The monitoring of opportunistic macroalgal blooms for the water framework directive.

Among the various quality elements which the Water Framework Directive requires should be monitored are macroalgae. One aspect of these is the presence, in transitional waters particularly, of large blooms of opportunistic macroalgae, such as Ulva and Enteromorpha. Within the United Kingdom (UK) and Republic of Ireland (RoI) there are currently no set ecological quality objectives or standards for macroalgae. Nor are there standard methods for monitoring macroalgal blooms, although various combinations of aerial photography, remote sensing and measurements on the ground are used. This paper attempts to set a logical framework for the prioritisation of sites for monitoring, the development of a tiered monitoring procedure and the derivation of thresholds for classification. Draft threshold limits for percentage cover and biomass of macroalgae have been derived from the literature. The importance of secondary effects and physico-chemical parameters is discussed.

Bacteriology of preserved stallion semen and antibiotics in semen extenders.

Three experiments were conducted to evaluate the effects of different antibiotics in a milk-glucose semen extender on motility of equine sperm and elimination of bacteria following storage of extended semen in vitro. In Experiment 1, 7 antibiotics were compared: amikacin, gentamicin, streptomycin, potassium penicillin, sodium penicillin, ticarcillin, and polymixin B. In Experiment 2, 3 antibiotic treatments were compared: potassium penicillin G, amikacin, or a combination of potassium penicillin G and amikacin. In Experiment 3, 3 antibiotic treatments were compared: potassium penicillin G-amikacin, ceptiofur, and a combination of ticarcillin and clavulanic acid (Timentin). Control treatments (antibiotic-free extender) were included in each experiment. Six motility variables were evaluated: percentage of motile sperm; percentage of progressively-motile sperm; percentage of rapidly-motile sperm; mean curvilinear velocity; mean average path velocity; and mean straight-line velocity. In Experiment 1, mean percentages of motile, progressively motile and rapidly motile sperm were lower (P < 0.05) in semen exposed to polymixin B then in other treatments. Mean average-path velocity of sperm in extender containing polymixin B was lower (P < 0.05) than that of all other treatments, with exception of control or ticarcillin. Mean straight-line velocity of sperm in extender containing polymixin B was lower (P < 0.05) than that of all other treatments, with exception of control, streptomycin or ticarcillin. Semen samples containing gentamicin, amikacin, streptomycin, or potassium penicillin were more effective (P < 0.05) at eliminating bacterial growth than those samples containing polymixin B. Semen samples containing gentamicin were also more effective (P < 0.05) at eliminating bacterial growth than those samples containing ticarcillin or sodium penicillin. In Experiment 2, mean percentage of rapidly-motile sperm, and mean curvilinear, average-path, and straight-line velocities were greater (P < 0.05) for potassium penicillin-amikacin than values for all other treatments. In 2 of 3 stallions, an effect of treatment on percentage of motile sperm was detected (P < 0.05). For one stallion, mean motility of potassium penicillin-amikacin was greater (P < 0.05) than that of all other treatment groups. For another stallion, mean motility of the control was lower (P < 0.05) than that of the other treatments. Following storage, potassium penicillin (16/18 [89%]) or potassium penicillin-amikacin (17/19 [94%]) were more effective (P < 0.05) at controlling aerobic and anaerobic bacterial isolates in semen specimens than was amikacin (10/18 [56%]). In Experiment 3, a difference among treatment groups for motility variables was not detected (P < 0.05). No bacterial growth was recovered in antibiotic-treated semen, with exception of Micrococcus sp. (2 colonies) which were isolated from one semen specimen treated with ceptiofur.

Current concepts of competitive exclusion cultures for the control of salmonellae in domestic poultry.

Two defined competitive exclusion (CE) cultures (CF-I and CF-II) and a characterized CE culture (CF-III), which are composed of mixtures of nonpathogenic bacteria, were developed from anaerobic continuous-flow (CF) cultures that had been inoculated with cecal contents from adult chickens. After the primary CF cultures attained homeostasis, 13 bacteria (11 facultative anaerobes and 2 obligate anaerobes) representing 6 genera were isolated from CF-I; II bacteria (9 facultative anaerobes and 2 obligate anaerobes) representing 7 genera were isolated from CF-II; and 29 bacteria (15 facultative anaerobes and 14 obligate anaerobes) representing 14 genera were isolated from CF-III. Newly hatched chicks were treated orally with each primary CF culture; challenged on day 3 with Salmonella typhimurium; and cultured on day 10. Each culture significantly (p < 0.05) reduced salmonellae intestinal colonization and organ invasion. From the reconstituted CF-I and CF-II cultures, all organisms were isolated and their fermentation parameters and efficacy against salmonellae challenge were similar, if not identical, to the primary cultures. The CF-I and CF-II cultures satisfied the 5 requirements for defined CE cultures: 1) the primary CE culture must be efficacious; 2) all bacteria must be isolated and identified; 3) the fermentation parameters of the reconstituted CE culture must be similar to those of the primary culture; 4) all bacteria from the reconstituted culture must be isolated and identified; and 5) the efficacy of the reconstituted culture must be very similar to the primary culture. From these integrated studies, 3 mechanisms were demonstrated for preventing the enteric colonization of salmonellae in newly hatched chicks that were pretreated with CE cultures. First, the component organisms in the CE culture establish a normal enteric flora prior to salmonellae exposure. Second, the CE organisms compete with salmonellae for essential nutrients. Third, the CE organisms produce concentrations of volatile fatty acids at low pH levels that are bacteriostatic for salmonellae.

Fermentation of radiolabeled carbohydrates by a reconstructed continuous-flow culture effective against Salmonella in broiler chicks.

An 81-d-old continuous-flow (CF) culture of broiler cecal bacteria was maintained in a lactose-based broth. The culture had been previously proven effective against Salmonella colonization in young chicks, especially when the chicks were provided dietary lactose. Portions of the CF culture were batch-cultured in glucose- and lactose-based broths containing 14C-labeled lactose, glucose, galactose, or lactic acid to determine the effect of media carbohydrate on fermentation products. Acetic and propionic acids were the major 14C-labeled fermentation products. 14C-Carbohydrates were fermented to lactic acid and then to acetic and propionic acids. Distribution of radiolabeled fermentation products was effected by the broth carbohydrate and the time postinoculation.

Treatment of commercial broiler chickens with a characterized culture of cecal bacteria to reduce salmonellae colonization.

The effect of treatment with a newly developed characterized continuous-flow (CCF) culture composed of 29 strains of cecal bacteria on salmonellae colonization was evaluated in commercially reared broiler chickens. Newly hatched chicks in three flocks were sprayed with CCF culture as they were placed in rearing houses and compared with untreated flocks on the same farm. Hatchery transport tray liners and rearing house feed, water, and little samples were cultured for the presence of salmonellae. Cecal samples were cultured after 3- and 6-wk growout. Skin-feather samples were cultured at 6-wk growout. Fifty-five percent of the transport tray liners were contaminated with salmonellae on the day of chick placement. At 3 wk, salmonellae serotypes present on the tray liners were widely distributed in the litter of the rearing houses. The results indicated that exposure to salmonellae occurred before culture treatment and continued in the rearing houses during the 6-wk growout period. Salmonellae cecal colonization was decreased (P < .05) in two of the treated flocks at 3 wk and diminished (P < .07) in the third treated flock compared with control flocks. At 6 wk, skin-feather contamination and cecal colonization were decreased (P < .05) in one of the treated flocks whereas no treatment effect occurred in two of the treated flocks compared with controls. The results clearly indicate the necessity of implementing integrated programs to control salmonellae in both the hatchery and rearing house environments. The CCF culture served to enhance salmonellae colonization resistance and may serve as a useful component of an integrated control program.

Fermentation of radiolabelled substrates by batch cultures of caecal microflora maintained in a continuous-flow culture.

Glucose- and lactose-based media containing either 14C-labelled glucose, galactose, lactose or lactic acid were inoculated with anaerobic cultures of chicken caecal bacteria maintained for 121 d in a continuous-flow (CF) culture. The culture was previously shown to reduce Salmonella colonization in the caeca of chicks inoculated with the culture and the reduction was associated with increases in volatile fatty acids. The distributions of 14C were determined among the fermentation products, especially acetic, propionic and lactic acids. After 12 h of incubation and fermentation, variations were observed in the total amounts of 14C, from each 14C-labelled substrate, detected as acetic and propionic acids in the glucose- and lactose-based media, respectively: 92.9 and 89.4% of 14C-glucose, 77.0 and 44.0% of 14C-galactose, 0.0 and 76.9% of 14C-lactose, and 76.5 and 93.2% of lactic acid in the two media, respectively. The results from this study indicate that carbohydrate metabolism by the CF culture was a major source of acetic and propionic acids and that lactic acid was an important metabolic intermediate for the synthesis of the volatile fatty acids.

Control of Salmonella typhimurium colonization in broiler chicks with a continuous-flow characterized mixed culture of cecal bacteria.

A continuous-flow culture system was used to isolate and maintain a mixed culture of cecal bacteria from adult broilers composed of 29 bacterial strains representing 10 genera. Broiler chicks were treated with the mixed culture in the drinking water on the day of hatch and challenged orally with 10(4) Salmonella typhimurium 2 d after treatment. The experiment was repeated in four separate trials using newly hatched chicks. The concentration of propionic acid and total volatile fatty acid (VFA) in the cecal contents was determined 2 d after treatment and at 10 d of age. Compared with controls, the number of treated chicks that were cecal culture-positive for Salmonella decreased (P < .01) in each of the trials. Additionally, the number of Salmonella in the cecal contents of the treated chicks at 10 d of age was decreased (P < .005) compared with controls in each trial. The decreased number of Salmonella in the cecal contents of the 10-d-old treated chicks was correlated with elevated concentrations of propionic acid (P < .05) and total VFA (P < .1) in the cecal contents of the treated chicks 2 d after treatment. The results indicated that VFA-producing bacteria present in the mixed culture became rapidly established in the ceca of the treated chicks and effectively increased resistance to S. typhimurium challenge.

Use of Differential Rumen Fluid-Based Carbohydrate Agar Media for Culturing Lactose-Selected Cecal Bacteria from Chickens.

A rumen fluid-based differential carbohydrate agar medium for enumerating chicken cecal carbohydrate-utilizing bacteria was tested. Eleven bacteria isolated from a continuous-flow culture that had been seeded with chicken cecal contents were inoculated on fifteen different media in an anaerobic environment. These media included various levels of rumen fluid (0%, 5%, 10%, 16%) and carbohydrate (glucose [GLU], galactose [GAL], lactose[LAC]). Viable cell recovery (colony-forming units [CFU]/direct cell counts × 100%) was higher for 16% rumen fluid-supplemented carbohydrate media than the lower concentrations of rumen fluid-supplemented carbohydrate media. In addition, average viable cell recovery of all 11 bacteria on differential carbohydrate media were significantly (P < 0.05) higher in galactose and lactose media than on the glucose media. Data indicated that 16% rumen-fluid based media can be used to enumerate anaerobic, lactose-fermenting subgroups of chicken cecal bacteria.

Resistance against Salmonella enteritidis cecal colonization in Leghorn chicks by vent lip application of cecal bacteria culture.

Leghorn chicks were treated with cultures of cecal bacteria from adult chickens by crop gavage, upper body spray, or vent lip application on the day of hatch. The chicks were challenged orally with 10(4) Salmonella enteritidis (SE) at 3 d of age and evaluated for SE cecal colonization at 10 d of age. The concentration of volatile fatty acids (VFA) in the cecal contents was determined on the day after culture treatment and at 10 d of age. Compared with controls, SE colonization was significantly decreased in each of the treatment groups. Vent lip application of a single .05-mL drop of cecal bacteria culture resulted in resistance against SE challenge comparable to crop gavage or spray treatment with .5 mL of culture. Resistance to SE challenge was directly associated with the concentrations of total VFA and propionic acid in the cecal contents of the treated chicks on the day after culture treatment. The results indicated that cecal bacteria from adult chickens that increase SE colonization resistance may rapidly become established in the ceca of newly hatched chicks following contact with the vent lips.

Competitive exclusion of Salmonella enteritidis in Leghorn chicks: comparison of treatment by crop gavage, drinking water, spray, or lyophilized alginate beads.

The protective effect of cecal bacteria cultures on Salmonella enteritidis cecal colonization was evaluated. Competitive-exclusion cultures were administered by crop gavage, in first drinking water, by whole body spray, or encapsulated in alginate beads and provided in feed pans. Leghorn chicks were treated with cultures of cecal bacteria on the day of hatch and challenged orally with 10(4) S. enteritidis 2 days after treatment. Salmonella cecal colonization was evaluated 7 days after challenge. No Salmonella organisms were detected in the ceca of chicks treated with cecal cultures by crop gavage. Chicks treated with cecal cultures in the drinking water or by spray application showed comparable protection and significant decreases (P < 0.05) in the number of Salmonella in the cecal contents compared with untreated controls. The consumption of cecal bacteria encapsulated in alginate beads significantly decreased (P < 0.05) Salmonella cecal colonization compared with control treatment, but it provided less protection than the other treatment methods evaluated.

Effect of dietary lactose and cell concentration on the ability of a continuous-flow-derived bacterial culture to control Salmonella cecal colonization in broiler chickens.

The effect of dietary lactose and of cell concentration of a continuous-flow (CF) derived bacterial culture on Salmonella typhimurium cecal colonization in 10-d-old broiler chickens was examined. One-day-old chicks were provided 1) no CF culture and the control diet; 2) no culture and a 2% lactose diet; 3) CF culture (10(8) or 10(11) anaerobic cfu) and control diet; or 4) CF culture (10(8) or 10(11) anaerobic cfu) and 2 to 4% lactose diet. All groups were challenged orally with 10(4) S. typhimurium at 3 d of age. Salmonella typhimurium growth in cecal contents was significantly decreased (P < .05) at 10 d of age when 2% lactose was used in combination with CF culture containing 10(8) or 10(11) anaerobic cfu. The protection factor (log10 Salmonella control diet divided by log10 Salmonella treatment group) for these treatment groups was 2.49 and 9.26, respectively. Results indicate that birds treated with CF culture and maintained on 2% dietary lactose are protected against S. typhimurium colonization. Additionally, inoculating birds with CF culture containing a higher concentration of anaerobic colony-forming units resulted in a substantially higher protection factor.

In vitro metabolism of radiolabeled carbohydrates by protective cecal anaerobic bacteria.

Cecal anaerobic bacteria from adult broilers were cultured in media containing .25% glucose or .25% lactose. Media also contained either [14C]-labeled lactose, glucose, galactose, or lactic acid as metabolic tracers. Cultures were analyzed at 4, 8, and 12 h for pH, radiolabeled and unlabeled volatile fatty acids, and lactic acid. The pH values of cultures containing .25% lactose were significantly (P < .05) higher than the pH values of cultures containing .25% glucose. Lactose cultures reached their lowest pH more slowly than glucose cultures. Concentrations of unlabeled volatile fatty acids increased and lactic acid decreased during incubation of the cultures. Radiolabeled sugars and lactic acid were more readily metabolized to volatile fatty acids in media containing lactose than in media containing glucose. The preferred metabolism of [14C]substrates, independent of media carbohydrate, was in the following order: lactic acid > galactose, lactose > glucose. The volatile fatty acids in which radiolabel was most concentrated were acetic acid, propionic acid, or butyric acid.

Effect of a defined continuous-flow derived bacterial culture and dietary lactose on Salmonella typhimurium colonization in broiler chickens.

A defined bacterial culture protective against Salmonella typhimurium cecal colonization in broiler chicks was derived utilizing a continuous-flow (CF) culture apparatus. Chicks receiving the CF culture in combination with a diet containing dietary lactose were protected against cecal colonization by S. typhimurium. The culture consisted of a mixture of gram-positive and gram-negative facultative and strictly anaerobic bacteria. The isolates were identified as Enterococcus avium, two strains of Enterococcus faecalis (designated A and B), Lactococcus lactis, Lactobacillus animalis, a Lactobacillus that could not be identified to species level (designated strain CMS), Citrobacter freundii, Escherichia coli, E. fergusonii, Bifidobacterium animals, and Propionibacterium acidipropionici. Results indicated that CF cultures can be used as a tool to identify bacteria which are antagonistic to S. typhimurium in the chick cecum.

Combined enzyme and antimicrobial susceptibility profiles of caprine Pasteurella haemolytica serovar 2 respiratory tract isolates.

The present investigation describes a novel method of demonstrating strain diversity among Pasteurella haemolytica biovar A, serovar 2 (PhA2) nasal turbinate isolates from a flock of 32 experimental goats during a naturally occurring outbreak of pasteurellosis. After a 21 day conditioning period in a feedyard, 51 PhA2 isolates from 27 culture-positive goats were identified including 1 on day 22, 14 on day 25, 21 on day 39, and 15 on day 66. Each PhA2 isolate was evaluated for its enzyme activity against 19 substrates with a commercial semiquantitative enzyme system and for its antimicrobial susceptibility with 12 drugs, resulting in 7 different enzyme profiles and 8 different antimicrobial susceptibility profiles. A total of 14 combined enzyme and antimicrobial susceptibility profiles were produced. The same PhA2 strain was isolated from only 4 of the 12 goats with 2 PhA2 isolations, while the same PhA2 strain was isolated from only 1 of the 6 goats with 3 PhA2 isolations. The data from this investigation demonstrated that the PhA2 upper respiratory tract flora from goats is highly heterologous.

Development of defined cultures of indigenous cecal bacteria to control salmonellosis in broiler chicks.

An in vitro continuous-flow (CF) culture system was utilized to isolate and maintain a defined mixed culture of indigenous cecal bacteria from adult broilers. The protective effects of the defined CF culture and dietary lactose on Salmonella typhimurium colonization were evaluated in broiler chicks. The CF culture was administered to chicks by crop gavage on the day of hatch. Lactose was provided as 5% (wt/wt) of the feed ration. The chicks were challenged orally with 10(4) S. typhimurium at 3 days of age and evaluated for Salmonella colonization 7 days after challenge. The experiment was repeated in six separate trials using newly hatched chicks and CF culture that was maintained in continuous steady-state conditions from 42 to 190 days. Compared with controls, the mean number of S. typhimurium in the cecal contents of the chicks given CF culture and dietary lactose decreased significantly (P < .01) by 4.2 log10 units. Similarly, the numbers of Salmonella cecal culture-positive chicks was significantly decreased (P < .01) by 55% in the chicks given CF culture and lactose. The results indicated that a defined culture of indigenous cecal bacteria isolated and maintained in CF culture, together with dietary lactose, effectively controlled S. typhimurium cecal colonization in newly hatched broiler chicks.

Research note: differential chemotaxis of Physarum polycephalum to Salmonella gallinarum and Salmonella pullorum.

The chemotactic and chemotropic responses of the plasmodial stage of the slime mold Physarum polycephalum were used to distinguish Salmonella gallinarum and Salmonella pullorum from 10 Salmonella serovars that are commonly isolated from domestic poultry. Utilizing an in vitro plasmodium agar plate assay method, P. polycephalum was attracted to S. gallinarum and S. pullorum, but the organism was repelled by Salmonella derby, Salmonella dublin, Salmonella enteritidis, Salmonella heidelberg, Salmonella minnesota, Salmonella montevideo, Salmonella newington, Salmonella newport, Salmonella reading, and Salmonella typhimurium.