R. Giglioti

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

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.

Efficacy of 11 Brazilian essential oils on lethality of the cattle tick Rhipicephalus (Boophilus) microplus

Herbal extracts have been investigated as an alternative for parasite control, aiming to slow the development of resistance and to obtain low-cost biodegradable parasiticides. The goal of this study was to evaluate the efficacy, in vitro, of 11 essential oils from Brazil on reproductive efficiency and lethality of the cattle tick Rhipicephalus (Boophilus) microplus. The effects of oils extracted from Curcuma longa, Zingiber officinale, Lippia alba, Lippia gracilis, Lippia origanoides, Lippia sidoides, Mentha arvensis, Mentha piperita, Croton cajucara (white and red), and Croton sacaquinha on ticks were investigated by the Immersion Test with Engorged Females (ITEF) and the modified Larval Packet Test (LPT). Distilled water and 2% Tween 80 were used as control treatments. Chemical analysis of the oils was done with gas chromatography coupled with mass spectrometry. Analysis of the in vitro tests using Probit (SAS program) allowed the calculation of lethal concentrations (LCs). Lower reproductive efficiency indexes and higher efficacy percentages in the ITEF were obtained with the oils extracted from C. longa (24 and 71%, respectively) and M. arvensis oils (27 and 73%, respectively). Lower LC50 was reached with C. longa (10.24 mg/mL), L. alba (10.78 mg/mL), M. arvensis (22.31 mg/mL), L. sidoides (27.67 mg/mL), and C. sacaquinha (29.88 mg/mL) oils. In the LPT, species from Zingiberaceae and Verbenaceae families caused 100% lethality at 25 mg/mL, except for L. sidoides. The most effective oils were from C. longa, L. gracilis, L. origanoides, L. alba, and Z. officinale. The LC50 and LC90 were, respectively: 0.54 and 1.80 mg/mL, 3.21 and 7.03 mg/mL, 3.10 and 8.44 mg/mL, 5.85 and 11.14 mg/mL, and 7.75 and 13.62 mg/mL. The efficacy was directly related to the major components in each essential oil, and the oils derived from Croton genus presented the worst performance, suggesting the absence of synergistic effect among its compounds. Since C. longa, containing 62% turmerone, was the one most efficient against ticks, this compound may be potentially used for tick control, but further research is needed, especially to assess toxicity of these compounds to the host. These new studies, together with the results presented here, may provide a strong rationale for designing pre-clinical and clinical studies with these agents.

Standardization of a SYBR Green Based Real-Time PCR System for Detection and Molecular Quantification of Babesia bovis and B. bigemina in Water Buffaloes (Bubalus bubalis)

Water buffalo ( Bubalus bubalis ) is a potential reservoir for Babesia bovis and B. bigemina in tropical regions , but the epidemiological evidence of their reservoir competence is limited, especially due to the lack of diagnostic tests capable of detecting and quantifying the low-level parasitemia present in the carrier animals. In this paper we present the standardization process of a SYBR Green based real-time PCR system (qPCR), consisting of two single qPCR assays, for the detection and quantification of B. bovis and/or B. bigemina . Both assays were optimized in similar protocols, including reagent concentrations and thermocycling parameters, so it is possible its use as a multiple qPCR in a single run. Both single assays showed a suitable analytical performance, especially by allowing detection of a greater number of carrier animals when compared with nested PCR assays (nPCR) against a reference panel of 60 DNA samples extracted from blood of both, infected- and non-infected buffaloes. Furthermore, a mathematical algorithm to convert the qPCR outcomes in percent of infected red blood cell was used, and was found that the estimated parasitemia in carrier buffaloes within the reference sample panels were close to those described in carrier cattle. This method could be a useful tool for epidemiological studies on the participation of the bubaline specie in the epidemic process of bovine babesiosis.