Monitoring Babesia bovis infections in cattle by using PCR-based tests

Abstract

The sensitivity and specificity of PCR tests based on the small-subunit rRNA gene sequence of Babesia bovis were compared in a blind study of experimentally infected cattle with the corresponding parameters of the complement fixation (CF) test currently used in the United States to screen for bovine babesiosis. Cattle were experimentally infected with a single inoculum of a cloned laboratory strain of B. bovis. Blood samples were collected and tested over a period covering from the day of infection to 10 months postinfection. The level of parasitemia (percent infected erythrocytes) present in each sample was estimated from test results and was plotted as a function of time postinfection. These data are the first describing the course of infection by methods capable of detecting parasitemias in the range of 10 ؊7 %, which frequently occur in the carrier state. Parasitemias in the samples tested strongly influenced the sensitivity and negative predictive value of the PCR-based tests which varied with time postinfection. The average sensitivities of the three PCR-based tests for B. bovis ranged from 58 to 70% for a single determination, while the sensitivity of the CF test was only 6%. Both PCR-based and CF tests for B. bovis had high specificity values ranging from 96 to 100%.

Simultaneous Detection of Bovine Theileria and Babesia Species by Reverse Line Blot Hybridization

“…Furthermore, in experimental animals, T. annulata could be detected simultaneously with T. mutans, T. orientalis, B. divergens, B. bovis, and B. bigemina. After Imidocarb treatment the B. bovis signal was completely lost, confirming the earlier findings of Calder et al (6). Our B. bigemina results are similar to those obtained with the previously published B. bigemina-specific PCR (15).…”

“…The detection limit of the T. annulata PCR based on the Tams1 gene (13) was 4.8 ϫ 10 Ϫ5 %, which is highly comparable with the sensitivity found for the RLB. For B. bovis, a detection of 10 Ϫ7 to 10 Ϫ8 % parasitemia was achieved by using 18S PCR and a specific probe followed by very sensitive phosphorimager detection (6). These investigators demonstrated that B. bovis parasitemia fluctuates and sometimes even escapes detection by their assay.…”

“…PCR detection of T. parva using repetitive DNA sequences or genespecific primers has also been developed (2). Sensitive PCR methods are available for B. bovis (6) and B. bigemina (15) but not yet for B. divergens. Assays to differentiate Theileria species on the basis of their rRNA genes have been described by Allsopp et al (1) and Bishop et al (3), but these assays did not include all Theileria species.…”

Molecular and serological detection of Babesia bovis- and Babesia bigemina-infection in bovines and water buffaloes raised jointly in an endemic field

“…The size of generated bands is indicated. (Calder et al, 1996;Gubbels et al, 1999). The newly presented nPCR assays may thus significantly reduce or eliminate false negative test results as compared to previously reported assays.…”

“…In contrast, PCR-based diagnostics has the potential to detect directly the parasite with high specificity and sensitivity (Mosqueda et al, 2007(Mosqueda et al, , 2012. Notwithstanding, it has been demonstrated that parasites of carrier animals often escape detection even from highly sensitive PCR-based diagnostic methods, posing a challenge to further optimize nPCR assay design and format in order to maximize sensitivity (Calder et al, 1996;Gubbels et al, 1999;Oliveira-Sequeira et al, 2005;Martins et al, 2008).…”