Simultaneous Detection of Both RNA and DNA Viruses Infecting Dry Bean and Occurrence of Mixed Infections by BGYMV, BCMV and BCMNV in the Central-West Region of Mexico.

A multiplex reverse transcription polymerase chain reaction (RT-PCR) assay was developed to simultaneously detect bean common mosaic virus (BCMV), bean common mosaic necrotic virus (BCMNV), and bean golden yellow mosaic virus (BGYMV) from common bean leaves dried with silica gel using a single total nucleic acid extraction cetyl trimethyl ammonium bromide (CTAB) method. A mixture of five specific primers was used to amplify three distinct fragments corresponding to 272 bp from the AC1 gene of BGYMV as well as 469 bp and 746 bp from the CP gene of BCMV and BCMNV, respectively. The three viruses were detected in a single plant or in a bulk of five plants. The multiplex RT-PCR was successfully applied to detect these three viruses from 187 field samples collected from 23 municipalities from the states of Guanajuato, Nayarit and Jalisco, Mexico. Rates of single infections were 14/187 (7.5%), 41/187 (21.9%), and 35/187 (18.7%), for BGYMV, BCMV, and BCMNV, respectively; 29/187 (15.5%) samples were co-infected with two of these viruses and 10/187 (5.3%) with the three viruses. This multiplex RT-PCR assay is a simple, rapid, sensitive, and cost-effective method for detecting these viruses in the common bean and can be used for routine molecular diagnosis and epidemiological studies.

A new allele of γ-kafirin gene coding for a protein with high lysine content in Mexican white sorghum germplasm.

BACKGROUND: Low protein digestibility and lysine content of white sorghum grain limit its use as a foodstuff. The increase in γ-kafirin cross-linking, has an important role in the reduction of protein digestibility. The objective of this study was to characterize the γ-kafirin gene in 12 Mexican tannin-free white sorghum genotypes and its relationship with protein digestibility and lysine content. RESULTS: Two alleles of γ-kafirin gene were identified: alleles 1 and 7. The predicted amino acid sequence of allele 7 showed seven point mutations; six were silent, and one missense (C235G), causing the substitution P79A in the deduced amino acid sequence. In silico analysis showed that γ-kafirin codified by allele 1 has five α-helixes without disulfide bonds, while γ-kafirin coding by allele 7 has four α-helixes and three disulfide bonds. Genotypes with allele 7 had higher lysine content than those with allele 1, showing no differences in the kafirin electrophoretic profile, neither a correlation with the protein content nor the in vitro pepsin digestibility. CONCLUSIONS: Mexican tannin-free white sorghum genotypes showed two γ-kafirin alleles, 1 and 7. Allele 7 was associated with higher lysine content; in silico analysis showed that the substitution of P79A in this allele could modify γ-kafirin secondary structure. © 2015 Society of Chemical Industry.

Prolactin stimulates the internalization of Staphylococcus aureus and modulates the expression of inflammatory response genes in bovine mammary epithelial cells.

The incidence of mastitis in dairy cattle is highest at the drying off period and parturition, which are characterized by high levels of the lactogenic hormone prolactin (PRL). One of the most frequently isolated contagious pathogens causing mastitis is Staphylococcus aureus. However, the role of PRL on S. aureus infection in mammary epithelium has not been studied. In this work we evaluated the effect of bovine PRL (bPRL) on S. aureus internalization in a primary culture of bovine mammary epithelial cells (bMEC) and on the expression of cytokine and innate immune response genes. Our data show that 5ng/mL bPRL enhances approximately 3-fold the internalization of S. aureus (ATCC 27543) into bMEC. By RT-PCR analysis, we showed that bPRL is able to up-regulate the expression of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and inducible nitric oxide synthase (iNOS) mRNAs. However, bPRL together with S. aureus did not modify the expression of TNF-alpha and iNOS mRNAs, while it down-regulated the expression of beta-defensin and IL-1beta mRNAs, as well as nitric oxide production, suggesting that infection and bPRL together can inhibit elements of the host immune response. To our knowledge, this is the first report that shows a role of bPRL during the internalization of S. aureus into bMEC.

Fungicidal and cytotoxic activity of a Capsicum chinense defensin expressed by endothelial cells.

Plant defensins are antimicrobial peptides that exhibit mainly antifungal activity against a broad range of plant fungal pathogens. However, their actions against Candida albicans have not been extensively studied. The mRNA for gamma-thionin, a defensin from Capsicum chinense, has been expressed in bovine endothelial cells. The conditioned medium of these cells showed antifungal activity on germ tube formation (60-70% of inhibition) and on the viability of C. albicans (70-80% of inhibition). Additionally, C. albicans was not able to penetrate transfected cells. Conditioned medium from these cells also inhibited the viability (80%) of the human tumor cell line, HeLa.

Invasive potential of bacterial isolates associated with subclinical bovine mastitis.

This work describes differences in the invasive ability of bacterial isolates associated with mastitis. Invasion ability was determined by the uptake and survival in a primary culture of bovine mammary epithelial cells (BMEC). BMEC were isolated from a healthy lactating cow and characterized by their morphology, immunostaining for cytokeratin and the detection of beta- and kappa-casein mRNAs. Ten bacterial isolates comprising the staphylococcal species Staphylococcus aureus (3), Staphylococcus epidermidis (1), Staphylococcus haemolyticus (1), Staphylococcus equorum (2), Staphylococcus xylosus (1) and Brevibacterium stationis (2) obtained from raw milk of cows with mastitis from backyard farms were assayed for their ability to invade BMEC. Only two S. aureus and one S. epidermidis isolates were able to invade BMEC, at similar levels to the S. aureus control strain ATCC 27543. In conclusion, using the in vitro model of infection used in this study, differences in bacterial invasion capability may be detected.