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Transboundary and Emerging Diseases

SHORT COMMUNICATION

Development of a Loop-mediated Isothermal Amplification


(LAMP) Assay for Rapid Diagnosis of Babesia canis
infections
H. Müller1, N. Aysul2, Z. Liu1, D. A. Salih1, T. Karagenc2, D. Beyer1, B. Kullmann1, J. S. Ahmed1 and
U. Seitzer1
1
Division of Veterinary Infection Biology and Immunology, Research Center Borstel, Borstel, Germany
2
Parazitoloji Anabilim Dali Adnan Menderes Universitesi Veteriner Fakultesi Aydin, Turkey

Keywords: Summary
babesia canis; diagnosis; loop mediated
isothermal amplification Vector-borne diseases are rising in interest due to global warming, which is
believed to impact on the distribution of vectors into new areas thus influenc-
Correspondence: ing the occurrence and epidemiology of vector-borne pathogens. Babesia canis
Ulrike Seitzer. Division of Veterinary Infection belongs to the Piroplasmidae and there are three described subspecies, namely
Biology and Immunology, Research Center B. canis canis, B. canis rossi and B. canis vogeli. They are each transmitted by a
Borstel, Parkallee 22, D-23845 Borstel,
different tick-species, Dermacentor reticulatus, Haemaphysalis leachi and Rhipi-
Germany. Tel.: + 49 (0)4537 188413;
Fax: + 49 (0) 4537 188627;
cephalus sanguineus, respectively. There are also differences in the geographical
E-mail useitzer@fz-borstel.de distribution and pathogenicity to dogs of each subspecies. In this study, we
aimed to establish a rapid and easy to perform DNA-based test using loop-
Received for publication July 3, 2009 mediated isothermal amplification to detect all three Babesia canis subspecies
in one assay.
doi:10.1111/j.1865-1682.2010.01113.x

Introduction Material and Methods


Babesia canis belongs to the Piroplasmidae and is classi- The Babesia canis putative (60.2) rhoptry protein gene
fied in three subspecies B. canis canis, B.canis rossi and (genbank accession no. M91168) was used to construct
B. canis vogeli (Uilenberg et al., 1989). Although they LAMP primers using the Primer Explorer Program
share a similar morphology, they have different vector (http://primerexplorer.jp/e/). The primers used were:
specificity (Dermacentor reticulatus, Haemaphysalis laechi BcanF3 5¢-ttcattatgcttttggcaca, BcanB3 5¢-gcaagaatcacgttaa
and Rhipicephalus sanguineus, respectively), geographical taaggta, BcanFIP 5¢-ccatccatcttttagacagcatctatcgcaatagtaatg
distribution, antigenicity and pathogenicity to dogs. The gaagatgag-3¢, BcanBIP 5¢-tcataacttcgcttgtggagggcaatgcttttca
definitive means of diagnosis of babesiosis is by the cccgct-3¢. To analyse the specificity, DNA isolated from
identification of Babesia parasites in Giemsa-stained B. canis canis, B. canis rossi, B. canis vogeli, B. motasi,
thin-film blood smears examined by microscopy. How- B. ovis, B. crassa and B. microti were subjected to LAMP
ever, the detection of Babesia parasites is difficult in exactly as described previously (Liu et al., 2008; Salih et
dogs with unapparent or chronic infections since the al., 2008). Specificity of the LAMP product was verified
level of parasitemia is very low. Therefore, the develop- by sequencing. A PCR amplified product using BcanF3
ment of a highly specific and sensitive system for the and BcanB3 primers was ligated into the pDrive vector
diagnosis of B. canis infection is required. In this study, (Qiagen, Hilden, Germany), transformed into Escherichia
we aimed to establish a rapid and easy to perform coli cells and selected clones were subjected to sequencing
test using loop-mediated isothermal amplification (Eurofins MWG Operon, Ebersberg, Germany). Sensitivity
(LAMP) to detect all three Babesia canis subspecies in was investigated using serial dilutions of genomic DNA
one assay. and comparing with detection by PCR using BcanF3 and

ª 2010 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 57 (2010) 63–65 63
LAMP Assay for Rapid Diagnosis of Babesia canis infections H. Müller et al.

(a)

(b)

Fig. 1. Specificity and sensitivity of LAMP assay for detection of B. canis DNA. (a) Only in Babesia canis canis, B. c. vogeli and B. c.rossi a positive
signal with the LAMP assay is obtained, all the Babesia species investigated are negative. M: Marker for lanes 1–5; 1: B. motasi (Turkey); 2: B. ovis;
3: B. crassa; 4: B. motasi (Texel); 5: B. canis vogeli; 6: B. c. rossi; 7: B. c. canis; 8: B. microti; M2: Marker for lanes 6–8. (b) Sensitivity of the LAMP
assay compared with PCR. M: Marker, 1: 2.5 ng; 2: 1 ng; 3: 500 pg; 4: 250 pg; 5: 100 pg; 6: 50 pg; 7: 25 pg; 8: 10 pg; 9: 5 pg; 10: 2.5 pg
gDNA of B. c. vogeli.

BcanB3 primers. Finally, 58 field samples collected from and the product could also be analyzed by the naked eye
dogs in Turkey were analysed. All DNA samples were also using SYBR green in the reaction as described by Liu
tested in reverse line blot (RLB) according to Schnittger et al. (2008) and Salih et al. (2008) (data not shown).
et al. (2004) to examine specificity of detection. The sensitivity of the test compared with PCR demon-
strated that LAMP was less sensitive, with a signal being
obtained from 25 pg gDNA and with PCR at 5 pg gDNA
Results and Discussion
(Fig. 1b). The sensitivity of the assay needs to be investi-
The established LAMP for the detection of the Babesia gated further in relation to parasitaemia. Regarding the
canis putative (60.2) rhoptry protein gene was shown to 58 field samples, although both RLB and LAMP detected
be applicable for the diagnosis of canine babesiosis caused 15 positive samples, 12 were congruent positive samples,
by infection with B. canis canis, B. canis vogeli or B. canis 3 were positive in RLB but negative in LAMP and vice
rossi. The specificity of the established LAMP is depicted versa, resulting in a calculated sensitivity of 80% and
in Fig. 1a. All B. canis subspecies are detected but none of specificity of 93% using RLB as the reference test. Further
the other Babesia. The optimal reaction time and temper- investigations are planned to evaluate the application of
ature for LAMP were determined to be 45 min at 64C, the LAMP assay in the field.

64 ª 2010 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 57 (2010) 63–65
H. Müller et al. LAMP Assay for Rapid Diagnosis of Babesia canis infections

Acknowledgements Salih, D. A., Z. Liu, M. A. Bakheit, A. M. Ali, A. M. El


Hussein, H. Unger, G. Viljoen, U. Seitzer, and
We thank Prof. Eberhard Schein, FU Berlin, and Dr Erich J. S. Ahmed, 2008: Development and evaluation of a
Zweygarth, LMU Munich, for providing control DNA of loop-mediated isothermal amplification method for
B. c. canis, B. c. rossi. This study was supported in part diagnosis of tropical theileriosis. Transbound. Emerg. Dis.
by the EU Inco-Dev projects INCOME (515915) and 55, 238–243.
ICTTD-3 (510561). Schnittger, L., H. Yin, B. Qi, M. J. Gubbels, D. Beyer, S.
Niemann, F. Jongejan, and J. S. Ahmed, 2004: Simultaneous
detection and differentiation of Theileria and Babesia
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ª 2010 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 57 (2010) 63–65 65