Detection of carbon nanotubes in bovine raw milk through Fourier transform Raman spectroscopy.

The potential use of carbon-based methodologies for drug delivery and reproductive biology in cows raises concerns about residues in milk and food safety. This study aimed to assess the potential of Fourier transform Raman spectroscopy and discriminant analysis using partial least squares (PLS-DA) to detect functionalized multiwalled carbon nanotubes (MWCNT) in bovine raw milk. Oxidized MWCNT were diluted in milk at different concentrations from 25.00 to 0.01 µg/mL. Raman spectroscopy measurements and PLS-DA were performed to identify low concentrations of MWCNT in milk samples. The PLS-DA model was characterized by the analysis of the variable importance in projection (VIP) scores. All the training samples were correctly classified by the model, resulting in no false-positive or false-negative classifications. For test samples, only one false-negative result was observed, for 0.01 µg/mL MWCNT dilution. The association between Raman spectroscopy and PLS-DA was able to identify MWCNT diluted in milk samples up to 0.1 µg/mL. The PLS-DA model was built and validated using a set of test samples and spectrally interpreted based on the highest VIP scores. This allowed the identification of the vibrational modes associated with the D and G bands of MWCNT, as well as the milk bands, which were the most important variables in this analysis.

Sex affects immunolabeling for histone 3 K27me3 in the trophectoderm of the bovine blastocyst but not labeling for histone 3 K18ac.

The mammalian embryo displays sexual dimorphism in the preimplantation period. Moreover, competence of the embryo to develop is dependent on the sire from which the embryo is derived and can be modified by embryokines produced by the endometrium such as colony stimulating factor 2 (CSF2). The preimplantation period is characterized by large changes in epigenetic modifications of DNA and histones. It is possible, therefore, that effects of sex, sire, and embryo regulatory molecules are mediated by changes in epigenetic modifications. Here it was tested whether global levels of two histone modifications in the trophectoderm of the bovine blastocyst were affected by sex, sire, and CSF2. It was found that amounts of immunolabeled H3K27me3 were greater (P = 0.030) for male embryos than female embryos. Additionally, labeling for H3K27me3 and H3K18ac depended upon the bull from which embryos were derived. Although CSF2 reduced the proportion of embryos developing to the blastocyst, there was no effect of CSF2 on labeling for H3K27me3 or H3K18ac. Results indicate that the blastocyst trophoctoderm can be modified epigenetically by embryo sex and paternal inheritance through alterations in histone epigenetic marks.

Changes in endometrial transcription of TLR2, TLR4, and CD14 during the first-week postpartum in dairy cows with retained placenta.

Changes in the endometrial transcription of pattern recognition receptors may increase the susceptibility to postpartum uterine infections in Holstein cows with retained placenta. To test this hypothesis, nine cows with retained placenta and ten cows without retained placenta were submitted to endometrial biopsies at the first and seventh days postpartum. Cows were monitored weekly with clinical and gynecological examinations until 42 days postpartum. Samples of the uterine contents were collected weekly for aerobic bacteria isolation. All cows had endometrial transcription of Toll-like receptors (TLRs) 1/6, 2, 4, 5, and 9; nucleotide-binding oligomerization domain (NOD)-like receptors 1 and 2; and the coreceptors cluster of differentiation 14 (CD14) and myeloid differentiation protein-2 (MD-2), as measured on the first and seventh days postpartum. Escherichia coli was the most common bacterium isolated from the uterine contents of cows with or without retained placenta until 21 days postpartum. Transcription levels of TLR2, TLR4, and CD14 in Holstein cows with retained placenta significantly decreased (P < 0.05) between the first and the seventh day postpartum. Conversely, cows without retained placenta did not have any significant changes in transcription levels between these time points.

Ovarian steroids do not affect bovine endometrial cytokine or chemokine responses to Escherichia coli or LPS in vitro.

The risk of bacterial infection of the endometrium causing uterine disease in cattle is increased in the progesterone-dominated luteal phase of the ovarian cycle, while oestrogens or oestrus are therapeutic or protective against disease. The first line of defence against bacteria, such as Escherichia coli that cause inflammation of the endometrium, is the innate immune system, which recognises bacterial lipopolysaccharide (LPS). This study tested the hypothesis that cyclic variation in ovarian hormone concentrations alters innate immune responses within the bovine endometrium. Ex vivo organ cultures of endometrium, and in vitro cultures of endometrial epithelial and stromal cells, and peripheral blood mononuclear cells (PBMCs), all mounted inflammatory responses to E. coli or LPS, with secretion of inflammatory mediators interleukin 1ß (IL1ß), IL6 and IL8, and increased expression of mRNA encoding IL1B, IL6, CXCL8 (IL8) and CCL5. However, these inflammatory responses, typical of innate immunity, were not affected by the stage of ovarian cycle in which the endometrium was collected for organ culture, or by exogenous oestradiol or progesterone. Although a dexamethasone-positive control reduced inflammation stimulated by E. coli or LPS, treatment with oestradiol or progesterone, or inhibitors of oestradiol or progesterone nuclear receptors, did not affect endometrial cell or PBMC secretion of IL1ß, IL6 or IL8, or IL1B, IL6, CXCL8 and CCL5 gene expression. In conclusion, the stage of the oestrus cycle or ovarian steroids did not modulate the innate immune response in the bovine endometrium in vitro.

Economic losses due to bovine brucellosis in Brazil / Perdas econômicas devidas à brucelose bovina no Brasil

Brucellosis is an important zoonosis of worldwide distribution. Reliable epidemiologic brucellosis data covering approximately 90% of the cattle population in Brazil have been recently published. Therefore, considering the scarcity of information regarding the economic impact of bovine brucellosis in Brazil, the goal of this study was to estimate economic impact of brucellosis on the Brazilian cattle industry. Several parameters including abortion and perinatal mortality rates, temporary infertility, replacement costs, mortality, veterinary costs, milk and meat losses were considered in the model. Bovine brucellosis in Brazil results in an estimated loss of R$ 420,12 or R$ 226,47 for each individual dairy or beef infected female above 24 months of age, respectively. The total estimated losses in Brazil attributed to bovine brucellosis were estimated to be approximately R$ 892 million (equivalent to about 448 million American dollars). Every 1% increase or decrease in prevalence is expected to increase or decrease the economic burden of brucellosis in approximately 155 million Reais.

A brucelose é uma zoonose de importância mundial. Recentes dados epidemiológicos dessa doença foram obtidos por meio de estudos que abrangeram em torno de 90% do rebanho bovino do Brasil. Observa-se escassez de informações sobre o impacto econômico causado pela brucelose bovina, portanto, o objetivo do presente estudo foi estimar as perdas econômicas na pecuária brasileira. Gastos relacionados com as ocorrências de abortos, natimortos, subfertilidade, descartes involuntários, mortalidade, intervenções veterinárias, diminuição da produção de leite e de carne, foram considerados nos cálculos. As perdas devidas à brucelose bovina no Brasil foram estimadas em R$ 420,12 ou R$ 226,47 para cada fêmea infectada acima de 24 meses de idade em rebanhos de leite ou corte, respectivamente. O prejuízo total estimado foi de, aproximadamente, R$ 892 milhões (equivalentes a $ 448 milhões de dólares americanos). A cada 1% de variação na prevalência, estima-se a variação de 155 milhões de reais no custo da brucelose bovina no Brasil.

NRAMP1 3' untranslated region polymorphisms are not associated with natural resistance to Brucella abortus in cattle.

The NRAMP1 gene encodes a divalent cation transporter, located in the phagolysosomal membrane of macrophages, that has been associated with resistance to intracellular pathogens. In cattle, natural resistance against brucellosis has been associated with polymorphisms at the 3' untranslated region (3'UTR) of the NRAMP1 gene, which are detectable by single-strand conformational analysis (SSCA). This study aimed to evaluate the association between NRAMP1 3'UTR polymorphisms and resistance against bovine brucellosis in experimental and natural infections. In experimentally infected pregnant cows, abortion occurred in 42.1% of cows with a resistant genotype (SSCA(r); n = 19) and in 43.1% of those with a susceptible genotype (SSCA(s); n = 23). Furthermore, no association between intensity of pathological changes and genotype was detected. In a farm with a very high prevalence of bovine brucellosis, the percentages of strains of the SSCA(r) genotype were 86 and 84% in serologically positive (n = 64) and negative (n = 36) cows, respectively. Therefore, no association was found between the NRAMP1-resistant allele and the resistant phenotype in either experimental or naturally occurring brucellosis. To further support these results, bacterial intracellular survival was assessed in bovine monocyte-derived macrophages from cattle with either the resistant or susceptible genotype. In agreement with our previous results, no difference was observed in the rates of intracellular survival of B. abortus within macrophages from cattle with susceptible or resistant genotypes. Taken together, these results indicate that these polymorphisms at the NRAMP1 3'UTR do not affect resistance against B. abortus in cattle and that they are therefore not suitable markers of natural resistance against bovine brucellosis.

Frequency of bovine Nramp1 (Slc11a1) alleles in Holstein and Zebu breeds.

Natural resistance against brucellosis in cattle is linked to the Nramp1 gene, which encodes a divalent cation transporter that localizes in the phagolysosome membrane in macrophages. Nramp1 gene in mouse plays a critical role in innate immunity favoring bacterial killing by macrophages in addition to its influence on adaptative immunity. Polymorphisms at the bovine Nramp1 3' untranslated region (3'UTR), detectable by Single Strand Conformational Analysis (SSCA), are associated with natural resistance against brucellosis. Such polymorphisms are associated with variation in the number of GT repeats. This study compared the frequency of Nramp1 3'UTR polymorphisms between Zebu and European bovine breeds. Eighty-one Holsteins (Bos taurus taurus) and 167 Zebu (Bos taurus indicus), including the following breeds: Nelore (n=95), Guzerá (n=37), and Gir (n=35), totaling 248 pure breed cattle studied. DNA extraction was performed using the guanidium protocol and genotyping was performed by SSCA. DNA from cattle considered genotypically resistant to brucellosis resulted in a single band (homozygous) with 175bp, corresponding to the 3'UTR with 13 GT pairs (GT13), whereas DNA from genotypically susceptible cattle generated one single band with 177bp (homozygous GT14) or double bands with both 175 and 177bp, or 175 and 179bp (heterozygous GT13/GT14 or GT13/GT15, respectively). A marked difference in the frequency of alleles was detected between the Zebu and Holstein cattle. Holsteins had an extremely homogeneous genotype, with 100% of the individuals with a GT13 genotype. In sharp contrast the Nelore breed had the most heterogeneous genotype with four allelic combinations, namely, homozygous GT13, homozygous GT14, heterozygous GT13/GT14, and heterozygous GT13/GT15. When the Zebu breeds were compared to each other, the only significant difference observed was the frequencies of the genotypes GT13 and GT14 between the Nelore and Guzerá breeds. The knowledge of allelic frequencies in different breeds of cattle may prove to be very useful in the future for planning breeding strategies for selection of resistant cattle.