population in India. These findings are the first report from India which would help in understanding the evolution and diversity of the T. annulata population in the country and can help in designing more effective strategies for controlling the disease.

A Real-Time PCR based assay for determining parasite to host ratio and parasitaemia in the clinical samples of Bovine Theileriosis.

Theileria annulata is an intracellular parasite that causes active and latent forms of bovine theileriosis. Diagnosis of the disease is primarily based on traditional methods such as microscopy, however, PCR based methods have proven to be superior in the absence of clear disease symptoms. However, diagnosis is difficult in cases of lower parasitaemia by conventional PCR. Hence, a rapid and sensitive method which can detect early infection and low parasite load is required. Therefore, we have developed an absolute quantification based real-time PCR (qPCR) assay. Reference standard curve using recombinant plasmids of a host (hprt) and a parasite gene (tasp) was constructed, and the assay was initially standardised using in vitro T. annulata cell lines. Further, 414 blood samples from suspected theileriosis cases were also evaluated using qPCR. The assay can estimate host to parasite ratios, calculate parasitaemia and treatment effectiveness in the clinical cases of theileriosis. In comparison with the conventional PCR results, 44 additional positive cases were found. Therefore, the assay holds importance in a clinical setting due to its ability to quantify the parasite load in clinical samples. It may be further used in distinguishing active and latent theileriosis infections and detection of drug resistance in the field.

Potential Sabotage of Host Cell Physiology by Apicomplexan Parasites for Their Survival Benefits.

Plasmodium, Toxoplasma, Cryptosporidium, Babesia, and Theileria are the major apicomplexan parasites affecting humans or animals worldwide. These pathogens represent an excellent example of host manipulators who can overturn host signaling pathways for their survival. They infect different types of host cells and take charge of the host machinery to gain nutrients and prevent itself from host attack. The mechanisms by which these pathogens modulate the host signaling pathways are well studied for Plasmodium, Toxoplasma, Cryptosporidium, and Theileria, except for limited studies on Babesia. Theileria is a unique pathogen taking into account the way it modulates host cell transformation, resulting in its clonal expansion. These parasites majorly modulate similar host signaling pathways, however, the disease outcome and effect is different among them. In this review, we discuss the approaches of these apicomplexan to manipulate the host-parasite clearance pathways during infection, invasion, survival, and egress.