Suzanne M. Rea

Shewanella gelidimarina sp. nov. and Shewanella frigidimarina sp. nov., novel Antarctic species with the ability to produce eicosapentaenoic acid (20:5 omega 3) and grow anaerobically by dissimilatory Fe(III) reduction

A polyphasic taxonomic study was performed to characterize dissimilatory iron-reducing strains mostly isolated from Antarctic sea ice. The strains were isolated from samples of congelated (land-fast) sea ice, grease ice, and ice algal biomass collected from the coastal areas of the Vestfold Hills in eastern Antarctica (68 degrees S 78 degrees E). The strains were facultatively anaerobic, motile, and rod shaped, were capable of anaerobic growth either by fermentation of carbohydrates or by anaerobic respiration, and utilized a variety of electron acceptors, including nitrate, ferric compounds, and trimethylamine N-oxide. A phylogenetic analysis performed with 16S rRNA sequences showed that the isolates formed two groups representing novel lineages in the genus Shewanella. The first novel group included seawater-requiring, psychrophilic, chitinolytic strains which had DNA G + C contents of 48 mol%. The members of the second strain group were psychrotrophic and did not require seawater but could tolerate up to 9% NaCl. The strains of this group were also unable to degrade polysaccharides but could utilize a number of monosaccharides and disaccharides and had G + C contents of 40 to 43 mol%. The whole-cell-derived fatty acid profiles of the sea ice isolates were found to be similar to the profiles obtained for other Shewanella species. The omega-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) (20:5 omega 3) was detected in all of the sea ice isolates at levels ranging from 2 to 16% of the total fatty acids. EPA was also found at high levels in Shewanella hanedai (19 to 22%) and Shewanella benthica (16 to 18%) but was absent in Shewanella alga and Shewanella putrefaciens. On the basis of polyphasic taxonomic data, the Antarctic iron-reducing strains are placed in two new species, Shewanella frigidimarina sp. nov. (type strain, ACAM 591) and Shewanella gelidimarina sp. nov. (type strain, ACAM 456).

A Vaccine against Rumen Methanogens Can Alter the Composition of Archaeal Populations

ABSTRACT The objectives of this study were to formulate a vaccine based upon the different species/strains of methanogens present in sheep intended to be immunized and to determine if a targeted vaccine could be used to decrease the methane output of the sheep. Two 16S rRNA gene libraries were used to survey the methanogenic archaea in sheep prior to vaccination, and methanogens representing five phylotypes were found to account for >52% of the different species/strains of methanogens detected. A vaccine based on a mixture of these five methanogens was then formulated, and 32 sheep were vaccinated on days 0, 28, and 103 with either a control or the anti-methanogen vaccine. Enzyme-linked immunosorbent assay analysis revealed that each vaccination with the anti-methanogen formulation resulted in higher specific immunoglobulin G titers in plasma, saliva, and rumen fluid. Methane output levels corrected for dry-matter intake for the control and treatment groups were not significantly different, and real-time PCR data also indicated that methanogen numbers were not significantly different for the two groups after the second vaccination. However, clone library data indicated that methanogen diversity was significantly greater in sheep receiving the anti-methanogen vaccine and that the vaccine may have altered the composition of the methanogen population. A correlation between 16S rRNA gene sequence relatedness and cross-reactivity for the methanogens (R2 = 0.90) also exists, which suggests that a highly specific vaccine can be made to target specific strains of methanogens and that a more broad-spectrum approach is needed for success in the rumen. Our data also suggest that methanogens take longer than 4 weeks to adapt to dietary changes and call into question the validity of experimental results based upon a 2- to 4-week acclimatization period normally observed for bacteria.

Reponses of sheep to a vaccination of entodinial or mixed rumen protozoal antigens to reduce rumen protozoal numbers

Two rumen protozoa vaccine formulations containing either whole fixed Entodinium or mixed rumen protozoa cells were tested on Merino sheep with the aim of decreasing the number and/or activity of protozoa in the rumen. Negative control (no antigen) and positive control (Tetrahymena corlissi antigens) treatments were also included in the experiment. Blood and saliva were sampled to measure the specific immune response. Protozoal numbers in the rumen were monitored by microscopic counts. Vaccination with protozoal formulations resulted in the presence of specific IgG in plasma and saliva, but saliva titres were low. Titres after secondary vaccination were higher (P 0.05) by the vaccination and there was also no difference (P>0.05) between treatments in rumen fluid ammonia-N concentration or wool growth. In vitro studies investigated the binding ability of the antibodies and estimated the amount of antibody required to reduce cell numbers in the rumen. The studies showed that the antibodies did bind to and reduced protozoa numbers, but the amount of antibody generated by vaccination was not enough to produce results in an in vivo system. It is suggested that the vaccine could be improved if specific protozoal antigens are determined and isolated and that improved understanding of the actions of protozoa antibodies in rumen fluid and the relationships between levels of antibodies and numbers of protozoa in the rumen is needed.

Technical note: Protozoa-specific antibodies raised in sheep plasma bind to their target protozoa in the rumen12

Binding of IgG antibodies to Entodinium spp. in the rumen of sheep (Ovis aries) was investigated by adding IgG, purified from plasma, directly into the rumen. Plasma IgG was sourced from sheep that had or had not been immunized with a vaccine containing whole fixed Entodinium spp. cells. Ruminal fluid was sampled approximately 2 h after each antibody dosing. Binding of protozoa by a specific antibody was detected using an indirect fluorescent antibody test. An antibody titer in the ruminal fluid was determined by ELISA, and the concentration of ruminal fluid ammonia-N and ruminal pH were also determined. Entodinium spp. and total protozoa from IgG-infused sheep were enumerated by microscopic counts. Two-hourly additions of IgG maintained a low antibody titer in the rumen for 12 h and the binding of the antibody to the rumen protozoa was demonstrated. Increased ammonia-N concentrations and altered ruminal fluid pH patterns indicated that additional fermentation of protein was occurring in the rumen after addition of IgG. No reduction in numbers of Entodinium spp. was observed (P>0.05). Although binding of antibodies to protozoa has been demonstrated in the rumen, it is unclear how much cell death occurred. On the balance of probability, it would appear that the antibody was degraded or partially degraded, and the impact of this on protozoal populations and the measurement of a specific titer is also unclear.