RESEARCH PAPER
Multiplex PCR for molecular screening of Borrelia burgdorferi sensu lato, Anaplasma spp. and Babesia spp.
 
More details
Hide details
1
‘Pedro Kourí’, Institute of Tropical Medicine, National Reference Laboratory of Spirochetes, Havana, Cuba
 
2
Institute of Biology, Laboratory of Eco-Epidemiology of Parasites, University of Neuchâtel, Neuchâtel, Switzerland
 
 
Ann Agric Environ Med. 2015;22(4):642-646
 
KEYWORDS
ABSTRACT
Introduction:
Ticks transmit a great variety of pathogenic microorganisms to humans and animals. The detection of tick-borne pathogens (TBP) is mainly by molecular techniques based on polymerase chain reactions (PCR).

To design and evaluate a multiplex PCR for the molecular screening of zoonotic TBP for exploratory studies.

Material and Methods:
Control DNA from reference strains, DNA from experimentally-infected biological specimens, and from Rhipicephalus sanguineus ticks collected from domestic and homeless dogs were used. A multiplex PCR assay to detect the presence of Borrelia burgdorferi sensu lato, Anaplasma spp. and Babesia spp. was designed and optimized using primers previously reported for B. burgdorferi sensu lato and Anaplasma spp., while for Babesia spp. they were designed in silico. The multiplex PCR was evaluated on the DNA from biological samples.

Results:
A new set of specific primers for Babesia spp. was designed. Adjustment of the master mix reactive concentrations and amplification conditions for the multiplex PCR allowed the successful amplification of the specific amplicons for each microbial group from the control DNA and experimentally-infected biological specimens. The efficiency of the multiplex PCR amplifying three DNA targets was confirmed. Individual and co-infection of Anaplasma spp. and Babesia spp. were detected in the R. sanguineus ticks from dogs.

Conclusions:
A multiplex PCR assay for the screening of three TBP is available. By using it, B. burgdorferi sensu lato, Anaplasma spp. and Babesia spp. can be detected accurately in one PCR reaction.

 
REFERENCES (38)
1.
Heyman P, Cochez C, Hofhuis A, van der Giessen J, Sprong H, Porter SR, Losson B, Saegerman C, Donoso-Mantke O, Niedrig M, Papa A. A clear and present danger: tick-borne diseases in Europe. Expert Rev Anti Infect Ther. 2010; 8(1): 33–50.
 
2.
De la Fuente J, Estrada-Peña A. Ticks and tick-borne pathogens on the rise (Editorial). Ticks Tick-Borne Dis. 2012; 3: 115–116.
 
3.
Jongejan F, Uilenberg G. The global importance of ticks. Parasitology. 2004; 129: s3-s14.
 
4.
Rajput ZI, Hu SH, Chen WJ, Arijo AG, Xiao CW. Importance of ticks and their chemical and immunological control in livestock. J Zhejiang Univ Sci B. 2006; 7(11): 912–921.
 
5.
Gray J, Zintl A, Hildebrandt A, Hunfeld KP, Weiss L. Zoonotic babesiosis: Overview of the disease and novel aspects of pathogen identity. Ticks Tick-Borne Dis. 2010; 1: 3–10.
 
6.
Lommano E, Bertaiola L, Dupasquier C, Gern L. Infections and co-infections of questing Ixodes ricinus ticks by emerging zoonotic pathogens in Western Switzerland. Appl Exp Microbiol. 2012; 78: 4606–4612.
 
7.
Piesman J, Eisen L. Prevention of tick-borne diseases. Annu Rev Entomol. 2008; 53: 323–343.
 
8.
Berggoetz M, Schmid M, Ston D, Wyss V, Chevillon C, Pretorius AM, Gern L. Tick-borne pathogens in the blood of wild and domestic ungulates in South Africa: Interplay of game and livestock. Ticks Tick- Borne Dis. 2014; 5: 166–175.
 
9.
Berggoetz M, Schmid M, Ston D, Wyss V, Chevillon C, Pretorius AM, Gern L. Protozoan and bacterial pathogens in tick salivary glands in wild and domestic animal environments in South Africa. Ticks Tick- Borne Dis. 2014; 5: 176–185.
 
10.
Chan K, Marras SA, Parveen N. Sensitive multiplex PCR assay to differentiate Lyme spirochetes and emerging pathogens Anaplasma phagocytophilum and Babesia microti. BMC Microbiology. 2013; 13(1): 295.
 
11.
Hojgaard A, Lukacikb G, Piesman J. Detection of Borrelia burgdorferi, Anaplasma phagocytophilum and Babesia microti, with two different multiplex PCR assays. Ticks Tick-Borne Dis. 2014; 5(3): 349–351.
 
12.
Liz, J. Ehrlichia phagocytophila: Aspects épidémiologiques, hématologiques et sérologiques de l’infection chez les bovins en Suisse. PhD Thesis, University of Neuchâtel, 1994; pp. 138.
 
13.
Gern L, Zhu Z, Aeschlimann A. Development of Borrelia burgdorferi in Ixodes ricinus females during blood feeding. Ann Parasitol Hum Comp. 1990; 65: 89–93.
 
14.
Guy EC, Stanek G. Detection of Borrelia burgdorferi in patients with Lyme disease by the polymerase chain reaction. J Clin Pathol. 1991; 44: 610–611.
 
15.
Rijpkema S, Golubic D, Molkenboer M, Verbeek-De Kruif N, Schellekens J. Identification of four genomic groups of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected in a Lyme borreliosis endemic region of northern Croatia. Exp Appl Acarol. 1996; 20: 23–30.
 
16.
Humair PF, Douet V, Moran-Cadenas F, Schouls L, Van de Pol I, Gern L. Molecular identification of blood meal source in Ixodes ricinus ticks using 12S rDNA as a genetic marker. J Med Entomol. 2007; 44: 869–880.
 
17.
Halos L, Jamal T, Vial L, Maillard R, Suau A, Le Menach A, Boulouis HJ, Vayssier-Taussat M. Determination of an efficient and reliable method for DNA extraction from ticks. Vet Res. 2004; 35: 709–713.
 
18.
Postic D, Assous MV, Grimont PAD, Baranton G. Diversity of Borrelia burgdorferi sensu lato evidenced by restriction fragment length polymorphism of rrf(5S)-rrl(23S) intergenic spacer amplicons. Int J Syst Bacteriol. 1994; 44(4): 743–752.
 
19.
Lee SH, Kim BJ, Kim JH, Park KH, Yeo SJ, Kim SJ et al. Characterization of Borrelia burgdorferi strains isolated from Korea by 16S rDNA sequence analysis and PCR-RFLP analysis of rrf(5S)-rrl(23S) intergenic spacer amplicons. Int J Syst Evol Microbiol. 2000; 50(Pt2): 857–863.
 
20.
Chu CY, Jiang BG, Liu W, Zhao QM, Wu XM, Zhang PH, Zhan L, Yang H, Cao WC. Presence of pathogenic Borrelia burgdorferi sensu lato in ticks and rodents in Zhejiang, south-east China. J Med Microbiol. 2008; 57(Pt8): 980–985.
 
21.
Pancholi P, Kolbert CP, Mitchell PD, Reed KD, Dumler JS, Bakken JS, Telford SR 3rd, Persing DH. Ixodes dammini as a potential vector of human granulocytic ehrlichiosis. J Infect Dis. 1995; 172: 1007–1012.
 
22.
Walls JJ, Caturegli P, Bakken JS, Asanovich KM, Dumler JS. Improved sensitivity of PCR for diagnosis of human granulocytic ehrlichiosis using epank 1 genes of Ehrlichia phagocytophila – group Ehrlichiae. J Clin Microbiol. 2000; 38: 354–356.
 
23.
Sirigireddy KR, Ganta RR. Multiplex detection of Ehrlichia and Anaplasma species pathogens in peripheral blood by real-time reverse transcriptase-polymerase chain reaction. J Molec Diag. 2005; 7(2): 308–316.
 
24.
Milutinović M, Masuzawa T, Tomanović S, Radulović Z, Fukui T, Okamoto Y. Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Francisella tularensis and their co-infections in host-seeking Ixodes ricinus ticks collected in Serbia. Exp Appl Acarol. 2008; 45(3–4): 171–183.
 
25.
Rymaszewska A. Divergence within the marker region of the groESL operon in Anaplasma phagocytophilum. Eur J Clin Microbiol Infect Dis. 2008; 27(11): 1025–1036.
 
26.
Poitout FM, Shinozaki JK, Stockwell PJ, Holland CJ, Shukla SK. Genetic variants of Anaplasma phagocytophilum infecting dogs in Western Washington State. J Clin Microbiol. 2005; 43(2): 796–801.
 
27.
Chen Z, Liu Q, Liu JQ, Xu BL, Lv S, Xia S, Zhou XN. Tick-borne pathogens and associated co-infections in ticks collected from domestic animals in central China. Parasit Vectors. 2014; 7: 237.
 
28.
Barros-Battesti DM, Reyes M, Famadas KM, Onofrio VC, Beati L, Guglielmone AA. The ixodid ticks (Acari: Ixodidae) of Cuba. Systematic Appl Acarol. 2009; 14: 101–128.
 
29.
Basu AK, Basu M, Adesiyun AA. A review on ticks (Acari: Ixodoidea: Ixodidae, Argasidae), associated pathogens and diseases of Trinidad and Tobago. Acarol. 2012; 52(1): 39–50.
 
30.
Savić S, Vidić B, Lazić S, Lako B, Potkonjak A, Lepsanović Z. Borrelia burgdorferi in ticks and dogs in the province of Vojvodina, Serbia. Parasite. 2010; 17(4): 357–361.
 
31.
Santamaria A, Calzada JE, Saldaña A, Yabsley MJ, Gottdenker NL. Molecular diagnosis and species identification of Ehrlichia and Anaplasma infections in dogs from Panama, Central America. Vector Borne Zoonotic Dis. 2014; 14(5): 368–370.
 
32.
Vairamuthu S, Ranju RS, Latha BR, Dhivya B, Balachandran C. A six year (2006–2011) retrospective study of hemoprotozoan parasites affecting dogs in Chennai, Tamil Nadu, India. J Parasit Dis. 2014; 38(2): 193–195.
 
33.
León A, Demedio J, Márquez Mario, Castillo E, Perera A, Zuaznaba O et al. Diagnosis of canine Ehrlichiosis in Havana city. RECVET. 2008; III(5). http://www.veterinaria.org/rev....
 
34.
Ramos R, Ramos C, Araujo F, Oliveira R, Souza I, Pimentel D, Galindo M, Santana M, Rosas E, Faustino M, Alves L. Molecular survey and genetic characterization of tick-borne pathogens in dogs in metropolitan Recife (north-eastern Brazil). Parasitol Res. 2010; 107(5): 1115–1120.
 
35.
Singh A, Singh H, Singh NK, Singh ND, Rath SS. Canine babesiosis in northwestern India: molecular detection and assessment of risk factors. Biomed Res Int. 2014; 2014: 1–5.
 
36.
Parola P, Socolovschi C, Jeanjean L, Bitam I, Fournier PE, Sotto A, Labauge P, Raoult D: Warmer weather linked to tick attack and emergence of severe rickettsioses. PLoS Negl Trop Dis. 2008, 2: e338.
 
37.
Rodríguez ON, Espaine L, Rivas A, Rodríguez P. Epidemiology of cattle diseases caused by haemoparasites in Cuba. Rev Cubana Cienc Vet. 1989; 20: 37–56.
 
38.
Corona B, Martínez S. Detection of Anaplasma marginale in bovine, using the msp5 gene amplification by PCR. Rev Salud Anim. 2011; 33(1): 24–31.
 
eISSN:1898-2263
ISSN:1232-1966
Journals System - logo
Scroll to top