Genetic Variation of the -tubulin Gene of Babesia caballi Strains

  • María Guadalupe Montes-Cortés Parasitology and Parasitological Diseases, Veterinary Faculty, Universidad de Extremadura, Cáceres, Spain
  • José Luis Fernández-García Genetics and Animal Breeding, Veterinary Faculty, Universidad de Extremadura, Cáceres, Spain
  • Miguel Ángel Habela Martínez-Estéllez Parasitology and Parasitological Diseases, Veterinary Faculty, Universidad de Extremadura, Cáceres, Spain
Keywords: Babesia caballi, -tubulingene, Equine piroplasmosis, PCR-RFLP marker

Abstract

Background: Equine piroplasmosis is caused by two haemoprotozoan parasites: Babesia caballi and Theileria equi. Negative economic impact on international trade has been associated to endemic sites. This is the reason why carrier detection requires reliable diagnostic methods. Various diagnostic modalities can be used alone or in combination including PCR. However, genetic variation of commonly used genes is still of debate. The aim of this research was to sequence the b-tubulin gene of a B. caballi strain from Spain and to compare it with known b-tubulin sequences.Methods: DNA was isolated from a cryopreserved strain from Spain and acute and chronic carrier horses. Firstly, degenerated primer pairs were designed based on GenBank sequences of different Babesia and Theileria species for sequencing. The primers were redesigned to amplify both parasites, simultaneously. Finally, a species-specific pri­mer pair for B. caballi was designed and a Restriction Fragment Length Polymorphism-PCR (PCR-RFLP) assay performed to know the difference of known B. caballi strains.Results: We provided new insights of the b-tubulin gene and a good molecular coverage of this gene, contributing with a number of useful primers to amplify T. equi and B. caballi. Moreover, PCR-RFLP assays based on the exon II of this gene confirmed the causative B. caballi strain in Spanish horses.Conclusion: We reported useful primer pairs for diagnostic and a new sequence of the b-tubulin gene of B. caballi, which will facilitate the development of future assays and the detection of infected horses, preventing thus the spread of this disease worldwide.

References

Rodríguez Bautista JL, Ikadai H, You M, Battsetseg B, Igarashi I, Nagasawa H, Fujisaki K (2001) Molecular evidence of Babesia caballi (Nuttall and Strick-land, 1910) parasite transmission from experimentally-infected SCID mice to the ixodid tick, Haemaphysalis longi-cornis (Neuman, 1901). Vet Parasitol. 13: 185–191.

Mehlhorn H, Schein E (1998) Description of Babesia equi (Laveran, 1901) as Theil-eria equi (Mehlhorn, Schein 1998). Par-asitol Res. 84: 467–475.

Uilenberg G (2006) Babesia: A historical overview. Vet Parasitol. 138: 3–10.

De Waal DT, Van Heerden J (1994) Eq-uine Piroplasmosis. In: Coetzer JAW, Tustin RC (Eds) Infectious Diseases of Livestock. Oxford University Press, New York, pp. 295–304.

Friedhoff KT, Tenter AM, Müller I (1990) Haemoparasites of equines: impact on international trade of horses. Rev Sci Tech Off Int Epiz. 9: 1187–1194.

Bashiruddin JB, Cammà C, Rebelo E (1999) Molecular detection of Babesia equi and Babesia caballi in horse blood by PCR amplification of part of the 16S rRNA gene. Vet Parasitol. 84: 75–83.

Wise LN, Kappmeyer LS, Mealey RH, Knowles DP (2013) Review of equine piroplasmosis. J Vet Intern Med. 27: 1334–1346.

Böse R, Jorgesen WK, Dalgliesh RJ, Friedhoff KT, de Vos AJ (1995) Cur-rent state and future trends in the diag-nosis of babesiosis. Vet Parasitol. 57: 61–74.

Mans BJ, Pienaar R, Latif AA (2015) A review of Theileria diagnostics and ep-idemiology. Int J Parasitol Parasites Wildl. 4: 104–118.

Alhassan A, Pumidonming W, Okamura M, Hirata H, Battsetseg B, Fujisaki K, Yokoyama N, Igarashi I (2005) Devel-opment of a single-round and multi-plex PCR method for the simultaneous detection of Babesia caballi and Babe-sia equi in horse blood. Vet Parasitol. 129: 43–49.

Sumbria D, Singla LD, Sharma A (2016b) Theileria equi and Babesia caballi in-fection in equines in Punjab: A study on serological and molecular prevalence. Trop Anim Health Prod. 48: 45–52.

Nicolaiewsky TB, Richter MF, Lunge VR, Cuhna CW, Delagostin O, Ikuta N, Fon-seca AS, Silva SS, Ozaki LS (2001) De-tection of Babesia equi (Laveran, 1901) by nested polymerase chain reaction. Vet Parasitol. 101: 9–21.

Rampersad J, Cesar E, Campbell MD, Samlal M, Ammons D (2003) A field evaluation of PCR for the routine de-tection of Babesia equi in horses. Vet Parasitol. 114: 81–87.

Sumbria D, Singla LD, Kumar S, Sharma A, Dahiya RK, Setia R (2016a) Spatial distribution, risk factors and haemato-biochemical alterations associated with Theileria equi infected equids of Pun-jab (India) diagnosed by indirect ELI-SA and nested PCR. Acta Tropica. 155: 104–112.

Kim C, Conza LB, Alhassan A, Iseki H, Yokoyama N, Xuan X, Igarashi I (2008) Diagnostic real-time PCR assay for the quantitative detection of Theil-eria equi from equine blood samples. Vet Parasitol. 151: 158–163.

Ueti MW, Palmer GH, Kappmeyer LS, Scoles GA, Knowles DP (2003) Ex-pression of equi merozoite antigen 2 during development of Babesia equi in the midgut and salivary gland of the vector tick Boophilus microplus. J Clin Microbiol. 41: 5803–5809.

Bhoora R, Quan M, Zweygarth E, Guthrie AJ, Prinsloo SA, Collins NE (2010) Sequence heterogeneity in the gene encoding the rhoptry-associated protein-1 (RAP-1) of Babesia caballi isolates from South Africa. Vet Parasi-tol. 169: 279–288.

Zamoto A, Tsuji M, Wei Q, Cho SH, Shin EH, Kim TS, Leonova GN, Hag-iwara K, Asakawa M, Kariwa H, Ta-kashima I, Ishihara C (2004) Epizooti-ologic survey for Babesia microti among small wild mammals in North-eastern Eurasia and geographic diver-sity in the-tubuline gene sequences. J Vet Med Sci. 66: 785–792.

García-Bocanegra I, Arenas-Montes A, Hernández E, Adaszek L, Carbonero A, Almería S, Jaén-Téllez JA, Gutiér-rez-Palomino P, Arenas A (2013) Se-roprevalence and risk factors associat-ed with Babesia caballi and Theileria equi infection in equids. Vet J. 195: 172–178.

Habela MA, Gragera-Slikker A, Moreno A, Montes G, Sevilla R (2005) Piro-plasmosis equina: conocimiento y gra-do de concienciación de los producto-res de caballos Pura Raza Española. Revista Equinus 11: 17–34.

Zweygarth E, Lopez-Rebollar LM, Nur-ton J, Guthrie AJ (2002) Culture, isola-tion and propagation of Babesia cabal-li from naturally infected horses. Para-sitol Res. 88: 460–462.

Camacho AT, Guitian FJ, Pallas E, Gestal JJ, Olmeda AS, Habela MA, Telford SR, Spielman A (2005) Theileria (Babe-sia) equi and Babesia caballi Infec-tions in Horses in Galicia, Spain. Trop Anim Health Prod. 37: 293–302.

Millar SA, Dykes DD, Polesky HF (1988) A simple salting out procedure for ex-tracting DNA from human nucleated cells. Nucleic Acids Res. 16: 1215.

Fernández-García JL (2012) The endan-gered Dama dama mesopotamica: ge-netic variability, allelic loss and hy-bridization signals. Contrib. Zool. 81: 223–233.

Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolution-ary Genetics Analysis (MEGA) soft-ware version 4.0. Mol Biol Evol. 24: 1596–1599.

Barbosa AM, Fernández-García JL, Car-ranza J (2009) A new Marker for rapid sex identification of red deer (Cervus elaphus). Hystrix It J Mamm. 20: 169–172.

Cacciò S, Cammà C, Onuma M, Severini C (2000) The -tubulin gene of Babe-sia and Theileria parasites is an in-formative marker for species discrimi-nation. Int J Parasitol. 30: 1181–1185.

Altschul SF, Madden TL, Schaffer AA,Zhang J, Zhang Z, Miller W, Lipman DJ (1997) BLASTN 2.2.20 (Ver. Feb-08-2009) Gapped BLAST and PSI-BLAST a new generation of protein da-tabase search programs. Nucleic Acids Res. 25: 3389–3402.

Sivakumar T, Tattiyapong M, Okubo K, Suganuma K, Hayashida K, Igarashi I, Zakimi S, Matsumoto K, Inokuma H, Yokoyama N. (2014) PCR detection of Babesia ovata from questing ticks in Japan. Ticks Tick Borne Dis. 5: 305–310.

Criado-Fornelio A, González-del-Río MA, Buling-Saraña A, Barba-Carretero JC (2004) The “expanding universe” of piroplasms. Vet Parasitol. 119: 337–345.

Nagore D, García-Sanmartín J, García-Pérez AL, Juste RA, Hurtado A (2004) Detection and identification of equine Theileria and Babesia species by re-verse line blotting: epidemiological Survey and phylogenetic analysis. Vet Parasitol. 123: 41–54.

Bhoora R, Franssen L, Oosthuizen MC, Guthrie AJ, Zweygarth E, Penzhorn BL, Jongejan F, Collins NE (2009) Se-quence Heterogeneity in the 18S rRNA gene within Theileria equi and Babesia caballi from horses in South Africa. Vet Parasitol. 159: 112–120.

Munkhjargal T, Sivakumar T, Battsetseg B, Nyamjargal T, Aboulaila M, Purevtseren B, Bayarsaikhan D, By-ambaa B, Terkawi MA, Yokoyama N, Igarashi I (2013) Prevalence and ge-netic diversity of equine piroplasmos in Tov province, Mongolia. Infect Genet Evol. 16: 178–185.

Alanazi AD, Said AE, Morin-Adeline V, Alyousif MS, Slapeta J (2014) Quanti-tative PCR detection of Theileria equi using laboratory workflows to detect asymptomatic persistently infected hors-es. Vet Parasitol. 206: 138–145.

Baneth G, Florin-Christensen M, CardosoL, Schnittger L (2015) Reclassification of Theileria annae as Babesia vulpes sp. nov. Parasit Vectors. 8: 207.

El-Sayed SAES, Rizk MA, Terkawi MA, Mousa A, El Said ESES, Elsayed G, Fouda M, Yokoyama N, Igarashi I (2015) Cocktail of Theileria equi anti-gens for detecting infection in equines. Asian Pac J Trop Biomed. 5: 977–981.

Mahmoud MS, El-Ezz NT, Abdel-Shafy S, Nassar SA, El Namaky AH, Khalil WK, Knowles D, Kappmeyer L, Silva MG, Suarez CE (2016) Assessment of Theileria equi and Babesia caballi in-fections in equine populations in Egypt by molecular, serological and haematological approaches. Parasit Vectors. 9: 260.

Published
2017-10-08
How to Cite
1.
Montes-Cortés MG, Fernández-García JL, Martínez-Estéllez M Ángel H. Genetic Variation of the -tubulin Gene of Babesia caballi Strains. J Arthropod Borne Dis. 11(3):344-353.
Section
Original Article