Babesia bovis and Babesia bigemina infection levels estimated by qPCR in Angus cattle from an endemic area of São Paulo state, Brazil

Abstract

The levels of infection by Babesia bovis and Babesia bigemina were estimated by absolute quantification through the quantitative PCR technique (qPCR). Fifty-one contemporaneous Angus cattle were evaluated on two occasions. The number of standard female Rhipicephalus microplus ticks present on the left side of the body was counted and blood samples were drawn from the tail vein into tubes containing the anticoagulant EDTA. The blood samples were submitted to DNA extraction and used to quantify the number of copies (NC) of DNA from B. bovis and B. bigemina by qPCR. The data on tick count and number of DNA copies were transformed for normalization and analyzed by a mixed model method. A multivariate model with repeated measures of the same animal, including the effects of collection, parasite species and their interaction, was used. The repeatability values were obtained from the matrix of (co)variances and were expressed for each species. The correlations between the counts of different species on the same animal, in the same collection or different collections, were also estimated. The results showed the qPCR could distinguish the two between infection by the two Babesia species. Infection levels by B. bovis and B. bigemina were detected in 100% and 98% of the animals, respectively. Significant differences were found (P<0.05) between the NC of the two Babesia species, B. bovis 1.49±0.07 vs. B. bigemina 0.82±0.06. Low repeatabilities were found for the counts of R. microplus and NC of B. bovis and B. bigemina: 0.05, 0.10 and 0.02, respectively. The correlations between R. microplus count and NC of B. bovis and B. bigemina were both very near zero. However, an association was observed between the NC of the two species, with a correlation coefficient of 0.30 for measures from the same collection. The absence of associations between the quantity of DNA from B. bovis and B. bigemina and the tick counts suggests that the variation of parasitemia by the hemoparasites did not depend on the tick infestation levels at the moment of each collection. The repeatability values estimated indicate that under the study conditions, the variations in the tick infestation levels and of parasitemia by B. bovis and B. bigemina depend more on factors related to each collection than on intrinsic factors of the animal.

Neither quantification by qPCR nor quantitative Elisa can be used to discriminate Angus cattle for resistance/susceptibility to Babesia bovis

“…All the animals were positive for B. bovis in both collections. The high sensitivity of qPCR to assess the level of infection by B. bovis has also been reported by Bilhassi et al (2014) and Giglioti et al (2016).…”

“…With respect to the fluctuation in the quantity of DNA from the hemoparasite in the animals' blood, it is hard to determine the cause. Considering the repeatability values found for the NC metric, we can infer that a large part of this variation can be attributed to environmental factors that act on the animal at the collection time, and only a small part can be attributed to the animals' ability to maintain low infection levels, as also verified by Giglioti et al (2016). Although tick infestation can vary between collections, previous studies have shown no association between tick count and NC (Giglioti et al, 2016) and indicate this cannot be reported as the cause of the variation.…”

“…Considering the repeatability values found for the NC metric, we can infer that a large part of this variation can be attributed to environmental factors that act on the animal at the collection time, and only a small part can be attributed to the animals' ability to maintain low infection levels, as also verified by Giglioti et al (2016). Although tick infestation can vary between collections, previous studies have shown no association between tick count and NC (Giglioti et al, 2016) and indicate this cannot be reported as the cause of the variation. The greater repeatability of the serological tests in relation to quantification of DNA of parasites in the bloodstream shows the existence, at least in peripheral blood, of a greater individual variation in the NC than in the level of antibodies.…”

“…No studies have been published reporting the repeatability and correlation between the Babesia infection levels quantified by qPCR and ELISA. The study by Giglioti et al (2016) was the first to estimate the repeatability of B. bovis infection levels in Angus cattle quantified by qPCR. The authors observed low repeatability for both collections analyzed.…”

Estimates of repeatability and correlations of hemoparasites infection levels for cattle reared in endemic areas for Rhipicephalus microplus

“…The diagnosis of these diseases in clinically affected animals is usually made through of microscopic examination of peripheral blood smears (Bock et al, 2004;Calder et al, 1996;Sahinduran, 2012). The detection of carrier animals requires the use of more sensitive techniques as PCR and qPCR Oliveira-Sequeira et al, 2005;Buling et al, 2007;Ramos et al, 2011;Bilhassi et al, 2014;Giglioti et al, 2016;Giglioti et al, 2017).…”