Detection of Babesia rossi genotypes using real-time PCR

Abstract

Babesia rossi is the most virulent Babesia species in dogs occurring in South Africa and is associated with severe clinical manifestation and mortalities. Babesia rossi is highly pathogenic and reacting dogs requires treatment to prevent mortalities. Mild, uncomplicated forms of the disease are effectively treated with antibabesial drugs. Complicated forms of the disease are difficult to treat with high mortality rate.This results in the disease being of economic importance in South Africa. There is a lack of information regarding the relationship between parasite genotype and disease phenotype. The broad objective of this study is to detect B. rossi genotypes using real-time PCR. This test is a method that can be used to monitor amplicon formation throughout the PCR reaction and to estimate the initial concentration of target DNA in samples. Polymerase chain reaction, sequencing and phylogenetic analysis were used to determine the genotype detection of Babesia rossi isolated from infected dogs in South Africa. The correlation between the parasite genotype and disease phenotype was also investigated. Correlation between B. rossi Erythrocyte Membrane Antigen (BrEMA1) genotypes and age of dogs was studied in 44 cases. A total of 101 blood samples were tested using the reverse line blot (RLB) assay. Ninety six percent hybridized to the Babesia rossi species-specific probe. Our findings demonstrate that the most encountered (BrEMA1) genotype is genotype29, followed by genotype28 and genotype19, with genotype29 associated with most of the severe clinical signs diagnosed compared to genotype19. The number of cases caused by genotype19 was low, constituting 22% of the cases. This is comparable with the 2009 report where genotype19 appeared highly prevalent and virulent, whereas the prevalence of genotype28/29 appeared moderate. In this dissertation, we present the first report on the detection of an amplification product of BrEMA1 genotypes using real-time PCR. Samples which were below the detectable limit of conventional PCR and could not be sequenced probably due to low parasitaemia were also used and real-time PCR provided the ability to detect B. rossi positive animals. This was able to detect 10 BrEMA1 genotypes. However, it was not reliable enough in differentiating between various BrEMA1 genotypes. When evaluating the relationship between BrEMA1 genotypes, clinical manifestation and age of the dogs, collapse was found to be a poor prognostic sign in dogs with babesiosis. Genotype29 was associated with most of the collapsed cases and with high number of the dogs that died. Although B. rossi can infect dogs of all ages, young dogs showed to be more susceptible to canine babesiosis than older dogs. This is in agreement with the survey carried out from the Onderstepoort Veterinary Academic Hospital (OVAH) in 1994. Since B. rossi is the most pathogenic species of the large babesias of dogs, the ability to manage the disease is dependent on rapid detection of the organism. Real-time PCR test is indeed a quicker method to confirm diagnoses of B. rossi infected dogs. It can detect B. rossi infection at a low DNA concentration (0.185 ng/μl) which provides a major advantage in detecting B. rossi infection in field blood samples.