Development of a real-time PCR method for rapid diagnosis of canine babesiosis and anaplasmosis

• Published in: Parasites & vectors Vol. 14; no. 1; p. 266
• Main Authors: Kivrane, Agnija, Namina, Agne, Seleznova, Maija, Akopjana, Sarmite, Capligina, Valentina, Ranka, Renate
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 20.05.2021; BioMed Central; BMC
• Subjects: Anaplasma phagocytophilum; Anaplasmosis; Arachnids; Assaying; Babesia canis; Babesiosis; Bc28.1 gene; Detection; Diagnosis; Diseases; DNA; DNA polymerase; Dogs; Domestic animals; Gene polymorphism; Genes; Genetic variability; Genotyping; Geographical distribution; Malate dehydrogenase; Maximum likelihood method; Mdh gene; Methods; Molecular diagnostic techniques; Nucleotide sequence; Parasitic diseases; Pathogens; PCR; Phylogenetics; Polymerase chain reaction; Polymorphism; Real time; Real-time PCR; Selectivity; Sequencing; Specificity; Tick-borne diseases; Variability; Veterinarians; Veterinary medicine; Latvia; Lithuania; Europe; Germany; Anaplasma phagocytophilum; Bc28.1 gene; Mdh gene; Genotyping; Real-time PCR; Babesia canis
• ISSN: 1756-3305; 1756-3305
• DOI: 10.1186/s13071-021-04756-9

Canine babesiosis and anaplasmosis, caused by Babesia canis and Anaplasma phagocytophilum, respectively, are significant tick-borne diseases in Baltic countries. Both diseases can be diagnosed on the basis of clinicopathological findings, by direct pathogen detection in blood smears or by indirect pathogen detection; however, because of high selectivity and specificity, molecular methods may be advantageous. The goal of this study was to develop a duplex real-time polymerase chain reaction (RT-PCR) method for the detection of B. canis and A. phagocytophilum in canine clinical samples. Sequence-based polymorphism analysis of genes encoding B. canis-specific merozoite surface protein Bc28.1 (Bc28.1) and A. phagocytophilum malate dehydrogenase (mdh) was performed on pathogen isolates present in Latvian domestic dogs. The obtained results were used to design a species-specific duplex RT-PCR assay. The presence of three B. canis Bc28.1 gene sequence types was revealed in canine samples with a nonuniform geographical distribution, and two types of A. phagocytophilum mdh genes were detected. The novel duplex RT-PCR assay provided correct classification of samples positive and negative for B. canis and A. phagocytophilum. The analytical sensitivity of this assay was ten gene copies/ reaction for both pathogens. A novel duplex RT-PCR molecular method was developed for the detection of B. canis and A. phagocytophilum in canine clinical samples. Sequence variability of Bc28.1 and mdh genes indicated the genetic variability of B. canis and A. phagocytophilum isolates occurring in Latvian domestic dogs.