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First Molecular Detection of Babesia Canis Vogeli in Dogs and Rhipicephalus Sanguineus from Mexico

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First Molecular Detection of Babesia Canis Vogeli in Dogs and Rhipicephalus Sanguineus from Mexico Central Archives of Palliative Care Bringing Excellence in Open Access Research Article *Corresponding author Julio V. Figueroa Millan, National Research Center for Veterinary Parasitology, INIFAP, Boulevard Cuauhnahuac No. 8534, Jiutepec, Morelos, 62550, First Molecular Detection of Mexico; Tel: 52-777-320-5544; Fax: 52-777-319-2850 ext 129; Email: Babesia canis vogeli in Dogs Submitted: 09 October 2017 Accepted: 11 November 2017 and Rhipicephalus sanguineus Published: 12 November 2017 Copyright © 2017 Figueroa-Millan et al. from Mexico ISSN: 2573-1165 1 1 Jose J. Lira-Amaya , Carmen Rojas-Martinez , Antonio Alvarez- OPEN ACCESS Martinez1, Alfredo Pelaez-Flores2, Francisco Martinez-Ibañez2, 2 1 Keywords Diego Perez-de la Rosa , and Julio V. Figueroa-Millan * • Canine babesiosis; PCR-RFLP; nPCR; Babesia 1 National Research Center for Veterinary Parasitology, INIFAP, Mexico canisvogeli; DNA sequencing 2National Center for Validation Services on Animal Health, SENASICA, Mexico Abstract At the global level, three distinct subspecies for B. canis have been identified:B. caniscanis in Europe; B. canisrossi in Africa, while in America the presence of B. canis vogeli has been demonstrated. However, B. canisrossi has recently been reported in the USA and B. canis vogeli in South Africa and Europe. In order to optimize two methods for the molecular detection of the subspecies of canine babesiosis in Mexico from blood samples and Rhipicephalus sanguineus ticks, 30 blood samples and 18 tick specimens were collected in dogs with clinical manifestations compatible with canine babesiosis and a history of exposure to ticks. The analysis of Restriction Fragment Length Polymorphisms in PCR-amplified and digested DNA with restriction enzymeTaq I and HinfI (PCR-RFLP) allowed detection of the corresponding pattern for the B. canis vogeli subspecies (203 bp, 171 bp and 26 bp) in two blood samples and in one of the specimens of ticks subject to the PCR-RFLP analysis. With the nested PCR assay, the fragment of interest (192 bp) was detected in three blood samples and in three of the ticks that were analyzed for the B. canis vogeli subspecies. The presence of B. canis in Mexico had already been demonstrated microscopically, however, so far this is the first report on the molecular detection of the subspeciesB. canis vogeli in our country and confirmed by DNA sequencing. ABBREVIATIONS clinical manifestations may also include anorexia, Weight loss, jaundice, hematuria, depression, ascites, lymphadenopathy, PCR: Polymerase Chain Reaction; nPCR: nested Polymerase splenomegaly, central nervous system abnormalities and renal Chain Reaction; RFLP: Restriction Fragment Length complications [7]. Polymorphism; DNA: Deoxyribonucleic Acid In Mexico, the molecular diagnosis for canine babesiosis INTRODUCTION has not been implemented, therefore, there are no reports Canine babesiosis is a tick-borne cosmopolitan disease that demonstrate and identify what subspecies of the B. canis affecting domestic dogs, and its distribution is is related to protozoan is present in the blood of infected domestic dogs vector presence being more prevalent in regions with tropical or in the tick vector. The polymerase chain reaction (PCR) has and subtropical climates. The disease is caused by B. canis and B. gibsoni In traerythrocytic parasites [1]. The species of B. canis B. canis rossi, and B.been canis used vogeli for inamplification samples of naturally of the 18S infected rRNA dogs gene, in whichregions has of that transmits the pathogen and its distribution: B. caniscanis, facilitated the detection and identification of whosehas been vector re-classified for transmission into three issubspecies the Dermacentor based on reticulatusthe vector Portugal, suggesting the presence and distribution of the vector tick; B. canisrossi transmitted by Haemaphysalis leachi; and B. tickItaly [5] [4], whereas these subspecies in Brazil haveB. canis also vogeli been isdescribed the only in subspecies Northern canisvogeli transmitted by Rhipicephalus sanguineus in tropical and subtropical areas. These tick species are distributed in Europe, South Africa and America, respectively [2], although the (PCR-RFLP),that has been is identified an alternative [8,9]. Thefor theanalysis discrimination of polymorphisms among the in presence of B. canis rossi in the USA and B. canis vogeli in South subspecieslength of amplified of B. canis. and digested restriction enzyme fragments Africa and Europe has recently been reported [3-5]. The direct diagnosis can be made through the microscopic observation of of the B. canis canis, B. In canis France vogeli and and Italy, B. canisrossiPCR amplification subspecies of the parasites in blood smears stained with Giemsa dye, and the usingthe 18S the rRNA Taq Igene and Hinffragment (400 bp) allowed the differentiation indirect type using commercial serological packages. The main B. canis vogeli and 227 subsets of B. canis signs of the disease are characterized by fever, lethargy, vomiting, I B.enzymes, canis rossi obtaining. In the casefragments of B. canis of 203bp, canis, hemoglobinuria and anemia [1,6], whereas in more severe cases 171 bp and 26 bp for [BCC] bp and 174 bp for and since the amplified sequence does not have restriction sites Cite this article: Lira-Amaya JJ, Rojas-Martinez C, Alvarez-Martinez A, Pelaez-Flores A, Martinez-Ibañez F, et al. (2017) First Molecular Detection of Babesia canis vogeli in Dogs and Rhipicephalus sanguineus from Mexico. Arch Palliat Care 2(2): 1013. Figueroa-Millan et al. (2017) Email: Central Bringing Excellence in Open Access for both enzymes [10,11], the fragment is not digested with any of these enzymes, in this way it is possible to discriminate the digested with the restriction enzymes Taq Hinf subspecies of B. canis canis. Currently in Mexico there is little differentiationThe PCR products of the species amplified of Babesia with the sp. Forgeneric the analysis primers of were PCR information about infection of Rhipicephalus sanguineus ticks and PCR-RFLP products, 3% agarose gels wereI and electrophoresed I for the with the protozoan parasite Babesia canis, however, in other countries such as Australia, France and Tunisia, they have been able to implement the molecular characterization of the parasite intofor 45 a Tmin vector at 100 and V theand transformationvisualized in a wastransilluminator carried out with using the PCR assay [12-14]. The objective of this work was chemicallyultraviolet competentlight (UV). E. The coli obtained amplicons were cloned to optimize two methods for the molecular detection of the subspecies of the etiological agent of canine babesiosis in Mexico (recombinant) colonies were cells selected, using awhich commercial were kitscored (pGEM in from blood samples and Rhipicephalus sanguineus ticks collected -T Easy Vector System II; PROMEGA). Two representative white from dogs. boxes with LB medium and ampicillin. Purification of the plasmid MATERIALS AND METHODS DNA was performed with a commercial kit (PureYield Plasmid A total of 30 blood samples were collected in dogs by Miniprep System Protocol; PROMEGA), for later use as a template puncture of the cephalic vein in tubes with anticoagulant (EDTA). for sequencing by the fluorinated dideoxynucleotide termination The dogs were referred to a particular veterinary clinic in the sequencemethod inidentity an automated in the databases sequencer using (Applied the blastn Biosystems). application city of Cuautla, Morelos, Mexico with clinical manifestations, (The DNA sequences obtained were analyzed). by searching for including fever, anemia, lethargy or epistaxis, consistent and suspicious of canine babesiosis and history of previous exposure RESULTShttp://blast.ncbi.nlm.nih.gov/Blast/blastn AND DISCUSSION or presence of ticks. An aliquot was taken and spread on a slide analysis, with predominance of trophozoite stages in one sample was observed by microscopy for at least 20 minutes. The cellular andTwo trophozoites positive and samples merozoites were consistent identified with by Babesia microscopic spp. in packageto be fixed containing with methanol red and and white stained blood with cells Giemsa was separated dye, each fromslide the second sample. However, the parasitemias were very low to the plasma by centrifugation at 4,000 rpm and stored at -20°C until use. At the time of the physical inspection of the dogs, 18 of the PCR test with the generic primers for the B. canis species be quantified (< 0.01%), (Figure 1). DNA amplification by means taxonomic codes as R. sanguineus [15], only one of them could total of samples analyzed, whereas from the tick samples analyzed bespecimens checked byof lightticks microscopywere collected, from awhich dog infected were identified with Babesia by inallowed this study the identification it was demonstrated of two positive consistently blood samples the presence from the of canis for which a parasitemia of about 80% was estimated. a 400 bp fragment in three samples (Figure 2). The results of Extraction of the genetic material from the cellular package enzymes could demonstrate partial digestion with the enzyme Taqthe amplified products that were digested with the restriction was done with a commercial kit (ZR Genomic DNA II Kit; ZYMO subject to the reaction, which corresponded to the expected size this study, the mortar crushing method was used prior to the I in two blood samples and only one of the tick samples RESEARCH). For the DNA extraction of the ticks analyzed in of the fragments with bands of
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