•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.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The main objective of cattle breeders in tropical and subtropical regions is to acquire animals with taurine-productive traits adapted to the broad weather range of these regions. However, one of the ...main challenges on using taurine genetics in these areas is the high susceptibility of these animals to tick-borne diseases. Consequently, the present study evaluated from 10 November 2021–19 April 2022, the over 13 assessments, the Babesia bovis and Babesia bigemina DNA loads and the IgG anti-B. bovis and anti-B. bigemina levels in Angus (n = 17, 100% Taurine) and Ultrablack (n = 14, ∼82% taurine and 18% Zebu) calves. Data were analyzed using a multivariate mixed model with repeated measures of the same animal including the fixed effects of evaluation, genetic group, sex, Babesia spp., and their interactions. The repeatability values were estimated from the (co)variances matrix and expressed for each species. The correlations between the DNA loads (CNlog) and IgG titers (S/P) values for the two species were also estimated using the same model. Regarding the specific IgG antibody titers for both Babesia spp., no significant differences were observed between the two genetic groups. However, for B. bovis and B. bigemina DNA loads, Ultrablack calves presented significantly higher values than Angus calves. Under the conditions evaluated in this study, our findings suggest that the low percentage of Zebu genetic in the Ultrablack breed was insufficient to improve resistance against babesiosis. Further studies must demonstrate if the low percentages of Zebu genetics in Taurine breeds can modify the susceptibility to babesiosis infections.
•qPCR enabled to determine the Babesia infection levels in Angus and Ultrablack calves•Ultrablack calves showed higher infection levels than Angus calves•Zebu blood in Ultrablacks calves was not enough to enhance resistance to babesiosis
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The rising consumption of buffalo milk derivatives in recent years has highlighted the search for buffaloes in Brazilian livestock farming. These products have greater added value compared with ...common dairy milk products. Consequently, addition of variable amounts of cows' milk during manufacture of buffalo dairy products may occur, which constitutes fraud by product adulteration. Thus, the present study developed and standardised a DNA extraction protocol for application on different dairy derivatives and two methods based on real-time PCR for fraud identification: high-resolution melting (HRM) and rhAmp® SNP system. The extraction method allowed to extraction of DNA from eleven different dairy products. The sensitivity of the rhAmp method (1%) was five times higher than HRM assay (5%) and may be considered a better choice for identification of product adulteration when high sensitivity levels are required.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
•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.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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.
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.
Full text
Available for:
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 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.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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.
Full text
Available for:
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
Gastrointestinal parasites cause high economic losses in small ruminant production and this problem has worsened because helminths have developed resistance to many of the commercial anthelmintics. ...In the state of São Paulo, studies have detected this problem in approximately 90% of the farms. Haemonchus contortus is the most prevalent and resistant helminth, and causes the greatest loss to small ruminant production. Albendazole (ABZ) is an anthelmintic which, although the related efficacy is below the expected values, is still widely used for the control of gastrointestinal parasites. The identification of the sensitivity and/or resistance profile of the strain for a specific commercial anthelmintic is an essential step for standardization of in vitro diagnosis tests. The objective of this work was to evaluate the sensitivity/resistance profile of two strains of H. contortus against ABZ at a concentration of 0.1 mg mL-1 in the in vitro egg hatch test. One of the two tested strains is resistant (previously validated as resistant to albendazole, ivermectin, levamisole, trichlorfon and closantel). This strain was compared with a sensitive strain (which requires validation). About 100 eggs of each strain were exposed to ABZ diluted in distilled water at pH 7. Each strain was tested with 6 replicates in a 48-well polystyrene plate and incubated for 24 hours at a temperature of 27°C. The hatchability was evaluated under an inverted microscope to count the number of hatched and non-hatched eggs. Data were evaluated by analysis of variance analysis to compare the efficacy of the strains. Data presented normality (Kolmogorov-Smirnov test) and homogeneity of variance (Levene test). The resistant strain was different from the susceptible strain (P<0.01). The mean efficacy of ABZ on the susceptible strain was 85.25 ± 4.75% and the mean efficacy on the resistant strain was 12.81 ± 7.48% at 0.1 mg mL-1. These data indicate the resistence of one susceptible and one resistant strain, which is important to be considered as positive and negative standard controls for the development of commercial tests to detect anthelmintic resistance to ABZ.
This study aimed to evaluate the efficacy of albendazole (ABZ) at different pH levels and different concentrations. ABZ is a broad-spectrum anthelmintic of the benzimidazole class, widely used to ...control gastrointestinal nematodes in animal production. Studies have shown that at low pH values the availability rate of ABZ increases. In order to evaluate whether pH could influence the efficacy of ABZ, in vitro hatchability tests were performed. The ABZ was diluted in distilled water previously prepared at the intended pH levels (pH 5, pH 5.5, pH 6, pH 6.5, pH 7, pH 7.5 and pH 8), as confirmed by a pH meter. The eggs were collected from artificially infected with Haemonchus contortus, a strain sensitive to ABZ. For each pH solution evaluated, different concentrations of ABZ (0.1, 0.3, 0.6, 1.2 and 2.5 mg mL-1) were tested. Approximately 100 eggs were exposed to these concentrations and pHs, in six replicates, using a 48-well cell culture plates. The numbers of hatched and intact eggs were counting after incubation for 24 h at 27 °C. To verify if there was a difference between the tested pH values and concentrations, analysis of variance and homogeneity of variance (Levene test) were performed. There was no difference between the pHs tested at different concentrations of ABZ (P>0.05). The mean efficacy of the negative control (considering all pHs) was 10.36 ± 6.85% and the mean ABZ efficacy at the concentration 2.5 mg mL-1 (considering all pHs tested) was 90.86 ± 4.19%. We conclude that solutions of ABZ diluted at different pHs did not alter the anthelminthic efficacy at the different concentrations tested.
PDF
