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Aims: To prevent the spread of brucellosis, a simple and rapid vertical flow technology (RVFT) for the detection of antibodies targeting brucellosis was developed., Methods and Results: In this study, Brucella sp. lipopolysaccharide was purified and used to detect brucellosis antibodies. Sheep IgG was used as a negative control. Colloidal gold-labeled recombinant staphylococcus aureus protein A was sprayed on a fibreglass membrane to prepare immunogold pads. Rapid vertical flow technology was used to detect Brucella in 1668 Sheep, 2743 bovine, 674 red deer and 420 human samples. The results indicated that the accuracy of this assay can reach 98%., Conclusions: The established RVFT uses a single multifunctional buffer that can be used to detect antibodies in serum, plasma, whole blood and other biological samples while preserving the advantages of lateral-flow immunoassays., Significance and Impact of the Study: This technology would be of great use in primary medical units and veterinary stations, and it is of great significance for the control of epidemic diseases., (© 2019 The Society for Applied Microbiology.)

Aims: The aim of this study was to design an assay for the identification of Mycobacterium avium subsp. paratuberculosis (MAP) to be used in faeces and milk samples of small ruminants with a loop-mediated isothermal amplification (LAMP) system, as a time-saving and user-friendly method in contrast to real-time PCR., Methods and Results: For the detection of MAP in milk and faeces of small ruminants, we developed a set of primers, specific for the target gene ISMap02. The analytical sensitivity of LAMP, when targeting ISMap02, showed a DNA detection limit of 10 fg μl -1 . After performing spiking experiments with two MAP reference strains, DSM 44133 and ATCC 19698 T , the limit of detection, using the LAMP protocol described herein were 3·8 MAP CFU per ml milk and 12·5 MAP CFU per gram faeces. All LAMP results during the establishment of the assay were compared to those of the real-time PCR results. An internal amplification control was incorporated into the assay to exclude false-negative results produced and had no significant negative impact on the analytical sensitivity. Validation of the assay was confirmed by testing field samples of faeces and revising the results with real-time PCR., Conclusion: Our study conducted the first MAP detection system with a LAMP targeting ISMap02. Due to the positive results we encourage the use of LAMP in combination with ISMap02, when detecting MAP in faeces samples, as an alternative to targeting other genes as f57 or IS900. Further research on MAP detection in different matrices like raw milk, tissue or sperm with this system is recommended., Significance and Impact of the Study: This study provides new achievements in MAP diagnostic. Especially small ruminants do not show signs of diarrhoea until the terminal stage of the illness. The greatest task in fighting MAP is to rule out animals, which shed MAP with faeces and milk before showing symptoms of Johne's disease. Worldwide there is a need to eradicate animals, which are low MAP shedders to stop the illness spreading in animal holdings. MAP detection with LAMP is time saving, easy to use, does not need expensive equipment, as, for example, PCR kits and can be used without access to laboratories. The target gene ISMap02 was shown to be a specific insertion element for MAP and is a reliable aim in future MAP detection studies., (© 2019 The Society for Applied Microbiology.)

Aims: This study aimed to verify the formation of biofilms by Moraxella bovis, Moraxella ovis and Moraxella bovoculi isolates from ruminants. In addition, the lysozyme activity against the isolates of M. bovis, M. ovis and M. bovoculi in free form and in biofilms was determined., Methods and Results: In this study, 54 isolates of Moraxella sp. obtained from bovine and ovine clinical samples were evaluated in vitro for capacity of biofilm formation and lysozyme susceptibility in planktonic and sessile cells. In addition, biofilms produced by four Moraxella sp. isolates were visualized under scanning electron microscope (SEM). It was possible to demonstrate, for the first time, the ability to form biofilms by M. ovis and M. bovoculi. The isolates of Moraxella sp. have the capacity to form biofilms in different intensities, varying among weak, moderate and strong. It was verified that the lysozyme shows activity on Moraxella sp. in planktonic form. However, on biofilms there was a reduction in the production, but without impairing its formation, and on consolidated biofilms the lysozyme did not have the capacity to eradicate the preformed biofilms., Conclusions: This work shows the capacity of biofilm formation by Moraxella sp. of veterinary importance. The lysozyme susceptibility of Moraxella sp. in planktonic form shows that this enzyme has bacteriostatic activity on this micro-organism and it reduced the production of biofilms., Significance and Impact of the Study: Based on the results, it is possible to infer that the biofilm formation capacity by Moraxella sp. and the resistance to lysozyme concentrations equal to or greater than the physiological levels of the ruminant tear may be linked not only to the capacity to colonize the conjunctiva, but also to remain in this place even after healing of the lesions, being a reservoir of Moraxella sp. in a herd., (© 2018 The Society for Applied Microbiology.)

Aims: The probiotic Bacillus amyloliquefaciens H57 increased weight gain, increased nitrogen retention and increased feed intake in ruminants when administered to the diet. This study aims to develop a better understanding of this probiotic effect by analysing changes in the rumen prokaryotic community., Methods and Results: Sequencing the 16S rRNA gene PCR amplicons of the rumen microbiome, revealed that ewes fed H57 had a significantly different rumen microbial community structure to Control sheep. In contrast, dairy calves showed no significant differences in rumen community structure between treatment groups. In both instances, H57 was below detection in the rumen community profile and was only present at low relative abundance as determined by qPCR., Conclusions: The altered rumen microbial community in sheep likely contributes to increased weight gain through more efficient digestion of plant material. As no change occurred in the rumen community of dairy calves it is suggested that increased weight gain may be due to changes in community function rather than structure. The low relative abundance of H57 as determined by qPCR, suggests that weight gain was not directly mediated by the probiotic, but rather by influencing animal behaviour (feed consumption) and/or altering the native rumen community structure or function., Significance and Impact of the Study: This study provides a novel look at the rumen prokaryotic community in both sheep and dairy calves when fed H57. These findings improve our understanding for the potential rumen community involvement in H57-enabled weight gain. The study reveals that the probiotic B. amyloliquefaciens H57 is capable of benefiting ruminants without colonizing the rumen, suggesting an indirect mechanism of action., (© 2018 The Society for Applied Microbiology.)

Aims: This work aims to determine the factors which play a role in establishing the microbial population throughout the digestive tract in ruminants and is necessary to enhance our understanding of microbial establishment and activity., Methods and Results: This study used Terminal Restriction Fragment Length Polymorphism (TRFLP) to investigate the microbial profiles of 11 regions of the digestive tract of two breeds of sheep (Beulah and Suffolk). TRFLP data revealed that the regions of the digestive tract were highly significantly different in terms of the composition of the bacterial communities within three distinct clusters of bacterial colonization (foregut, midgut and hindgut). The data also show that breed was a significant factor in the establishment of the bacterial component of the microbial community, but that no difference was detected between ciliated protozoal populations., Conclusions: We infer that not only are the different regions of the tract important in determining the composition of the microbial communities in the sheep, but so too is the breed of the animal., Significance and Impact of the Study: This is the first time that a difference has been detected in the digestive microbial population of two different breeds of sheep., (© 2016 The Society for Applied Microbiology.)

Aims: To investigate, using culture-independent techniques, the presence and diversity of methanogenic archaea in the foregut of kangaroos., Methods and Results: DNA was extracted from forestomach contents of 42 kangaroos (three species), three sheep and three cattle. Four qualitative and quantitative PCR assays targeting the archaeal domain (16S rRNA gene) or the functional methanogenesis gene, mcrA, were used to determine the presence and population density of archaea in kangaroos and whether they were likely to be methanogens. All ruminal samples were positive for archaea, produced PCR product of expected size, contained high numbers of archaea and high numbers of cells with mcrA genes. Kangaroos were much more diverse and contradictory. Fourteen kangaroos had detectable archaea with numbers 10- to 1000-fold fewer than sheep and cattle. Many kangaroos that did not possess archaea were positive for the mcrA gene and had detectable numbers of cells with this gene and vice versa. DNA sequence analysis of kangaroos' archaeal 16S rRNA gene clones show that many methanogens were related to Methanosphaera stadmanae. Other sequences were related to non-methanogenic archaea (Thermoplasma sp.), and a number of kangaroos had mcrA gene sequences related to methane oxidising archaea (ANME)., Conclusions: Discrepancies between qualitative and quantitative PCR assays for archaea and the mcrA gene suggest that the archaeal communities are very diverse and it is possible that novel species exist., Significance and Impact of the Study: Archaea (in general) were below detectable limits in many kangaroos, especially Red kangaroos; when present they are in lower numbers than in ruminants, and the archaea are not necessarily methanogenic. The determination of why this is the case in the kangaroo foregut could assist in reducing emissions from other ecosystems in the future., (© 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.)

Aims:   To investigate the influence of yeast extract, peptone, temperature and pH upon protease productivity by Bacillus sp. HTS102--a novel wild strain isolated from wool of a Portuguese sheep breed (Merino)., Methods and Results:   A 2(4) full factorial, central composite design together with response surface methodology was used to carry out the experiments and analyse the results, respectively. Among the individual parameters tested, temperature and peptone concentration produced significant effects upon protease productivity. A high correlation coefficient (R(2 ) = 0·994, P < 0·01) indicated that the empiric second-order polynomial model postulated was adequate to predict said productivity, with the optimum loci characterized by: temperature of 43°C, peptone content of 1·4 g l(-1) , pH of 5·1 and yeast extract concentration of 10·0 g l(-1) ., Conclusions:   Protease synthesis depends chiefly on temperature and peptone level. The maximum protease activity was more than twice that obtained with the basal medium, so the experimental design and analysis undertaken were effective towards process optimization., Significance and Impact of the Study:   Rational choice of processing conditions for maximum protease productivity will be relevant if an economically feasible fermentation process based on Bacillus sp. HTS102 is intended., (© 2012 The Authors. Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.)

Aims:  The objective of this study was to isolate, identify and characterize a collection of lytic bacteriophages capable of infecting enterohaemorrhagic Escherichia coli (EHEC) serotypes., Methods and Results: Phages were isolated from dairy and cattle feedlot manure using E. coli O157, O26 and O111 strains as hosts. Phages were enriched from faecal slurries by culture in 10× trypticase soy broth at 37°C overnight. Phage plaques were obtained by mixing the filtered culture supernatant with molten tryptone agar containing the phage E. coli host strain, pouring the inoculated agar on top of cooled TS agar and incubating the culture overnight. Phages were purified from plaques and screened against additional E. coli and EHEC strains by the efficiency of plating method (EOP). Phage CEV2, and five other phages previously isolated, were able to lyse all of the 15 O157 strains tested with EOP values consistently above 0·001. Two phages were found to be highly effective against strains of E. coli O157 through EOP tests and against O26 strains through spot tests, but not against the O serogroup 111 strains. A cocktail of eight phage that lyse E. coli O157 strains resulted in >5 log CFU ml(-1) reductions at 37°C. Multiplex-PCR revealed that none of these eight phages carried stx1, stx2, hlyA or eaeA genes., Conclusions: A cocktail of bacteriophages was capable of lysing most strains of two EHEC serotypes., Significance and Impact of the Study: This collection of phages can be combined and potentially used as an antimicrobial cocktail to inactivate E. coli strains from O serogroups 157 and 26 and reduce their incidence in the food chain., (© 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.)

Aims: To assess whether flies and slugs acquire strains of Campylobacter jejuni and Campylobacter coli present in local ruminant faeces., Methods and Results: Campylobacter was cultured from flies, slugs and ruminant faeces that were collected from a single farm in Scotland over a 19-week period. The isolates were typed using multi-locus sequence typing (MLST) and compared with isolates from cattle and sheep faeces. Campylobacter jejuni and Camp. coli were isolated from 5·8% (n=155, average of 75 flies per pool) and 13·3% (n=15, average of 8·5 slugs per pool) of pooled fly and slug samples, respectively. The most common sequence type (ST) in flies was Camp. coli ST-962 (approx. 40%) regardless of the prevalence in local cattle (2·3%) or sheep (25·0%) faeces. Two positive slug pools generated the same ST that has not been reported elsewhere., Conclusions:   Despite their low carriage rate, flies are able to acquire Campylobacter STs that are locally present, although the subset carried may be biased when compared to local source. Slugs were shown to carry a previously unreported Campylobacter ST., Significance and Impact of the Study: This study has demonstrated that flies carry viable Campylobacter and may contribute to the transfer of STs within and between groups of animals on farms. Further, they may therefore present a risk to human health via their contact with ready-to-eat foods or surfaces., (© 2010 University of Aberdeen. Journal compilation © 2010 The Society for Applied Microbiology.)

Aims: To develop a novel molecular tool for the quantitative detection of the ruminant pathogen Listeria ivanovii in different biological matrices., Methods and Results: A real-time PCR (RTi-PCR) for the quantitative and species-specific identification of L. ivanovii was designed to target the region of the smcL gene. The assay includes an internal amplification control (IAC) to avoid false-negative results. The smcL-IAC RTi-PCR assay was 100% selective and allowed the detection of as little as one genome equivalent in 45% of reactions. The quantification accuracy was excellent, as demonstrated by its high linearity (R(2)>0·9989) and PCR efficiency (E>0·984) over a 6-log dynamic range, down to 10 genome equivalents. Finally, the applicability of this assay was evaluated with artificially contaminated biological matrices implicated in the transmission of this bacterium such as sheep raw milk, blood and amniotic fluid. The smcL-IAC RTi-PCR assay allowed the detection of as few as 50 colony forming unit numbers (CFUs) per 25 ml of raw milk, 43 CFUs per 1 ml of blood or 50 CFUs per 1 ml of amniotic fluid., Conclusions: This method can be an adequate alternative for the identification of L. ivanovii and for complete diagnosis of animal and human listeriosis. significance and impact of the study: We present an alternative for the detection of another pathogenic member of Listeria genus, which can help to distinguish from Listeria monocytogenes and therefore facilitates the establishment of preventive and prophylactic measures in food and farm environments., (© 2010 The Authors. Journal compilation © 2010 The Society for Applied Microbiology.)

Aims: To demonstrate that a thorough characterization and virulotyping of Escherichia coli strains isolated from sheep over time leads to new insights into ovine E. coli potentially becoming human pathogens through horizontal gene transfer., Methods and Results: One hundred and fifty E. coli isolates from two sheep, sampled over 3 weeks, were characterized by serotyping, virulotyping, genotyping using multiple locus variable number tandem repeats analysis (MLVA) and susceptibility to phage infection in vitro. The 35 MLVA profiles and the serotype and virulotypes of the strains were closely associated. Many MLVA profiles differed in one locus independent of serotypes. Escherichia coli isolates of the same serotype or virulotype had identical or very similar MLVA profiles. No transductants that incorporated the bacteriophages were found in vivo, but six E. coli isolates were susceptible to the phage infection in vitro. Changes in MLVA profiles were seen after acquisition of Stx phages in vitro only., Conclusions: The sheep carried Stx phage susceptible E. coli that possessed virulence markers associated with human pathogenicity. Changes in bacterial genomes by phage transfer may complicate outbreak source investigations. Serotype has to be taken into account when evaluating strain relationships by MLVA., Significance and Impact of the Study: Sheep carry E. coli that encode for virulence markers and belong to serogroups known to be human pathogens. In addition, a selection of isolates was found to be susceptible to horizontal transfer of Shiga toxin genes by means of bacteriophages in vitro, and the transfer resulted in a discernible change of the MLVA patterns of E. coli.

Aims: The aims of this study were to evaluate the host-specific distribution of Bacteroidales 16S rRNA gene sequences from human- and animal-related effluents and faeces, and to define a ruminant-specific marker., Methods and Results: Bacteroidales 16S rRNA gene clone libraries were constructed from samples of effluent (sewage, bovine manure and pig slurry) and faeces (human, bovine, pig and wild bird), using PCR primers targeting order Bacteroidales. The phylogenetic analysis revealed six main distinct human-, bovine-, pig- and wild bird-specific clusters. From the bovine-specific cluster II, we designed a ruminant-specific marker, Rum-2-Bac, and this showed 97% sensitivity (n=30) and 100% specificity (n=40) when tested by TaqMan real-time PCR. Average concentrations of this marker in bovine and sheep faeces and in bovine manure were 8.2+/-0.5, 8.4+/-1.3 and 7+/-0.5 log10 copies per gram, respectively. It was also quantified in samples of runoff water impacted by bovine manure, with average concentrations of 5.1+/-0.3 log10 copies per millilitre water., Conclusions: Our results confirmed that some members of Bacteroidales isolated from effluents and faeces had host-specific distributions. Identification of a bovine-specific cluster made it possible to design a reliable ruminant-specific marker., Significance and Impact of the Study: The host-specific distribution of Bacteroidales sequences from effluents mirrored the host-specific distribution of sequences observed in individual faeces. This efficient new ruminant-specific Bacteroidales 16S rRNA marker represents a useful addition to the microbial source tracking toolbox.

Aims: To investigate the inter-strain variation in (i) substrate utilization and (ii) the restriction fragment length polymorphism (RFLP) pattern based on the distribution of an insertion element (IS1550) in Mycoplasma fermentans strains, and to establish any correlation between subgroups within the species and their source or habitat., Methods and Results: Using a sensitive dynamic pH method, the pattern and kinetics of substrate utilization by a panel of 17 M. fermentans strains from various sources was determined. This study correlated the biochemical characteristics of these strains with RFLP patterns based on the distribution of an insertion sequence (IS1550) with the sources of the strains. The test isolates were divided into four major groups according to the pattern of substrates metabolized. Interestingly, two strains isolated from cell lines in RFLP cluster I failed to utilize arginine. Ovine strains showed distinct substrate utilization patterns and produced RFLP patterns not previously encountered., Conclusions: All strains utilized glucose, but the ability to utilize arginine, fructose and N-acetyl glucosamine varied. There was also some correlation evident between the metabolic data and the RFLP clusters., Significance and Impact of the Study: This study has provided a better understanding of the biochemical and genetic diversity of M. fermentans strains from various sources.

Aims: To determine possible preslaughter and processing sources of psychrophilic and psychrotolerant clostridia causing spoilage of vacuum-packed chilled meats., Methods and Results: Molecular methods based on the polymerase chain reaction (PCR) amplification of specific 16S rDNA fragments were used to detect the presence of Clostridium gasigenes, Clostridium estertheticum, Clostridium algidicarnis and Clostridium putrefaciens in a total of 357 samples collected from ten slaughter stock supply farms, slaughter stock, two lamb-processing plants, their environments, dressed carcasses and final vacuum-packed meat stored at -0.5 degrees C for 5(1/2) weeks. Clostridium gasigenes, C. estertheticum and C. algidicarnis/C. putrefaciens were commonly detected in farm, faeces, fleece and processing environmental samples collected at the slaughter floor operations prior to fleece removal, but all these micro-organisms were detected in only 4 out of 26 cooling floor and chiller environmental samples. One out of 42 boning room environmental samples tested positive for the presence of C. gasigenes and C. estertheticum, but 25 out of 42 of these samples were positive for C. algidicarnis/C. putrefaciens. Nearly all of the 31 faecal samples tested positive for the presence of C. gasigenes and C. estertheticum; however, only two of these samples were positive for C. algidicarnis and/or C. putrefaciens. Clostridial species that were subject to this investigation were frequently detected on chilled dressed carcasses., Conclusions: The major qualitative and quantitative differences between the results of PCR detection obtained with the primers specific for 'blown pack' -causing clostridia (C. gasigenes and C. estertheticum) and those obtained with primers specific for C. algidicarnis and C. putrefaciens suggest that the control of meat spoilage caused by different groups of meat clostridia is best approached individually for each group., Significance and Impact of the Study: This paper provides information significant for controlling meat spoilage-causing clostridia in the meat-processing plants.

Aims: Development of a novel inoculation technique to improve the current methods of determining the leaching of Escherichia coli O157:H7 from faeces., Methods and Results: Ruminant faeces were inoculated with a high [c. 10(7) colony forming units (CFU) g(-1)] or low (c. 10(4) CFU g(-1)) load of a lux-marked strain of E. coli O157:H7 via injection, and subjected to four simulated heavy rainfall events. The population density and metabolic activity of E. coli O157:H7 recovered within the leachate was determined following each simulated rain event and compared with the indigenous E. coli population. The concentration of E. coli O157:H7 in the leachates followed a similar trend to that of nonpathogenic E. coli. Significantly greater densities of generic and pathogenic E. coli were recovered in the leachates generated from sheep faeces compared with cattle faeces. Pathogen metabolic activity was also significantly greater in sheep faeces., Conclusions: Our findings show that E. coli O157:H7 may readily leach from ruminant faeces during rain events. The bacterium leaches more freely from sheep faeces than from cattle faeces and displays greater metabolic activity within sheep leachate., Significance and Impact of the Study: A novel inoculation technique was developed that allowed the determination of both population density and cellular activity of E. coli O157:H7 in leachate derived from faeces.

Aims: To investigate the short- and long-term effects of an extract of Sapindus rarak saponins (SE) on the rumen fibrolytic enzyme activity and the major fibrolytic micro-organisms., Methods and Results: Two feeding trials were conducted. In the short-term trial, four fistulated goats were fed a basal diet containing sugar cane tops and wheat pollard (65:35, w/w) and were supplemented for 7 days with SE at a level of 0.6 g kg(-1) body weight. Rumen liquor was taken before, during and after SE feeding. In the long-term trial, 28 sheep were fed the same basal diet as the goats and were supplemented for 105 days with 0.24, 0.48 and 0.72 g kg(-1) body mass of the extract. Rumen liquor was taken on days 98 and 100. Protozoal numbers were counted under the microscope. Cell wall degradation was determined by enzyme assays and the major fibrolytic micro-organisms were quantified by dot blot hybridization. Sapindus extract significantly depressed rumen xylanase activity in both trials and carboxymethylcellulase activity in the long-term trial (P < 0.01). Fibrobacter sp. were not affected by the SE in both trials, while ruminococci and the anaerobic fungi showed a short-term response to the application of saponins. Protozoal counts were decreased only in the long-term trial with sheep., Conclusion: These data suggest that there is an adaptation of Ruminococcus albus, Ruminococcus flavefaciens and Chytridiomycetes (fungi) to saponin when fed over a long period. The fact that no correlation between the cell wall degrading enzyme activities and the cell wall degrading micro-organisms was observed suggests that the organisms tracked in this experiment are not the only key players in ruminal cell wall degradation., Significance and Impact of the Study: Sapindus rarak saponins partially defaunate the rumen flora. Their negative effect on cell wall degradation, however, is not related to rumen organisms currently recognized as the major cell wall degrading species. The adaptation of microbes in the long-term feeding experiment suggests that the results from short-term trial on the ruminal microbial community have to be interpreted carefully.

Aims: To investigate if cattle on the same farm as sheep are a possible risk factor for stx in sheep and to determine whether or not sheep and cattle on the same farm share the same stx pool., Methods and Results: Faecal samples from sheep and cattle were screened for stx by polymerase chain reaction (PCR). Of these samples, 87.6 and 64.6% were stx positive in sheep and cattle, respectively. There was no difference in stx occurrence in sheep from farms with or without cattle. From stx positive samples, 118 Shiga toxin-producing Escherichia coli (STEC) isolates were recovered by a filter-hybridization method. Serotyping, PCR and pulsed-field gel electrophoresis (PFGE) showed that there was a distinct association between serotypes, stx profiles and animal species., Conclusions: Keeping animals together in pens, which enhances faecal-oral contact, is suggested as a possible explanation for the differences seen in stx occurrence. Sheep and cattle isolates are distinctly different in serotype and stx profile although isolated from the same farm, and are more related to isolates within the same serotype with the same stx profile than to isolates with different serotype from the same farm., Significance and Impact of Study: The study supports the animal-host relationship hypothesis suggested in other studies and indicates that the STEC sheep reservoir in Norway may not pose a serious public health risk.

Aims: Comparison of enterocins produced by six Enterococcus faecium strains and one Ent. faecalis strain isolated from different origin with regard to their microbiological and biochemical characteristics in view of their technological potential and practical use., Methods and Results: The seven enterococci were sensitive to the glycopeptide antibiotics vancomycin and teicoplanin and did not show haemolytic activity. The absence of the glycopeptide-resistant genotypes and the genes involved in the production of the lantibiotic cytolysin was confirmed by PCR. The enterocins were active towards Listeria innocua and other lactic acid bacteria. Their temperature stability was dependent on the pH and their activity was higher at acidic pH. A bactericidal and bacteriolytic effect was shown. PCR analyses revealed that the gene of enterocin A was present in the genome of Ent. faecium CCM 4231, Ent. faecium 306 I.2.20 and Ent. faecalis Y; both enterocin A and B genes were present in the genome of Ent. faecium LMG 11423T, Ent. faecium RZS C5 and Ent. faecium RZS C13. Enterocin P was detected in the genome of Ent. faecium RZS C5 and Ent. faecium RZS C13. No signal was found for Ent. faecium SF 68. Enterocins from Ent. faecium RZS C5, Ent. faecium RZS C13 and Ent. faecium SF 68 were purified to homogeneity., Conclusions: Ent. faecium RZS C5 and Ent. faecium RZS C13 produced an enterocin with a molecular mass of 5460 and 5477 Da, respectively, which was in the range of that of enterocin B. The amino acid sequence analysis of the enterocin from Ent. faecium RZS C13 revealed 24 N-terminal residues, which were identical to those of enterocin B. The enterocin from Ent. faecium SF 68 had a molecular mass of 4488 Da, which did not correspond to any enterocin known so far., Significance and Impact of the Study: The number of characterized enterocins is increasing. As this type of work is tedious and time-consuming, it may be interesting to include PCR as a first step to know if the Enterococcus strain in study produces either a known or a new enterocin. Also, it is important to check the absence of cytolysin and resistance to vancomycin for a further application of the Enterococcus strain in food or health applications.

Aims: To determine the effect of condensed tannins in Calliandra calothyrsus (calliandra) on rumen microbial function., Methods and Results: Microbial populations, ruminal protein synthesis and fermentation end-products were measured in sheep fed roughage hay supplemented with calliandra (30%), with and without inclusions of polyethylene glycol (PEG) to counteract the effect of tannin. Molecular and conventional enumeration techniques were used to quantify rumen bacteria, fungi and protozoa, and protein synthesis was predicted from estimates of urinary purine excretion. The total number of cellulolytic bacteria, including populations of Fibrobacter succinogenes and Ruminococcus spp., was significantly lower in sheep supplemented with calliandra and these populations increased when animals were treated with PEG. By contrast, protozoa and fungi and the microbial group containing Bacteroides-Porphyromonas-Prevotella bacteria appeared to be less affected. The efficiency of microbial protein synthesis in the rumen was not altered significantly., Conclusion: Calliandra caused significant shifts in rumen microbial populations without changing the efficiency of protein synthesis., Significance and Impact of the Study: The effect of calliandra tannins on rumen digestion may result more from complexing with nutrients than direct inhibition of micro-organisms.

The rates at which sheep on different types of pasture shed campylobacters in their faeces were measured over 12 months. Overall, shedding of campylobacters at pasture was between a third and a half of the carriage rate (92%) of the intestines of sheep at slaughter. Shedding was highest during saltmarsh grazing, followed by upland fell and farm grazing. The rate of shedding varied at different times of the year, with the highest rates (100%) coinciding with lambing, weaning, and movement onto new pasture. The lowest rates (0%) occurred when sheep were fed on hay and silage. On the farm, low rates occurred during the whole of gestation, both when the sheep were indoors and outdoors. Campylobacter jejuni was the main species isolated and survived for up to 4 d in sheep faeces. Lambs became colonized by Campylobacter within 1-5 d of being born. Ewes, which were not shedding campylobacters prior to lambing, started to shed after lambing, and ewes which were shedding low numbers of Campylobacter before lambing, increased the numbers of bacteria being shed after lambing.

A survey of the isolation rate and population size of thermophilic campylobacters in lambs at slaughter was carried out to determine the seasonal variation of thermophilic campylobacters in ovine hosts. Isolation rates determined by enrichment methods were always higher than those using direct plating onto selective agar and showed that Campylobacter could be isolated from 91.7% (n = 360) of samples from the small intestine of the lambs. Enumerations (MPN), done monthly over a 2-year period, averaged 4.00 log 10 (n = 1080, S.D. 0.16) campylobacters g-1 fresh weight (fw) intestinal contents with some samples giving values higher than 7 log MPN gfw-1. These results show that the prevalence of thermophilic campylobacters in sheep intended for slaughter is much higher than previously reported. Statistical analyses showed that there was a significant seasonal periodicity in the Campylobacter populations in the small intestines of lambs at slaughter (P = 0.004) but that there was no statistical relationship with environmental parameters such as minimum and maximum temperature, rainfall or hours of sunshine. In an analysis along the length of the intestinal tract of lambs, campylobacters were isolated from 80% of small intestine and 30% of rumen samples, but not from the true stomach, large intestine or caecal samples. A survey of sheep grazing on salt marsh, fell (upland) and farm pasture showed that the isolation rate of thermophilic campylobacters in fresh faeces was substantially lower (29.3% (n = 420)) than that from the small intestine of lambs at slaughter. No significant difference (> 0.05) was found between the isolation rate of campylobacters from faeces sampled in late spring/early summer and autumn, nor between the different types of grazing.