A novel pathway producing dimethylsulphide in bacteria is widespread in soil environments.

The volatile compound dimethylsulphide (DMS) is important in climate regulation, the sulphur cycle and signalling to higher organisms. Microbial catabolism of the marine osmolyte dimethylsulphoniopropionate (DMSP) is thought to be the major biological process generating DMS. Here we report the discovery and characterization of the first gene for DMSP-independent DMS production in any bacterium. This gene, mddA, encodes a methyltransferase that methylates methanethiol and generates DMS. MddA functions in many taxonomically diverse bacteria including sediment-dwelling pseudomonads, nitrogen-fixing bradyrhizobia and cyanobacteria, and mycobacteria including the pathogen Mycobacterium tuberculosis. The mddA gene is present in metagenomes from varied environments, being particularly abundant in soil environments, where it is predicted to occur in up to 76% of bacteria. This novel pathway may significantly contribute to global DMS emissions, especially in terrestrial environments and could represent a shift from the notion that DMSP is the only significant precursor of DMS.

The dddP gene, encoding a novel enzyme that converts dimethylsulfoniopropionate into dimethyl sulfide, is widespread in ocean metagenomes and marine bacteria and also occurs in some Ascomycete fungi.

The marine alphaproteobacterium Roseovarius nubinhibens ISM can produce the gas dimethyl sulfide (DMS) from dimethylsulfoniopropionate (DMSP), a widespread secondary metabolite that occurs in many phytoplankton. Roseovarius possesses a novel gene, termed dddP, which when cloned, confers on Escherichia coli the ability to produce DMS. The DddP polypeptide is in the large family of M24 metallopeptidases and is wholly different from two other enzymes, DddD and DddL, which were previously shown to generate DMS from dimethylsulfoniopropionate. Close homologues of DddP occur in other alphaproteobacteria and more surprisingly, in some Ascomycete fungi. These were the biotechnologically important Aspergillus oryzae and the plant pathogen, Fusarium graminearum. The dddP gene is abundant in the bacterial metagenomic sequences in the Global Ocean Sampling Expedition. Thus, dddP has several novel features and is widely dispersed, both taxonomically and geographically.

Molecular genetic analysis of a dimethylsulfoniopropionate lyase that liberates the climate-changing gas dimethylsulfide in several marine alpha-proteobacteria and Rhodobacter sphaeroides.

The alpha-proteobacterium Sulfitobacter EE-36 makes the gas dimethylsulfide (DMS) from dimethylsulfoniopropionate (DMSP), an abundant antistress molecule made by many marine phytoplankton. We screened a cosmid library of Sulfitobacter for clones that conferred to other bacteria the ability to make DMS. One gene, termed dddL, was sufficient for this phenotype when cloned in pET21a and introduced into Escherichia coli. Close DddL homologues exist in the marine alpha-proteobacteria Fulvimarina, Loktanella Oceanicola and Stappia, all of which made DMS when grown on DMSP. There was also a dddL homologue in Rhodobacter sphaeroides strain 2.4.1, but not in strain ATCC 17025; significantly, the former, but not the latter, emits DMS when grown with DMSP. Escherichia coli containing the cloned, overexpressed dddL genes of R. sphaeroides 2.4.1 and Sulfitobacter could convert DMSP to acrylate plus DMS. This is the first identification of such a 'DMSP lyase'. Thus, DMS can be made either by this DddL lyase or by a DMSP acyl CoA transferase, specified by dddD, a gene that we had identified in several other marine bacteria.

Evidence that the Rhizobium regulatory protein RirA binds to cis-acting iron-responsive operators (IROs) at promoters of some Fe-regulated genes.

Mutations in rirA of Rhizobium have been shown to deregulate expression of several genes that are normally repressed by iron. A conserved sequence, the iron-responsive operator (IRO), was identified near promoters of vbsC (involved in the synthesis of the siderophore vicibactin), rpoI (specifies an ECF sigma factor needed for vicibactin synthesis) and the two fhuA genes (encoding vicibactin receptor). Removal of these IRO sequences abolished Fe-responsive repression. Most of these genes were constitutively expressed in the heterologous host, Paracoccus denitrificans, but introduction of the cloned rirA gene repressed expression of these Rhizobium genes in this heterologous host if the corresponding IRO sequences were also intact. These observations are the first to examine the mechanisms of RirA, which has no sequence similarity to well-known iron-responsive regulators such as Fur or DtxR. They provide strong circumstantial evidence that RirA is a transcriptional regulator that binds to cis-acting regulatory sequences near the promoters of at least some of the genes whose expression it controls in response to Fe availability.

The Fur-like protein Mur of Rhizobium leguminosarum is a Mn(2+)-responsive transcriptional regulator.

In wild-type Rhizobium leguminosarum, the sitABCD operon specifies a Mn(2+) transporter whose expression is severely reduced in cells grown in the presence of this metal. Mutations in the R. leguminosarum gene, mur (manganese uptake regulator), whose product resembles the Fur transcriptional regulator, cause high-level expression of sitABCD in the presence of Mn(2+). In gel-shift mobility assays, purified R. leguminosarum Mur protein bound to at least two regions near the sitABCD promoter region, although this DNA has no conventional consensus Fur-binding sequences (fur boxes). Thus, in contrast to gamma-proteobacteria, where Fur binds Fe(2+), the R. leguminosarum Fur homologue, Mur, act as a Mn(2)-responsive transcriptional regulator.

Fur is not the global regulator of iron uptake genes in Rhizobium leguminosarum.

Rhizobium leguminosarum fur mutants were unaffected in Fe-dependent regulation of several operons that specify different Fe uptake systems, yet cloned R. leguminosarum fur partially corrected an Escherichia coli fur mutant and R. leguminosarum Fur protein bound to canonical fur boxes. The lack of a phenotype in fur mutants is not due to functional redundancy with Irr, another member of the Fur superfamily found in the rhizobia, since irr fur double mutants are also unaffected in Fe-responsive regulation of several operons involved in Fe uptake. Neither Irr nor Fur is needed for symbiotic N(2) fixation on peas. As in Bradyrhizobium japonicum, irr mutants accumulated protoporphyrin IX. R. leguminosarum irr is not regulated by Fur and its Irr protein lacks the motif needed for haem-dependent post-translational modification that occurs in B. japonicum Irr. The similarities and differences in the Fur superfamily in the rhizobia and other Gram-negative bacteria are discussed.

Preventing tractor-related injuries and deaths in rural populations: using a persuasive health message framework in formative evaluation research.

Though agriculture is the most dangerous occupation in the United States, two key issues impede the effectiveness of farm safety interventions. First, little is known about what farmers think about farm equipment accidents and safety procedures. Second, current safety interventions are typically atheoretical and focus on information exchange, instead of persuasion. Formative evaluation is desperately needed, but rarely used in farm safety campaigns. The study reported here represents a formative evaluation based on a theoretically-grounded persuasive health message framework. The goal of this formative evaluation was to discover farmers' safety practices, as well as their beliefs about farm equipment accidents and safety. Methodological triangulation was achieved by assessing farmers' beliefs, attitudes, and behaviors via face-to-face interviews (N = 46), telephone interviews (N = 48), and mailed surveys (N = 177). The formative evaluation revealed that farmers believe farm equipment accidents to be severe and dangerous, yet believe themselves to be invulnerable to these accidents.

Canine parvovirus serum neutralizing antibody assay: assessment of factors responsible for disparity of results between tests.

Canine parvovirus (CPV) serum neutralization (SN) test components were evaluated to determine their effect on antibody titer results. The use of different strains of CPV and different cell substrates had little effect on assay results. Variations in SN antibody titer results were associated with the use of challenge virus preparations that differed in the ratio of hemagglutination units (HAU) to infectivity units (FAID50). Sensitivity and reproducibility can be achieved by using a standardized challenge virus preparation containing a low HAU/FAID50 ratio.

Intranasal vaccination of cattle against IBR and PI3: field and laboratory observations in dairy, beef, and neonatal calf populations.

The author first summarizes the development of intranasal vaccination by Nasalgen P and particularly Nasalgen IP, previously reported, and then reviews present knowledge regarding safety and efficacy of Nasalgen IP in feeder calves, dairy animals and neonates. The minimal immunogenic dose is evaluated, as well as the secretory antibody response and the development of interferon. Two studies are reported on the transmission from vaccinates to susceptible non-vaccinates and the evaluation of the potential to revert to a more virulent form. An important part is devoted to the latest studies carried out which confirm that wide acceptance should be given to the intranasal vaccination of cattle.

Evaluation of a bovine viral diarrhea vaccine produced in a porcine kidney cell line.

An attenuated bovine viral diarrhea (BVD) live-virus vaccine, produced in a continuous porcine cell line, evoked an immune response which produced a protective level of serum antibodies in vaccinated cattle. Post-vaccinal reactions to the vaccinal virus were not observed in cattle vaccinated at 5 feedlots or in cattle vaccinated in experimental tests. The vaccinal virus did not produce demonstrable viremia or detectable excretion of virus from the respiratory or digestive tracts. Leukopenia or abnormality in differential leukocyte values did not occur as with virulent BVD viral infection. Transmission of vaccinal virus from vaccinated cattle to susceptible controls did not occur under conditions in which vaccinated and nonvaccinated cattle were in constant contact for 28 days.

Immune response of cattle to intranasally or parenterally administered parainfluenza type 3 virus vaccines.

The principal significance of PI-3 virus in the bovine respiratory disease complex would seem to be its role in predisposing the respiratory tract to bacterial infection by (5) disrupting the integrity of the superficial mucosa and (3) producing a more favourable milieu for bacterial growth. Attention has therefore been given to establishing local immunity to preclude infection of superficial cells of the mucosa. Criteria used to evaluate the degree of such immunity are (5) presence of specific antibody activity in nasal secretions and (3) extent of virus excretion in nasal secretions following challenge with low passage virus. Studies completed thus far demonstrate that modified, live PI-3 virus administered intranasally is more effective than either modified, live or inactivated PI-3 virusadministered by parenteral injection in providing antibody activity in respiratory tractsecretions and in reducing virus excretion following challenge. IgA antibodies, presumably synthesized locally, apparently contribute heavily to the total antibody activity present in respiratory tract secretions.

Immune response to infectious bovine rhinotracheitis virus (IBRV) following natural infection or vaccination by intranasally or parenterally administered vaccines.

Neutralizing antibody activity is present in serum and nasal secretions of cattle following IBR virulent virus challenge or intranasal vaccination with live, avirulent virus. Levels of nasal secretion antibody activity (NS-AA) are low and short-lived following initial exposure, but are considerably enhanced following reexposure provided infection occurs. NS-AA has not been detected in calves following parenteral administration of live virus vaccine. Such calves are subject to infection by challenge virus administered intranasally as early as three weeks after vaccination. Unlike parenterally administered vaccine, vaccine administeredintranasally promotes active immunity in young calves born of immune dams. Calves so vaccinated respond to later challenge with rapid, pronounced increases in both serum antibody and NS-AA without displaying overt signs of disease. Avirulent virus administered intranasally promotes release of interferon into nasal secretions for a period of six to eight days, coincident with continued virus replication. Classes of immunoglobulin involved in the various responses will be discussed.

Interferon in nasal secretions and sera of calves after intranasal administration of avirulent infectious bovine rhinotracheitis virus: association of interferon in nasal secretions with early resistance to challenge with virulent virus.

Calves which had received avirulent infectious bovine rhinotracheitis virus (AV-IBR) by intranasal (IN) administration developed detectable levels of interferon (IF) in nasal secretions as early as 40 hr later. Peak titers (1:640) of IF appeared in secretions 72 to 96 hr after administration of virus, and titers of 1:80 to 1:320 were maintained through the 8th day. Lower titers (1:5 to 1:10) of IF were detected in sera obtained on the 4th to 8th days after administration of virus. Peak titers of IF in respiratory tract secretions were accompanied by a 100- to > 1,000-fold reduction in the levels of AV-IBR present in the secretions. Serum antibody was not detected prior to the 8th day after administration of AV-IBR. Calves which received AV-IBR by the IN route 72 or 96 hr earlier were refractory to challenge with virulent infectious bovine rhinotracheitis virus (IBR), whereas calves receiving AV-IBR 18 or 40 hr earlier became clinically ill following challenge. The temporal association between appearance of IF in respiratory tract secretions and onset of protection against challenge suggests a cause and effect relationship. No IF was detected in either nasal secretions or sera of calves receiving modified IBR virus by intramuscular injection. Following subsequent IN challenge of these calves, IF was detected in nasal secretions as early as 24 hr postchallenge and was maintained at titers of 1:40 to 1:80 for approximately 4 days, even in the absence of virus recovery. Greater ease of local IF induction with IBR virus in calves previously sensitized with that virus is suggested.

Epidemiological studies of A-Hong Kong-68 virus infection in dogs.

Experimental inoculation of dogs with the A/Hong Kong/68 influenzavirus resulted in subclinical infection. The virus was readily passed to contacts in the same cage when the latter were exposed in the same inoculation room 24 hours after experimental infection. Removing the site of contact to a noncontaminated room or delaying contact until 48 hours after experimental inoculation greatly reduced the possibility of infection in contact animals. A survey of 271 canine serum samples obtained after a human epidemic from different geographical areas of the USA and the United Kingdom showed that 5.9% of the samples were positive; no positive reactions were found among 111 pre-epidemic samples. These studies demonstrated the laboratory and natural susceptibility of dogs to the Hong Kong variant and suggest the possible role of dogs in the epidemiology of human influenza.