Roger J. Merry

PATHOGENS IN LIVESTOCK WASTE, THEIR POTENTIAL FOR MOVEMENT THROUGH SOIL AND ENVIRONMENTAL-POLLUTION

Abstract Livestock wastes contain many pathogenic microorganisms including bacteria, viruses and protozoa. Following the application of these wastes to land the potential exists for environmental contamination. Plants, soil and ultimately water courses which may subsequently be used as catchments for public water supplies may all be affected. Research attention is now being focused on this possibility, especially in the case of protozoan pathogens which may be the most important as they are often resistant to current methods used in public water treatment. In this review we highlight some of the many factors that are likely to influence the degree of pollution by their effect on both the vertical and horizontal transport of microorganisms through soil. Soil pH, temperature, the presence of plants, microbial surface properties, type of waste, soil type and soil water content and flow may all affect the rate and extent of vertical transport, with the latter two generally considered to be the most important. Lateral movement is a particular problem in soils with impermeable substrata or in waterlogged conditions and in these cases the major factors affecting movement include rainfall rate, topography of the land and the rate at which microorganisms partition into the runoff.

Microbial protein supply from the rumen.

Abstract This paper considers the theory, assessment and prediction of microbial protein synthesis in the rumen. The difficulties of techniques for assessing microbial protein synthesis, as well as the complexity of the rumen ecosystem, have limited progress. Inconsistencies in the literature undermine the value of incorporating advanced rumen models into rationing schemes and limit the exploitation of microbial protein as an important protein resource for ruminants. The paper gives examples of situations in which particular factors have significant effects on microbial protein synthesis, but moves on to discuss the development of new less-invasive approaches for estimating microbial protein synthesis. The latter approaches have the attraction of offering in-built technology transfer through the development of diagnostic tests, based on samples of milk or urine. Some of these techniques offer a description of rumen function that is less rigorously quantitative (in terms of microbial protein synthesis), but more usefully qualitative (in terms of microbial populations, substrates and interactions).

Movement of the protozoan pathogen Cryptosporidium parvum through three contrasting soil types

The potential for transfer of the protozoan pathogen Cryptosporidium parvum through soil to land drains and, subsequently, water courses following the application of livestock waste to land was monitored in the laboratory using simulated rainfall and intact soil cores. Following irrigation over a 21-day period, Cryptosporidium parvum oocysts applied to the surface of soil cores (initial inoculum concentration 1×108 oocysts core−1) were detected, albeit in low numbers, in the leachates from clay loam and silty loam soils but not in that from a loamy sand soil. Variations in leaching patterns were recorded between replicate cores. At the end of the study soil cores were destructively sampled to establish the location of oocysts remaining within the soil. Distribution within cores was similar in all three soil types. The majority (72.8+-5.2%) of oocysts were found in the top 2 cm of soil, with numbers decreasing with increasing depth to 13.2±2.8%, 8.39±1.4%, and 5.36±1.4% at depths of 10, 20, and 30 cm, respectively.

The effect of clover silages on long chain fatty acid rumen transformations and digestion in beef steers

Lee, M. R. F., Harris, L. J., Dewhurst, R. J., Merry, R., Scollan, N. D. (2003). The effect of clover silages on long chain fatty acid rumen transformations and digestion in beef steers. Animal Science, 76, (3), 491-501.

An automated system for measuring gas production from forages inoculated with rumen fluid and its use in determining the effect of enzymes on grass silage.

This paper describes an automated system that has been developed to measure the production of fermentation gas from ruminant livestock feeds inoculated with rumen fluid. The design of the apparatus and its method of use enables gas production to be determined from fresh, unprocessed plant material, as well as the more commonly used ground, particulate substrates, thus representing a closer simulation of forages consumed in vivo. The system consists of 48×140 ml bottles containing 100 ml buffered rumen fluid and 1 g of test substrate. Gas is produced as a consequence of the fermentation of the substrate. Gas, accumulating in the head-space of bottles, is released automatically, by use of pressure sensitive switches and solenoid valves, when a pre-determined pressure is reached. This prevents any build up of pressure in the fermentation bottle, which can affect the behaviour of the gas and the fermentation process. Gas accumulation profiles are produced as the fermentation proceeds and give information on forage digestibility and fermentation kinetics. In this paper, we describe the principles of the gas production technique and provide examples of how the automated system has been used in the evaluation of forages for ruminants. The results obtained show that the automated system is a useful tool for the determination of fermentation kinetics of ruminant feeds. It is simple to use and is considerably less labour intensive than manual gas measurement techniques.

Use of a novel soil tilting table apparatus to demonstrate the horizontal and vertical movement of the protozoan pathogen Cryptosporidium parvum in soil

A novel greenhouse based soil tilting table apparatus was used to investigate the potential for movement of the protozoan pathogen Cryptosporidium parvum both through and across a low permeability soil following the application of contaminated livestock waste to land. Soil blocks supported at an angle of 7.5% by the soil table were inoculated at one end with oocyst seeded slurry and subsequently irrigated at regular intervals over a 70-day period. Movement of the pathogen in runoff was demonstrated for at least 21 days and in one case in excess of 70 days from the time of inoculation. Water was also lost following percolation down through the soil profile and significant numbers of oocysts were also lost via this route, average numbers leached decreasing from 8.36±0.56×106 at day 1 to 2.27±0.73×104 at day 70. At the end of the study cores were removed from the soil blocks to determine the location of oocysts remaining within the soil. Numbers decreased down through the soil profile and as the distance from the point of inoculation increased so that 70 cm from the point of inoculation no oocysts could be detected in the soil at any depth. This implies that oocysts contained in runoff stay in the aqueous phase and do not precipitate out onto the soil surface, suggesting that even if the distances travelled are increased there may still be a significant pollution threat.

Effect of different levels of phosphorus on rumen microbial fermentation and synthesis determined using a continuous culture technique.

A continuous culture technique was used to study the phosphorus requirements of rumen micro-organisms. Solutions of artificial saliva containing 120, 80, 40 and 0 mg inorganic phosphorus (Pi)/l were infused into the reaction vessels previously inoculated with rumen contents, resulting in Pi concentrations in the vessel contents of 48, 28, 4 and less than 1 mg/l respectively. Various fermentative and synthetic characteristics were examined. In the vessel contents, concentrations of protozoa (about 0.9 X 10(5)/ml) were not significantly affected by Pi concentration. Total volatile fatty acids (VFA) produced averaged about 6.83 mmol/h with Pi levels of 48 and 28 mg/l. Reduction in Pi concentrations to 4 and less than 1 mg/l resulted in significant reductions in total VFA to approximately 6.25 and 3.75 mmol/h respectively, accompanied by a rise in pH from 6.5 to 7.3. Ammonia-nitrogen values, which averaged about 131 mg/l at the higher Pi concentrations, also increased with the lowest level of Pi to about 240 mg/l. ATP concentrations averaged about 14 mumol/l at the highest Pi concentration and fell progressively with each reduction in Pi concentration to a final value of 2.5 mumol/l with the Pi level less than 1 mg/l. At Pi concentrations of 48 and 28 mg/l, the digestibilities of xylose, arabinose and cellulose-glucose were maintained at about 0.90, 0.62 and 0.70 g/g input respectively. At lower Pi concentrations these digestibilities fell significantly and corresponding values at Pi less than 1 mg/l were 0.73, 0.41 and 0.31 respectively. Starch digestion was unaffected by Pi concentration and remained at about 0.90 g/g input. The amount of microbial-N synthesized averaged 0.48 g/d and was maintained with Pi concentrations down to 4 mg/l. There was, however, a significant reduction to 0.26 g/d with Pi concentrations of less than 1 mg/l. The efficiency of microbial protein synthesis was variable but averaged approximately 25 g N/kg total carbohydrate fermented. It was estimated that the minimum Pi concentrations required in rumen fluid in vivo to maintain maximum degradative and synthetic microbial activities was in the range 75-100 mg/l and that over-all P requirement of the microbes was of the order of 5.1 g/kg apparently digested organic matter intake.

Contribution of rumen protozoa to duodenal flow of nitrogen, conjugated linoleic acid and vaccenic acid in steers fed silages differing in their water-soluble carbohydrate content

The present experiment was designed to estimate the quantitative contribution of rumen protozoa to the total N, conjugated linoleic acid (CLA) and vaccenic acid (VA; trans-11-18 : 1) flow to the duodenum of steers fed two silage diets: control silage (CS) and silage high in water-soluble carbohydrates (HS). Protozoal duodenal flows were estimated using a real-time PCR assay to quantify the genes encoding protozoal 18S ribosomal RNA. Denaturing gradient gel electrophoresis was used to confirm that the rumen protozoa populations were similar to the protozoal population flowing to the duodenum. Estimated duodenal flow of protozoal N was 14.2 and 18.2 g/d (P>0.05) for animals fed the CS and HS diets respectively. Protozoal flow thus represented between 12 and 15 % of the total N duodenal flow. In terms of fatty acid flow, protozoa accounted for between 30 and 43 % of the CLA and 40 % of the VA reaching the duodenum. The contribution of protozoa to 16 : 0 and 18 : 0 flows to the duodenum was less than 20 and 10 %, respectively. These results show that the fatty acids within protozoa make up a significant proportion of the CLA and VA reaching the duodenum of ruminants.

High-throughput metabolic fingerprinting of legume silage fermentations via Fourier transform infrared spectroscopy and chemometrics

ABSTRACT Silage quality is typically assessed by the measurement of several individual parameters, including pH, lactic acid, acetic acid, bacterial numbers, and protein content. The objective of this study was to use a holistic metabolic fingerprinting approach, combining a high-throughput microtiter plate-based fermentation system with Fourier transform infrared (FT-IR) spectroscopy, to obtain a snapshot of the sample metabolome (typically low-molecular-weight compounds) at a given time. The aim was to study the dynamics of red clover or grass silage fermentations in response to various inoculants incorporating lactic acid bacteria (LAB). The hyperspectral multivariate datasets generated by FT-IR spectroscopy are difficult to interpret visually, so chemometrics methods were used to deconvolute the data. Two-phase principal component-discriminant function analysis allowed discrimination between herbage types and different LAB inoculants and modeling of fermentation dynamics over time. Further analysis of FT-IR spectra by the use of genetic algorithms to identify the underlying biochemical differences between treatments revealed that the amide I and amide II regions (wavenumbers of 1,550 to 1,750 cm−1) of the spectra were most frequently selected (reflecting changes in proteins and free amino acids) in comparisons between control and inoculant-treated fermentations. This corresponds to the known importance of rapid fermentation for the efficient conservation of forage proteins.

Evidence in support of a role for plant-mediated proteolysis in the rumens of grazing animals

The present work aimed to differentiate between proteolytic activities of plants and micro-organisms during the incubation of grass in cattle rumens. Freshly cut ryegrass was placed in bags of varying permeability and incubated for 16 h in the rumens of dairy cows that had previously grazed a ryegrass sward, supplemented with 4 kg dairy concentrate daily. Woven polyester bags (50 microm pore size) permitted direct access of the micro-organisms and rumen fluid enzymes to the plant material. The polythene was impermeable even to small molecules such as NH(3). Dialysis tubing excluded micro-organisms and rumen enzymes/metabolites larger than 10 kDa. DM loss was 46.3 % in polyester, 36.2 % in polythene and 38.1 % in dialysis treatments. It is possible that the DM loss within polythene bags occurred due to a solubilisation of plant constituents (e.g. water-soluble carbohydrates) rather than microbial attachment/degradation processes. The final protein content of the herbage residues was not significantly different between treatments. Regardless of bag permeability, over 97 % of the initial protein content was lost during incubations in situ. Electrophoretic separation showed that Rubisco was extensively degraded in herbage residues whereas the membrane-associated, light-harvesting protein remained relatively undegraded. Protease activity was detected in herbage residues and bathing liquids after all incubation in situ treatments. Although rumen fluid contains proteases (possibly of plant and microbial origin), our results suggest that, owing to cell compartmentation, their activity against the proteins of intact plant cells is limited, supporting the view that plant proteases are involved in the degradation of proteins in freshly ingested herbage.