•A novel qPCR method was able to detect and quantify cow, buffalo, goat and sheep DNA in milk and dairy products.•Established detection limit was 0.016 ng for the four species.•The limit of detection ...of cow DNA in buffalo, goat and sheep DNA samples was 0.1% (0.01 ng)•This method is able to detect and quantify adulteration between cows, buffaloes, goats and sheep dairy samples.
Species identification in dairy products has a notable importance in food traceability and adulteration control and consequently has a significant effect on the final economic value of foods. In the present study, we developed a method based on real-time quantitative PCR (qPCR) for detection and quantification of cow DNA in DNA samples from milk and dairy products from buffaloes, goats, and sheep. The qPCR reactions showed high specificity, and the amplifications only occurred to species-specific primers. The calibration curves allowed for the quantification of the amount of DNA of each species-specific primer, and the established detection limit was 0.016 ng for the four species. The detection limit of cow DNA in buffalo, goat and sheep DNA samples was 0.1% (0.01 ng). Although the present study aimed to detect and quantify cow DNA in buffalo, goat, and sheep dairy products, we believe that the qPCR assays can also be directed to differentiate and quantify goat × sheep, and/or buffalo × goat/sheep.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Cattle babesiosis is a tick-borne disease responsible for significant losses for the livestock industries in tropical areas of the world. These piroplasms are under constant control of the host ...immune system, which lead to a strong selective pressure for arising more virulent or attenuated phenotypes. Aiming to better understand the most critical genetic modifications in Babesia bovis genome, related to virulence, an in silico analysis was performed using DNA sequences from GenBank. Fourteen genes (sbp-2, sbp-4, trap, msa-1, msa-2b, msa-2c, Bv80 (or Bb-1), 18S rRNA, acs-1, ama-1, β-tub, cp-2, p0, rap-1a) related to parasite infection and immunogenicity and ITS region were selected for alignment and comparison of several isolates of Babesia bovis from different geographic regions around the world. Among the 15 genes selected for the study of diversity, only 7 genes (sbp-2, sbp-4, trap, msa-1, msa-2b, msa-2c, Bv80) and the ITS region presented sufficient genetic variation for the studies of phylogeny. Despite this genetic diversity observed into groups, there was not sufficient information available to associate molecular markers with virulence of isolates. However, some genetic groups no were correlated with geographic region what could indicate some typical evolutionary characteristics in the relation between parasite-host. Further studies using these genes in herds presenting diverse clinical conditions are required. The better understanding of evolutionary mechanisms of the parasite may contribute to improve prophylactic and therapeutic measures. In this way, we suggest that genes used in our study are potential markers of virulence and attenuation and have to be analyzed with the use of sequences from animals that present clinical signs of babesiosis and asymptomatic carriers.
•Bovine babesiosis is a tick-borne disease of cattle caused by the protozoan parasites of the genus Babesia, mainly Babesia bovis;•These parasites are under constant control of the host immune system;•There is selective pressure for the emergence of more virulent or attenuated phenotypes of B. bovis;•The molecular markers can contribute for better understanding of B. bovis evolutionary profile.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Background
High quality and quantity of messenger RNA (mRNA) are required for accuracy of gene expression studies and other RNA-based downstream applications. Since RNA is considered a labile ...macromolecular prone to degradation, which may result in falsely altered gene expression patterns, several commercial stabilizing reagents have been developed aiming to keep RNA stable for long period. However, for studies involving large number of experimental samples, the high costs related to these specific reagents may constitute a barrier.
Methods and results
In this context the present study was designed aiming to evaluate the stability of mRNA in whole bovine blood collected in EDTA tubes during storage at common fridge (4 °C). Whole blood samples were collected from six Holstein calves and submitted to RNA extraction in each different interval: immediately after blood sampling (< 2 h), at 1-day post-sampling (dps), 2 dps, 3 dps, 7 dps and 14dps intervals. RNA integrity and purity were evaluated, and RT-qPCR assays were run using seven different genes (B2M, ACTB, PPIA, GAPDH, YWHAZ, CD4 and IFN-γ) aiming to evaluate the presence of altered gene transcription during storage. All extracted RNA samples presented high purity, while optimal integrity and unaltered gene expression were observed in whole experimental group up to 3 days of storage.
Conclusion
Bovine blood RNA remained stable in K3EDTA tubes for 3 days stored at common fridge and can be successfully and accurately used for gene expression studies.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Bovine babesiosis is a tick-borne disease caused by intraerythrocytic protozoa and leads to substantial economic losses for the livestock industry throughout the world.
is considered the most ...pathogenic species, which causes bovine babesiosis in Brazil. Genomic data could be used to evaluate the viability of improving resistance against
infection level (IB) through genomic selection, and, for that, knowledge of genetic parameters is needed. Furthermore, genome-wide association studies (GWAS) could be conducted to provide a better understanding of the genetic basis of the host response to
infection. No previous work in quantitative genetics of
infection was found. Thus, the objective of this study was to estimate the genetic correlation between IB and tick count (TC), evaluate predictive ability and applicability of genomic selection, and perform GWAS in Hereford and Braford cattle. The single-step genomic best linear unbiased prediction method was used, which allows the estimation of both breeding values and marker effects. Standard phenotyping was conducted for both traits. IB quantifications from the blood of 1,858 animals were carried using quantitative PCR assays. For TC, one to three subsequent tick counts were performed by manually counting adult female ticks on one side of each animal's body that was naturally exposed to ticks. Animals were genotyped using the Illumina BovineSNP50 panel. The posterior mean of IB heritability, estimated by the Bayesian animal model in a bivariate analysis, was low (0.10), and the estimations of genetic correlation between IB and TC were also low (0.15). The cross-validation genomic prediction accuracy for IB ranged from 0.18 to 0.35 and from 0.29 to 0.32 using k-means and random clustering, respectively, suggesting that genomic predictions could be used as a tool to improve genetics for IB, especially if a larger training population is developed. The top 10 single nucleotide polymorphisms from the GWAS explained 5.04% of total genetic variance for IB, which were located on chromosomes 1, 2, 5, 6, 12, 17, 18, 16, 24, and 26. Some candidate genes participate in immunity system pathways indicating that those genes are involved in resistance to
in cattle. Although the genetic correlation between IB and TC was weak, some candidate genes for IB were also reported in tick infestation studies, and they were also involved in biological resistance processes. This study contributes to improving genetic knowledge regarding infection by
in cattle.
Parasitemia generated by
Anaplasma marginale
causes significant losses in the cattle industry. A major constraint to the effective control and management of anaplasmosis in livestock is the lack of a ...rapid and reliable diagnostic test to identify the parasite and allow for immediate therapy. In the present study, we developed a novel DNA-based assay for the detection of
A. marginale
in bovine blood samples, using loop-mediated isothermal amplification (LAMP). DNA from six cattle and hemoparasite samples (
Babesia bovis
,
Babesia bigemina
,
Anaplasma centrale
and
A. marginale
) were tested for specificity, sensitivity and cross-reactions. The developed LAMP procedures were also confirmed and compared with the qPCR method. The same gene sequence (major surface protein 1b,
msp1b
) of
A. marginale
was used to design a set of primers for the LAMP and qPCR assays. The results showed that LAMP is specific, as no positive signal was observed for the other tested hemoparasites. However, the sensitivity of the qPCR assay was ten times higher than LAMP. Our findings indicate that this LAMP method has a good sensitivity and high specificity for the detection of
A. marginale
and may have a potential application in the detection and differentiation of bovine anaplasmosis.
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•A novel LNA probe-based qPCR allowed the detection of A1 and A2 alleles from β -casein gene in bovine samples.•100% of agreement between results obtained by rHamp and LNA qPCR assays.•The limit of ...detection of A1 in A2 samples was 1% or 7.5 DNA copies.•This method is a highly sensitive and specific tool for detecting A1 and A2 alleles directly in milk.
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 β-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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Infectious bursal disease (IBD) is an immunosuppressive viral disease of chickens, associated with severe economic losses and major threats to poultry production worldwide. Disease prevention ...programs rely on unequivocal identification of the pathogen, as well as vaccination programs. This study developed a sensitive, one-step, real-time, quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay using a hydrolysis probe system for infectious bursal disease virus (IBDV, VP1 gene) detection and quantification, which was compared to other routinely used diagnostic methods. The assay successfully detected IBD reference viruses and field isolates. The absence of cross-reactivity was detected with negative samples or with other avian viruses in the analytical specificity test. The detection limit of this assay was 70 RNA copies. RT-qPCR was more sensitive in the detection of serially diluted IBDV isolates compared to virus isolation. For clinical samples, the sensitivity and specificity values of RT-qPCR compared to enzyme-linked immunosorbent assay (ELISA) were 97.5% and 100%, respectively, and compared to histopathology, these values were 100% and 93.94%, respectively. RT-qPCR can provide a simple and reliable assay for IBDV surveillance programs and for evaluation of control strategies.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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.
Proteins play different and essential roles in the human organism. Containing essential amino acids, proteins, and minerals, beef is considered the main source of protein in human nutrition. It is ...generally accepted that the protein profile is directly correlated to tenderness and beef pigmentation and is also related to its organoleptic properties. In the present work, it is demonstrated the changes in protein profile, differences of metal concentrations, and how metals bonded to proteins can vary during the ripening phase, evaluated over a 14-day beef aging period. The proposed extraction procedure indicated 85% efficiency, preserving the metal-protein structure. Seventeen protein bands were detected using SDS-PAGE, and a 43-kDa band was found to be the most intense. The arrangement of SDS-PAGE and SEC-ICP-MS results indicated the possible links between minerals and organic functional molecules, such as Ca to troponin, Cu and Zn to albumin, and Fe to myoglobin and hemoglobin.
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