Dairy cattle farmers' preferences for different breeding tools.

Breeding technologies play a significant role in improving dairy cattle production. Scientifically proven tools for improved management and genetic gain in dairy herds, such as sexed semen, beef semen, genomic testing, dairy crossbreeding, and multiple ovulation embryo transfer (MOET), are readily available to dairy farmers. However, despite good accessibility, decreasing costs, and continuous development of these tools, their use in Sweden is limited. This study investigated Swedish dairy farmers' preferences for breeding tools through a survey including a discrete choice experiment. The survey was distributed online to 1 521 Swedish farmers and by an open link published through a farming magazine. In total, the study included 204 completed responses. The discrete choice experiment consisted of 10 questions with two alternative combinations, which gave 48 combinations in total. Utility values and part-worth values were computed using a conditional logit model based on the responses in the discrete choice experiment for nine groups of respondents: one group with all respondents, two groups based on respondents using dairy crossbreeding or not within the past 12 months, two based on herd size, two based on respondent age, and two based on whether respondents had used breeding advisory services or not. The strongest preferences in all groups were for using sexed semen and beef semen. Genomic testing was also significantly preferred by all groups of respondents. Except in large herds, MOET on own animals was significantly and relatively strongly disfavoured by all groups. Buying embryos had no significant utility value to any group. Dairy crossbreeding had low and insignificant utility values in the group of all respondents, but it was strongly favoured by the group that had used dairy crossbreeding within the past 12 months, and it was disfavoured by the group that had not. Part-worth values of combined breeding tools showed that combinations of sexed and beef semen, alone or with genomic testing without dairy crossbreeding, were the most preferred tools. Compared with the most common combinations of breeding tools used in the past 12 months, the part-worth values indicated that Swedish dairy farmers may prefer to use breeding tools more than they do today. Statements on the different breeding tools indicated that the respondents agreed with the benefits attributed to the breeding tools, but these benefits may not be worth the cost of genomic testing and the time consumption of MOET. These valuable insights can be used for further development of breeding tools.

Characterization of the region downstream of the pilus biogenesis gene pilC1 in Neisseria gonorrhoeae.

The nucleotide sequence of a 3 kb region downstream of pilC1 in Neisseria gonorrhoeae MS11 was analyzed. This region contains two open reading frames, ORF1 and ORF2, and several repetitive DNA elements. ORF1 encodes an outer membrane protein that shows homology to orf98 of Pediococcus acidilactici. PCR with primers specific for ORF1 revealed that the gene is present in all gonococcal strains tested. The other open reading frame, ORF2, is highly homologous to the putative integral membrane protein HI1680 of Haemophilus influenzae.

A new coding mutation in the Tnf-alpha leader sequence in tuberculosis-sensitive I/St mice causes higher secretion levels of soluble TNF-alpha.

I/St and A/Sn mice are polar extremes in terms of several parameters defining sensitivity to Mycobacterium tuberculosis. TNF-alpha, mainly produced by activated macrophages, can mediate both physiological and pathophysiological processes. Adequate TNF-alpha levels are essential for a forceful protective response to M. tuberculosis. We have functionally characterized a nonsynonymous substitution, Arg8 His, in the highly conserved cytoplasmic domain of the pro-TNF-alpha leader peptide from extremely M. tuberculosis-sensitive I/St mice. This was compared to the common pro-TNF-alpha variant found in A/Sn mice. Using cDNA constructs, both variants were constitutively expressed in HEK293A cells. A significantly higher secretion level of Arg8 His TNF-alpha was shown using flow cytometry and ELISA analysis (P=0.0063), while intracellular levels were similar for both protein variants. An even TNF-alpha distribution throughout the cells was seen using confocal microscopy. This suggests that the Arg8 His substitution affects pro-TNF-alpha processing. The I/St mouse may serve as a model to further explore the function of the well-conserved cytoplasmic region of TNF-alpha. However, other identified substitutions in the I/St promoter, introns and 3'UTR of Tnf-alpha, as well as the cellular environment in vivo may affect the balance between soluble and intracellular Arg8 His TNF-alpha before and during M. tuberculosis infection.

PilC of pathogenic Neisseria is associated with the bacterial cell surface.

Adherence of pathogenic Neisseria to target host cells is mediated by pili. PilC1 and PilC2 are two high-molecular-weight proteins involved in pilus assembly and cellular adherence functions of the pili. Inactivation of pilC1 or pilC2 in N. meningitidis resulted in clones that expressed the same number of pili as the parent, contained no alterations in pilE and showed no detectable differences in PilE glycosylation. However, the PilC2+ pilC1- mutant showed much reduced adherence to target cells, indicating that production of PilC1 is essential for pilus-mediated adherence. To study further the functional differences between the meningococcal pilC genes, we determined the complete nucleotide sequence of pilC1 and pilC2 of N. meningitidis. Alignment of six PilC sequences demonstrated that PilC is composed of both conserved and variable regions. By immunogold labelling of bacterial sections we showed that PilC is present in the membranes of both piliated and non-piliated bacteria. Further, we demonstrated that PilC is associated with the bacterial cell surface.

Cholera toxin and extracellular Ca2+ induce adherence of non-piliated Neisseria: evidence for an important role of G-proteins and Rho in the bacteria-cell interaction.

In this study, we characterize the interaction between non-piliated (P-) Neisseria gonorrhoeae and human epithelial cells. P- mutants lacking the pilus subunit protein PilE attach at low levels to cells. Although the binding may not lead to heavy inflammatory responses, the interaction between P- Neisseria and host cells most probably play a role in colonization and asymptomatic carriage of the pathogen. Here we show that the adherence of P N. gonorrhoeae is blocked by GDP-beta-S [guanosine 5'-O(thio)diphosphate], a non-hydrolyzable GTP analogue, and by C3 exotoxin, an inhibitor of the small G-protein Rho. G-protein activators such as cholera toxin, that activates Gs, and fluoroaluminate, a general G-protein activator, induced bacterial adherence. Furthermore, increase of the extracellular free [Ca2+] dramatically enhanced adherence of non-piliated Neisseria. The pharynx and the urogenital tract are natural entry sites of the pathogenic Neisseria species, and at both sites the epithelial cells can be exposed to wide variations in Ca2+ concentration. Taken together, these data show the importance of extracellular Ca2+ in the pathogenic Neisseria-host interaction, and reveal a novel function of cholera toxin, namely induction of bacterial adherence.

Transcellular passage of Neisseria gonorrhoeae involves pilus phase variation.

Piliated and nonpiliated Neisseria gonorrhoeae organisms were added on top of confluent layers of HEC-1-B cells, each maintained on a microporous Transwell-COL membrane. The bacteria released into the lower chamber were characterized with respect to the following virulence determinants: pili, which mediate adherence to target host cells; PilE, the major pilus subunit protein; and PilC, which is involved in pilus biogenesis and adherence. Even if >99% of the added bacteria of N. gonorrhoeae MS11 were piliated, bacteria recovered on the other side of the cell layer were predominantly nonpiliated. The recovered clones still expressed unassembled PilE protein, but 50% had lost PilC production. Nonpiliated gonococci, in which the 5' end of pilE had been deleted, were released in reduced numbers, and piliated recA bacteria added to the cell layer were not released at all, at time points when piliated recA+ clones were found at high numbers in the lower chamber. Our data indicate that bacteria producing unassembled PilE protein are selected for during passage through an epithelial cell layer. The finding that the pilE gene sequence was altered in the transmigrants suggests that pilin sequence variation is involved in the transcellular passage of N. gonorrhoeae.

Membrane cofactor protein (MCP or CD46) is a cellular pilus receptor for pathogenic Neisseria.

Pili of Neisseria gonorrhoeae and Neisseria meningitidis mediate binding of the bacteria to human cell-surface receptors. We found that purified pili bound to a 55- to 60-kDa doublet band on SDS-PAGE of separated human epithelial cell extracts. This is a migration pattern typical of membrane cofactor protein (MCP or CD46). MCP is a widely distributed human complement regulatory protein. Attachment of the bacteria to epithelial cells was blocked by polyclonal and monoclonal antibodies directed against MCP, suggesting that this complement regulator is a receptor for piliated Neisseria. We proved this hypothesis by demonstrating that piliated, but not non-piliated, gonococci bound to CHO cells transfected with human MCP-cDNA. We also demonstrated a direct interaction between purified recombinant MCP and piliated Neisseria. Finally, recombinant MCP protein produced in E. coli inhibited attachment of the bacteria to target cells. Taken together, our data show that MCP is a human cell-surface receptor for piliated pathogenic Neisseria.

Cell signaling by the type IV pili of pathogenic Neisseria.

Neisseria gonorrhoeae and Neisseria meningitidis are Gram-negative bacterial pathogens that infect human mucosal epithelia. Type IV pilus-mediated adherence of these bacteria is a crucial early event for establishment of infection. In this work, we show that the type IV pili transduce a signal into the eucaryotic host cell. Purified adherent pili, but not pili from a low binding mutant, trigger an increase in the cytosolic free calcium ([Ca2+]i) in target epithelial cells, a signal known to control many cellular responses. The [Ca2+]i increase was blocked by antibodies against CD46, a putative pilus receptor, suggesting a role for this protein in signal transduction. Pilus-mediated attachment was inhibited by depletion of host cell intracellular Ca2+ stores but not by removal of extracellular Ca2+. Further, kinase inhibition studies showed that pilus-mediated adherence is dependent on casein kinase II. In summary, these data reveal a novel function of the type IV pili, namely induction of signal transduction pathways in host cells.

Attachment of Neisseria gonorrhoeae to the cellular pilus receptor CD46: identification of domains important for bacterial adherence.

Pili of Neisseria gonorrhoeae mediate binding of the bacteria to human host cells. Membrane cofactor protein (MCP or CD46), a human cell-surface protein involved in regulation of complement activation, acts as a cellular pilus receptor. In this work, we examined which domains of CD46 mediate bacterial adherence. The CD46 expression was quantified and characterized in human epithelial cell lines. N. gonorrhoeae showed the highest adherence to ME180 cells, which have BC1 as the dominant phenotype. The BC isoforms of CD46 were expressed in all cell lines tested. The adherence was not enhanced by high expression of other isoforms, showing that the BC domain of CD46 is important in adherence of N. gonorrhoeae to human cells. To characterize the pilus-binding site within the CD46 molecule, a set of CD46-BC1 deletion constructs were transfected into COS-7 cells. Piliated N. gonorrhoeae attached well to CD46-BC1-expressing COS-7 cells. We show that the complement control protein repeat 3 (CCP-3) and the serine-threonine-proline (STP)-rich domain of CD46 are important for efficient adherence to host cells. Further, partial deletion of the cytoplasmic tail of CD46 results in low bacterial binding, indicating that the cytoplasmic tail takes part in the process of establishing a stable interaction between N. gonorrhoeae and host cells.