Elsevier

Acta Tropica

Volume 191, March 2019, Pages 128-132
Acta Tropica

Evidence of multiple point mutations in Theileria annulata cytochrome b gene incriminated in buparvaquone treatment failure

https://doi.org/10.1016/j.actatropica.2018.12.041Get rights and content

Abstract

Drug resistance is one of the emerging and re-emerging epidemics affecting both veterinary and public health sectors. Buparvaquone provides the most satisfactory means in the treatment of bovine tropical theileriosis. However, recently there has been widespread reports of development of resistance of Theileria annulata to buparvaquone. To investigate the situation in Sudan where bovine tropical theileriosis is endemic, fifty blood samples from T. annulata-positive cattle. were used for DNA extraction, PCR and cytochrome b gene nucleotide sequencing. Analysis of the two buparvaquone binding site regions Q01 (130–148) and Q02 (244–266), revealed three non- synonymous mutations at codon 146; alanine (GCT) to threonine (ACT) within the Q01 region across all 50 isolates and the other mutation at codon 129; serine (AGC) to glycine (GGC) in 18 isolates which is very close to the Q01 binding site. However, we documented another mutation at position 227; valine (GTG) to methionine (ATG) close to the close to the Q02 binding site, in three isolates with mutation at codon 129. We concluded that this study has provided evidence of point mutations in the cytochrome b gene of T. annulata that might be associated with buparvaquone treatment failure in Sudan.

Introduction

Bovine tropical theileriosis caused by Theileria annulata has been reported in Sudan since the 1920’s (El Hussein et al., 2012). To date, it remains one of the most economically important tick-borne diseases and a major limitation to the expansion of dairy farming in Sudan that causes high mortalities in exotic and crossbreed cattle (Latif, 1994; Minjauw and McLeod, 2003; Abakar et al., 2018). Although the indigenous zebu breeds show resistance to clinical tropical theileriosis, the carrier state negatively impact the productivity of these animals (Bakheit and Latif, 2002). This has been exacerbated by that fact that no coordinated approach in the management of the disease is practiced and solely left to individual animal owners who depend on the use of anti-theilerial drugs and acaricide application to control the disease (El Hussein et al., 2004, 2012).

Drug resistance studies in antiprotozoal drugs that act on the cytochrome b gene have been extensively carried out and have demonstrated that a single mutation or gene deletion is sufficient for loss of sensitivity which may be followed by secondary mutations to enhance the resistance (Hyde, 2002; Munday et al., 2015). Moreover, the heavy usage and/ or underdosage of antiprotozoal drugs has resulted in selection pressure that has contributed to the development of resistance as reported by Hyde, (2007). Furthermore, it has been reported that mutations at the drug-binding site Q0 that substitute neutral and hydrophobic amino acid to a polar and hydrophilic amino acid could affect the drug binding efficiency (Mhadhbi et al., 2015). It has also been documented that mutations outside but close to the Q0 drug binding site can also result in treatment failure (Walker at al., 1998; Sharifiyazdi et al., 2012; Mhadhbi et al., 2015).

Mutations in the T. annulata cytochrome b gene that are associated with buparvaquone treatment failure have been demonstrated in vivo as well as in the field isolates from animals that did not respond to buparvaquone treatment at the normal dose in Iran, Turkey and Tunisia (Mhadhbi et al., 2010; Hacilarlioglu et al., 2012; Sharifiyazdi et al., 2012; Mhadhbi et al., 2015). In Khartoum state, veterinarians have been reporting cases where animals succumbed to bovine tropical theileriosis despite being treated with the normal dose of buparvaquone. In this study we have provided evidence of a new point mutation and other mutations in cytochrome b gene in T. annulata isolated from Sudan that are associated with resistance to buparvaquone described elsewhere.

Section snippets

Ethical consideration

Fifty cattle blood samples (approximately 5 ml) was drawn from the external jugular vein into EDTA vacutainer tubes from farms in Khartoum State, Sudan. The procedure was reviewed and approved by the University of Khartoum, Sudan (Approval no. 2017BS), and Sudan University of Science and Technology, Khartoum, Sudan (Approval no.28–46), and informed consent was sought from animal owners. These samples were collected from cattle clinically diagnosed and confirmed by Giemsa blood smears to be

Sequence processing and analysis

Two sequence fragments were generated for each sample and ATGC software version 9.1 (GENETYX Corporation, Tokyo, Japan) was used for manual editing to correct possible base calling errors and remove primer annealing sites. The regions of unambiguously aligned sequences were eventually joined to reconstruct sequences of 1442 bp of 18S rDNA gene and 1041 bp of the cytochrome b gene which were retained for final analysis.

The 18S rDNA gene was amplified from all 50 samples, but only 42 of them were

Discussion

Buparvaquone is the drug of choice for the control of tropical theileriosis, it is active against both schizont and piroplasm stages when administered intramuscularly at the dose of 2.5 mg/kg with success rate of as high as 92% (McHardy et al., 1985; Hashemi-Fesharki, 1991; Gharbi and Darghouth, 2015). It has been suggested that buparvaquone and other members of the hydroxynaphtoquinones act by competitively inhibiting the binding of ubiquinone (coenzyme Q) to the cytochrome b of the

Conclusion

In conclusion, this study has provided evidence of point mutations in the cytochrome b gene of T. annulata that might be associated with buparvaquone treatment failure in Sudan. We attributed this to the heavy usage of buparvaquone in the treatment of tropical bovine theileriosis in Sudan. However, further experimental studies are needed to provide conclusive evidence of the resistance of T. annulata to buparvaquone in Sudan.

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