An investigation into the pathogenic properties of Escherichia coli strains BLR, BL21, DH5 alpha and EQ1

Aims: To examine Escherichia coli strains EQ1, DH5 alpha, BLR and BL21 for known pathogenic mechanisms. Methods and Results: Using specific DNA probes, the strains were shown not to carry the genes encoding invasion, various adhesion phenotypes or expression of a range of enterotoxins. The strains were unable to express long-chain lipopolysaccharide and were susceptible to the effects of serum complement. Using a BALB/c mouse model, the strains were shown to be unable to survive in selected tissues or to persist in the mouse gut. Using a chick model, strains EQ1, BLR and BL21 invaded livers but not spleens; only strain EQ1 persisted in the chick gut. In Merino sheep, only strain EQ1 was detected 6 d postinfection. Conclusions: Escherichia coli strains EQ1, DH5 alpha, BLR and BL21 did not carry the well-recognized pathogenic mechanisms required by strains of E. coli causing the majority of enteric infections. Significance and Impact of the Study: Escherichia coli strains EQ1, DH5 alpha, BLR and BL21 were considered to be non-pathogenic and unlikely to survive in host tissues and cause disease.

Attaching and effacing lesions caused by Escherichia coli O157:H7 in experimentally inoculated neonatal lambs

Four 6-day-old conventionally reared lambs were inoculated orally with a total of 10(9) cfu comprising equal numbers of four enterohaemorrhagic Escherichia coli (EHEC) O157:H7 strains. All animals remained clinically normal. Tissues were sampled under terminal anaesthesia at 12, 36, 60 and 84 h post inoculation (hpi). EHEC O157:H7 was cultured from most gastrointestinal tract sites. Small, sparse attaching and effacing (AE) lesions were found in the caecum at 12 and 36 hpi and in the terminal colon and rectum at 84 hpi. Organisms in the lesions were labelled specifically by an O157 antiserum. The results indicate that the well-characterised mechanisms for intimate attachment encoded by the locus for enterocyte effacement (LEE) of EHEC O157:H7 may contribute to the initial events. at least, of colonisation of sheep.

Prevalence of Leptospira species among farmed and domestic animals in Greece

A total of 1527 serum samples from pigs, goats, sheep, cattle and dogs in Greece were examined by the microscopic agglutination test and 11.8 per cent of them had antibodies against one or more Leptospira serovars at titres of 1/100 or more. The predominant serovar affecting farm animal species was Bratislava, and Copenhageni was common among dogs and the second most important serovar when all animals were considered together. Another prevalent serovar was Australis, but antibodies to Pomona were detected only in goats and cattle.

Characterisation of attaching-effacing Escherichia coli isolated from animals at slaughter in England and Wales

Escherichia coli isolates were recovered from faecal samples taken from cattle, sheep and pigs at slaughter in England and Wales. Isolates (n = 1227) selected at random from this collection were each hybridised in colony dot-blot experiments with an eae gene probe that presumptively identified attaching-effacing E. coli (AEEC). Of the 99 (8.1%) eae positive isolates 72 were of ovine origin, 24 were of bovine origin and three of porcine origin. None were typed as O157:H7 whereas 78 were assigned to 23 serogroups and 21 were untypable. The most frequently isolated eae positive serogroups were O156 (10), O26 (8), O103 (8), O108 (7) O56 (6) and O168 (6) of which serogroups O103 and O156 only were recovered from all three animal species. In tissue culture adherence assays, 36 representatives of eae positive isolates of all serogroups and host of origin tested induced intimate attachment with varying degrees of actin accumulation and pedestal formation in the HEp-2 cells. The identity of the eae type for these 36 was determined by specific PCR and the most prevalent intimin types were caebeta (15), eaegamma (12) and eaeepsilon (4). Isolates were examined by PCR for the presence of other virulence determinants and five possessed stx1 but none possessed stx2. One O115 eaeepsilon isolate possessed cnf1 and 2, hlyA, etpD and katP genes which is a novel combination of virulence determinants.

Interaction between attaching and effacing Escherichia coli serotypes O157:H7 and O26:K60 in cell culture

Ruminants harbour both O157:H7 and non-O157 Attaching Effacing Escherichia coli (AEEC) strains but to date only nonO157 AEEC have been shown to induce attaching effacing lesions in naturally infected animals. However, O157 may induce lesions in deliberate oral inoculation studies and persistence is considered dependent upon the bacterially encoded locus for enterocyte effacement. In concurrent infections in ruminants it is unclear whether non-O157 AEEC contribute either positively or negatively to the persistence of E. coli O157:H7. To investigate this, and prior to animal studies, E. coli O157:H7 NCTC 12900, a non-toxigenic strain that persists in conventionally reared sheep, and non-toxigenic AEEC O26:K60 isolates of sheep origin were tested for adherence to Hep-2 tissue culture alone and in competition one with another. Applied together, both strains adhered in similar numbers but lower than when either was applied separately. Pre-incubation of tissue culture with either one strain reduced significantly (P < 0.05) the extent of adherence of the strain that was applied second. It was particularly noticeable that AEEC O26 when applied first reduced adherence and inhibited microcolony formation, as demonstrated by confocal microscopy, of E. coli 01 57:H7. The possibility that prior colonisation of a ruminant by non-O157 AEEC such as O26 may antagonise O157 colonisation and persistence in ruminants is discussed. (C) 2004 Elsevier B.V. All rights reserved.

Synergistic effects of mixing cocksfoot and sainfoin on in vitro rumen fermentation: role of condensed tannins

Some bioactive secondary metabolites in forage legumes can cause digestive interactions, so that the rumen fermentation pattern of a mixture of forages can differ from the average values of its components. The objective of this study was to investigate the potential role of condensed tannins (CT) on the synergistic effects between one grass species, cocksfoot, and one CT-containing legume species, sainfoin, on in vitro rumen fermentation characteristics. Cocksfoot and sainfoin in different proportions (in g/kg, 1000:0, 750:250, 500:500, 250:750 and 0:1000) were incubated under anaerobic conditions in culture bottles containing buffered rumen fluid from sheep. Incubations were carried out using artificial saliva with and without polyethylene glycol (PEG), which binds and thus inactivates CT. Rumen fermentation parameters describing the degradation and the fate of the energetic and nitrogenous substrates were measured at 3.5 and 24 h. At the early fermentation stage, when the sainfoin level increased from 0 to 1000 g/kg, the ammonia concentration in the medium quadratically decreased from 3.20 to 0.53 mmol/l in absence of PEG (P<0.01) but not in its presence. This result demonstrates that sainfoin CT decreased the rumen degradation of the proteins in the whole mixture, including the proteins in cocksfoot, rather than just the proteins in sainfoin. Interestingly, the total gas and methane productions were lower in mixtures incubated in absence of PEG than in presence of PEG (P<0.001) while no significant PEG effect was observed on digestibility. At the late fermentation stage, a positive quadratic effect on dry matter digestibility was detected without PEG (P<0.05), indicating a synergistic action of cocksfoot plus sainfoin on plant substrate degradation due to CT. The presence of PEG increased gas production (P<0.001) and NH3-N concentration in the medium (P<0.001). Our results suggest that CT could allow a better utilization of plant substrates in mixtures by the rumen ecosystem by improving the partitioning of degraded substrates toward lower gas losses, and decreasing the protein degradation.

A refined sampling strategy for intra-tooth stable isotope analysis of mammalian enamel

Serial sampling and stable isotope analysis performed along the growth axis of vertebrate tooth enamel records differences attributed to seasonal variation in diet, climate or animal movement. Because several months are required to obtain mature enamel in large mammals, modifications in the isotopic composition of environmental parameters are not instantaneously recorded, and stable isotope analysis of tooth enamel returns a time-averaged signal attenuated in its amplitude relative to the input signal. For convenience, stable isotope profiles are usually determined on the side of the tooth where enamel is thickest. Here we investigate the possibility of improving the time resolution by targeting the side of the tooth where enamel is thinnest. Observation of developing third molars (M3) in sheep shows that the tooth growth rate is not constant but decreases exponentially, while the angle between the first layer of enamel deposited and the enamel–dentine junction increases as a tooth approaches its maximal length. We also noted differences in thickness and geometry of enamel growth between the mesial side (i.e., the side facing the M2) and the buccal side (i.e., the side facing the cheek) of the M3. Carbon and oxygen isotope variations were measured along the M3 teeth from eight sheep raised under controlled conditions. Intra-tooth variability was systematically larger along the mesial side and the difference in amplitude between the two sides was proportional to the time of exposure to the input signal. Although attenuated, the mesial side records variations in the environmental signal more faithfully than the buccal side. This approach can be adapted to other mammals whose teeth show lateral variation in enamel thickness and could potentially be used as an internal check for diagenesis.

Using rain-use efficiency to explore livestock production trends in rangelands in the Transkei, South Africa

We qualitatively describe the condition of communally managed rangelands in the Transkei, South Africa, using GIS and high resolution near-infrared imagery. Using livestock census data from 28 magisterial districts in the Transkei, we explored the trends in livestock biomass from 1923–1998. The area had been subjected to intensive herbivory by domestic livestock during that period, and the high livestock biomass had been blamed for the perceived degradation or ‘overgrazing’ of the region. Our assessment used the concept rain-use efficiency (RUE) (kg dry matter ha–1 mm–1) to determine whether there is evidence of change in the efficiency of the system to produce domestic livestock. We calculated RUE from annual livestock numbers and the mean annual rainfall for each district. We found no evidence of a decline in rain-use efficiency between the two assessment periods (1923–1944, 1945–1998). There was evidence of a shift in the ratio of sheep to goats between 1923 and 1998, with goat numbers increasing (greater than twofold) relative to sheep in eight districts. This trend may be associated with changes in the structure of vegetation. We conclude that this region is not showing evidence of system run down that affects domestic livestock production.

Theca cells and the regulation of ovarian androgen production

Theca cells are essential for female reproduction being the source of androgens that are precursors for follicular oestrogen synthesis and also signal through androgen receptors (AR) in the ovary and elsewhere. Theca cells arise from mesenchymal cells around the secondary follicle stage. Their recruitment, proliferation and cytodifferentiation are influenced, directly or indirectly, by paracrine signals from granulosa cells and oocyte although uncertainty remains over which are the critically important signals at particular stages. In a reciprocal manner, theca cells secrete factors that influence granulosa cell proliferation and differentiation at different follicle stages. Differentiated theca interna cells acquire responsiveness to luteinizing hormone (LH) and other endocrine signals and express components of the steroidogenic machinery required for androgen biosynthesis. They also express insulin-like peptide 3 (INSL3) and its receptor (RXFP2), levels of which increase during bovine antral follicle development. INSL3 signaling may play a role in promoting androgen biosynthesis since knockdown of either INSL3 or its receptor (RXFP2) in bovine theca cells inhibits androgen biosynthesis while exogenous INSL3 can raise androgen secretion. Bone morphogenetic proteins (BMPs) of thecal or granulosal origin suppress thecal production of both INSL3 and androgen. Inhibin, produced in greatest amounts by granulosa cells of preovulatory follicles, reverses these BMP actions. Thus, BMP-induced inhibition of thecal androgen production may be mediated by reduced INSL3-RXFP2 signaling. Activins also inhibit androgen production in an inhibin-reversible manner and recent evidence in sheep indicates that theca cells synthesize and secrete activin, implying an autocrine role in suppressing androgen biosynthesis in smaller follicles, akin to that envisaged for BMPs.

Discovery of a mammalian splice variant of myostatin that stimulates myogenesis

Myostatin plays a fundamental role in regulating the size of skeletal muscles. To date, only a single myostatin gene and no splice variants have been identified in mammals. Here we describe the splicing of a cryptic intron that removes the coding sequence for the receptor binding moiety of sheep myostatin. The deduced polypeptide sequence of the myostatin splice variant (MSV) contains a 256 amino acid N-terminal domain, which is common to myostatin, and a unique C-terminus of 65 amino acids. Western immunoblotting demonstrated that MSV mRNA is translated into protein, which is present in skeletal muscles. To determine the biological role of MSV, we developed an MSV over-expressing C2C12 myoblast line and showed that it proliferated faster than that of the control line in association with an increased abundance of the CDK2/Cyclin E complex in the nucleus. Recombinant protein made for the novel C-terminus of MSV also stimulated myoblast proliferation and bound to myostatin with high affinity as determined by surface plasmon resonance assay. Therefore, we postulated that MSV functions as a binding protein and antagonist of myostatin. Consistent with our postulate, myostatin protein was co-immunoprecipitated from skeletal muscle extracts with an MSV-specific antibody. MSV over-expression in C2C12 myoblasts blocked myostatin-induced Smad2/3-dependent signaling, thereby confirming that MSV antagonizes the canonical myostatin pathway. Furthermore, MSV over expression increased the abundance of MyoD, Myogenin and MRF4 proteins (P,0.05), which indicates that MSV stimulates myogenesis through the induction of myogenic regulatory factors. To help elucidate a possible role in vivo, we observed that MSV protein was more abundant during early post-natal muscle development, while myostatin remained unchanged, which suggests that MSV may promote the growth of skeletal muscles. We conclude that MSV represents a unique example of intra-genic regulation in which a splice variant directly antagonizes the biological activity of the canonical gene product.

A dynamic mechanistic model of lactic acid metabolism in the rumen

Current feed evaluation systems for ruminants are too imprecise to describe diets in terms of their acidosis risk. The dynamic mechanistic model described herein arises from the integration of a lactic acid (La) metabolism module into an extant model of whole-rumen function. The model was evaluated using published data from cows and sheep fed a range of diets or infused with various doses of La. The model performed well in simulating peak rumen La concentrations (coefficient of determination = 0.96; root mean square prediction error = 16.96% of observed mean), although frequency of sampling for the published data prevented a comprehensive comparison of prediction of time to peak La accumulation. The model showed a tendency for increased La accumulation following feeding of diets rich in nonstructural carbohydrates, although less-soluble starch sources such as corn tended to limit rumen La concentration. Simulated La absorption from the rumen remained low throughout the feeding cycle. The competition between bacteria and protozoa for rumen La suggests a variable contribution of protozoa to total La utilization. However, the model was unable to simulate the effects of defaunation on rumen La metabolism, indicating a need for a more detailed description of protozoal metabolism. The model could form the basis of a feed evaluation system with regard to rumen La metabolism.

Preliminary ethnoarchaeological research on modern animal husbandry in Bestansur, Iraqi Kurdistan: integrating animal, plant and environmental data

This paper presents preliminary results from an ethnoarchaeological study of animal husbandry in the modern village of Bestansur, situated in the lower Zagros Mountains of Iraqi Kurdistan. This research explores how modern families use and manage their livestock within the local landscape and identifies traces of this use. The aim is to provide the groundwork for future archaeological investigations focusing on the nearby Neolithic site of Bestansur. This is based on the premise that modern behaviours can suggest testable patterns for past practices within the same functional and ecological domains. Semi-structured interviews conducted with villagers from several households provided large amounts of information on modern behaviours that helped direct data collection, and which also illustrate notable shifts in practices and use of the local landscape over time. Strontium isotope analysis of modern plant material demonstrates that a measurable variation exists between the alluvial floodplain and the lower foothills, while analysis of modern dung samples shows clear variation between sheep/goat and cow dung, in terms of numbers of faecal spherulites. These results are specific to the local environment of Bestansur and can be used for evaluating and contextualising archaeological evidence as well as providing modern reference material for comparative purposes.

Prediction of metabolisable energy concentrations of fresh cut grass using digestibility data measured with non-pregnant non-lactating cows

Pasture-based ruminant production systems are common in certain areas of the world, but energy evaluation in grazing cattle is performed with equations developed, in their majority, with sheep or cattle fed total mixed rations. The aim of the current study was to develop predictions of metabolisable energy (ME) concentrations in fresh-cut grass offered to non-pregnant non-lactating cows at maintenance energy level, which may be more suitable for grazing cattle. Data were collected from three digestibility trials performed over consecutive grazing seasons. In order to cover a range of commercial conditions and data availability in pasture-based systems, thirty-eight equations for the prediction of energy concentrations and ratios were developed. An internal validation was performed for all equations and also for existing predictions of grass ME. Prediction error for ME using nutrient digestibility was lowest when gross energy (GE) or organic matter digestibilities were used as sole predictors, while the addition of grass nutrient contents reduced the difference between predicted and actual values, and explained more variation. Addition of N, GE and diethyl ether extract (EE) contents improved accuracy when digestible organic matter in DM was the primary predictor. When digestible energy was the primary explanatory variable, prediction error was relatively low, but addition of water-soluble carbohydrates, EE and acid-detergent fibre contents of grass decreased prediction error. Equations developed in the current study showed lower prediction errors when compared with those of existing equations, and may thus allow for an improved prediction of ME in practice, which is critical for the sustainability of pasture-based systems.

Prediction of nutrient digestibility and energy concentrations in fresh grass using nutrient composition

Improved nutrient utilization efficiency is strongly related to enhanced economic performance and reduced environmental footprint of dairy farms. Pasture-based systems are widely used for dairy production in certain areas of the world, but prediction equations of fresh grass nutritive value (nutrient digestibility and energy concentrations) are limited. Equations to predict digestible energy (DE) and metabolizable energy (ME) used for grazing cattle have been either developed with cattle fed conserved forage and concentrate diets or sheep fed previously frozen grass, and the majority of them require measurements less commonly available to producers, such as nutrient digestibility. The aim of the present study was therefore to develop prediction equations more suitable to grazing cattle for nutrient digestibility and energy concentrations, which are routinely available at farm level by using grass nutrient contents as predictors. A study with 33 nonpregnant, nonlactating cows fed solely fresh-cut grass at maintenance energy level for 50 wk was carried out over 3 consecutive grazing seasons. Freshly harvested grass of 3 cuts (primary growth and first and second regrowth), 9 fertilizer input levels, and contrasting stage of maturity (3 to 9 wk after harvest) was used, thus ensuring a wide representation of nutritional quality. As a result, a large variation existed in digestibility of dry matter (0.642-0.900) and digestible organic matter in dry matter (0.636-0.851) and in concentrations of DE (11.8-16.7 MJ/kg of dry matter) and ME (9.0-14.1 MJ/kg of dry matter). Nutrient digestibilities and DE and ME concentrations were negatively related to grass neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents but positively related to nitrogen (N), gross energy, and ether extract (EE) contents. For each predicted variable (nutrient digestibilities or energy concentrations), different combinations of predictors (grass chemical composition) were found to be significant and increase the explained variation. For example, relatively higher R(2) values were found for prediction of N digestibility using N and EE as predictors; gross-energy digestibility using EE, NDF, ADF, and ash; NDF, ADF, and organic matter digestibilities using N, water-soluble carbohydrates, EE, and NDF; digestible organic matter in dry matter using water-soluble carbohydrates, EE, NDF, and ADF; DE concentration using gross energy, EE, NDF, ADF, and ash; and ME concentration using N, EE, ADF, and ash. Equations presented may allow a relatively quick and easy prediction of grass quality and, hence, better grazing utilization on commercial and research farms, where nutrient composition falls within the range assessed in the current study.

Diet and herding strategies in a changing environment: stable isotope analysis of Bronze Age and Late Antique skeletal remains from Ya'amūn, Jordan

Carbon and nitrogen stable isotope ratios of 45 human and 23 faunal bone collagen samples were measured to study human diet and the management of domestic herbivores in past Jordan, contrasting skeletal remains from the Middle and Late Bronze Age and the Late Roman and Byzantine periods from the site of Ya'amūn near Irbid. The isotope data demonstrate that the management of the sheep and goats changed over time, with the earlier animals consuming more plants from semi-arid habitats, possibly because of transhumant herding strategies. The isotope data for fish presented here are the first from archaeological contexts from the Southern Levant. Although fish of diverse provenance was available at the site, human diet was predominately based on terrestrial resources and there was little dietary variability within each time-period. Isotopic variation between humans from different time-periods can mostly be explained by ‘baseline shifts’ in the available food sources; however, it is suggested that legumes may have played a more significant role in Middle and Late Bronze Age diet than later on.