Correlations and repeatability between Babesia spp. infection levels using two dairy cattle breeding systems.

Babesia bovis and Babesia bigemina are tick-transmitted piroplasms that cause severe damage to the livestock industry in tropical regions of the world. Recent studies demonstrated differences in infection levels of these haemoparasites among bovine breeds and variation between individual cows regarding resistance to these diseases. This study aimed to estimate the repeatability and correlations between B. bovis and B. bigemina using two cattle breeding systems, an individual system (IS) and a collective paddock system (CPS). All animals were Holstein breed, and the levels of B. bovis and B. bigemina in blood samples were estimated by quantitative polymerase chain reaction (qPCR). The estimated correlations for the B. bigemina and B. bovis DNA copy number for IS and CPS were moderate and high, respectively, whereas repeatability estimates for both systems and both Babesia species were moderate. Although we cannot infer that the type of rearing system directly influenced the correlation and repeatability coefficients, it appears that the bovine parasitemia burden may be dependent on (or determine) the parasitemia burden on ticks because the bovines remained in the same place for a longer time in both systems. Thus, the babesiosis infection levels of the ticks may have been uniform, a phenomenon that also ensures greater uniformity in cattle infection. This factor may have favored the occurrence of infected ticks leading to higher repeatability estimates and correlations. Our study confirms high variability in resistance/susceptibility between breeds, and the high correlations found may be linked to this characteristic and the most intensive breeding type of dairy cattle. Besides, under the present study conditions, the estimated correlations suggest that measuring an infection level of one Babesia species can predict the level of infection of the other.

Selective control of Rhipicephalus microplus in a dairy cattle herd from different genetic groups.

Alternatives for Rhipicephalus microplus control are needed in the light of its resistance to acaricides. One of the ways to decrease the use of acaricides in a herd is selective control (SC). In the present study, SC was evaluated in a dairy herd consisting of different genetic groups: Holstein, Jersey, crossbreed and Girolando. Ticks were counted in the right anterior third region on around 90 cows, totaling nine evaluations at intervals of 21 days. Commercial pour-on acaricide was applied only when the infestation was greater than or equal to eight ticks larger than 4 mm in the anterior third region. Tick counts were transformed into log10 and analyzed using mixed models. There was significant difference among groups: Holstein had the highest averages of tick numbers, as expected, although 34.3% did not receive tick treatment. In the other groups, SC reduced the use of acaricides by 79.1% for crossbreed, 81.5% for Jersey and 94.9% for Girolando. The criterion used for applying the acaricide successfully kept the tick population under control. The great advantage of SC was savings to the system, without harming the animals, in addition to generate fewer residues in the animals and in the environment.

Novel LNA probe-based assay for the A1 and A2 identification of ß-casein gene in milk samples.

The rising consumption of A2 milk and its derivatives in recent years has garnered attention from both consumers and producers, mainly due its possible health benefits, such as enhanced digestion and easier absorption. Thus, a novel real-time PCR using a combination of locked nucleic acid modified (LNA) conjugated probes was developed to genotype A1 and A2 alleles of ß-casein gene (CSN2) and to detect and quantify the A1 presence in A2 samples. The limit of detection for each probe (A1 and A2) was evaluated using decreasing serial dilutions. Besides, the sensitivity of A1 allele detection in the A2 samples was also tested. The limits of detection of A1 and A2 alleles were 6 copies, while for A1 allele detection in A2 samples was 7.5 copies (1%). The LNA-probe based method was found to be rapid, robust, highly sensitive, cost effective, and can be employed as screening test to certificate the A2 dairy products.

Semi-quantitative evaluation of Babesia bovis and B. bigemina infection levels estimated by HRM analysis.

Bovine babesiosis is economically the most important arthropod-borne disease of cattle worldwide. The most significant damage caused by bovine babesiosis is attributed to Babesia bovis due to its higher pathogenicity. This study aimed to develop a real-time PCR method followed by HRM (high-resolution melting) analysis for the simultaneous detection of B. bovis and B. bigemina, enabling a semi-quantitative analysis of Babesia levels using a single-tube reaction. The HRM was compared with real-time PCR using species-specific hydrolysis probes. The HRM analysis allowed to differentiate both Babesia species and was sensitive in the detection and differentiation of 10% for each Babesia species in the sample. Our results suggest the use of this method to estimate the prevalence of infections by B. bovis or B. bigemina as an alternative to the methods of absolute quantification by real-time PCR since it neither requires precise estimates of the number of DNA loads nor the construction of calibration curves. The simultaneous detection of the two Babesia species can be used to characterise the infection levels in cattle populations from different geographical regions, allowing a better control of these diseases.

New high-sensitive rhAmp method for A1 allele detection in A2 milk samples.

Cows' milk may contain two types of ß-casein: A1 and A2. A1 digestion is associated with the release of ß-casomorphine-7 peptide, which can cause adverse gastrointestinal effects. Two methods - high-resolution melting (HRM) and rhAmp® SNP genotyping - were developed to identify the ß-casein gene (CSN2) A1 and A2 alleles directly in milk. DNA milk samples from 45 animals were examined and 10 samples were also sequenced to confirm the accuracy of the assays. The analytical sensitivities of both strategies for A1 allele identification were evaluated by testing decreasing dilutions of A1 allele DNA copies (500 - 5 copies) in the A2 sample. The limits of detection for A1 in A2 samples were 10% (100 copies) and 2% (10 copies) for HRM and rhAmp, respectively. Both techniques were specific, differentiating between A1 and A2 alleles. However, we recommend rhAmp genotyping testing over HRM because of its enhanced sensitivity for A1.

Cloning and endogenous expression of a Eucalyptus grandis UDP-glucose dehydrogenase cDNA.

UDP-glucose dehydrogenase (UGDH) catalyzes the oxidation of UDP-glucose (UDP-Glc) to UDP-glucuronate (UDP-GlcA), a key sugar nucleotide involved in the biosynthesis of plant cell wall polysaccharides. A full-length cDNA fragment coding for UGDH was cloned from the cambial region of 6-month-old E. grandis saplings by RT-PCR. The 1443-bp-ORF encodes a protein of 480 amino acids with a predicted molecular weight of 53 kDa. The recombinant protein expressed in Escherichia coli catalyzed the conversion of UDP-Glc to UDP-GlcA, confirming that the cloned cDNA encodes UGDH. The deduced amino acid sequence of the cDNA showed a high degree of identity with UGDH from several plant species. The Southern blot assay indicated that more than one copy of UGDH is present in Eucalyptus. These results were also confirmed by the proteomic analysis of the cambial region of 3- and 22-year-old E. grandis trees by 2-DE and LC-MS/MS, showing that at least two isoforms are present. The cloned gene is mainly expressed in roots, stem and bark of 6-month-old saplings, with a lower expression in leaves. High expression levels were also observed in the cambial region of 3- and 22-year-old trees. The results described in this paper provide a further view of the hemicellulose biosynthesis during wood formation in E. grandis.

Cloning and endogenous expression of a Eucalyptus grandis UDP-glucose dehydrogenase cDNA

UDP-glucose dehydrogenase (UGDH) catalyzes the oxidation of UDP-glucose (UDP-Glc) to UDP-glucuronate (UDP-GlcA), a key sugar nucleotide involved in the biosynthesis of plant cell wall polysaccharides. A full-length cDNA fragment coding for UGDH was cloned from the cambial region of 6-month-old E. grandis saplings by RT-PCR. The 1443-bp-ORF encodes a protein of 480 amino acids with a predicted molecular weight of 53 kDa. The recombinant protein expressed in Escherichia coli catalyzed the conversion of UDP-Glc to UDP-GlcA, confirming that the cloned cDNA encodes UGDH. The deduced amino acid sequence of the cDNA showed a high degree of identity with UGDH from several plant species. The Southern blot assay indicated that more than one copy of UGDH is present in Eucalyptus. These results were also confirmed by the proteomic analysis of the cambial region of 3- and 22-year-old E. grandis trees by 2-DE and LC-MS/MS, showing that at least two isoforms are present. The cloned gene is mainly expressed in roots, stem and bark of 6-month-old saplings, with a lower expression in leaves. High expression levels were also observed in the cambial region of 3- and 22-year-old trees. The results described in this paper provide a further view of the hemicellulose biosynthesis during wood formation in E. grandis.