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Shear stress adaptation of Listeria monocytogenes in mono and dual-species biofilms
(Elsevier Ltd, 2025-12-01) Pant K; Palmer J; Flint S
While the impact of stress on L. monocytogenes associated with food processing has been recognized in planktonic conditions, the available research overlooks the response of this pathogen in the multi-species biofilm, commonly found in food processing and manufacture. The objective of this study was to understand the effect of shear stress on L. monocytogenes in single and dual-species (with P. fluorescens) biofilm formed in a continuous turbulent flow system. In the single-species biofilm, L. monocytogenes was able to form a biofilm under the turbulent flow with cell concentration reaching 5.1 log CFU/cm2 after 48 h, where filamentous cells (27.7 μm in length) were observed. In contrast, there were no visible filaments in the dual-species biofilm, and L. monocytogenes cell concentration was significantly higher (p < 0.001) at 8.7 log CFU/cm2. The cells harvested from single-species L. monocytogenes biofilm formed under turbulent flow showed significantly (p < 0.001) lower motility and higher adhesion compared with cells harvested from planktonic and static conditions. Gene expression analysis showed significant (p < 0.001) downregulation of motB (motility), sigB (stress), and cell division (ftsX and ftsW), and upregulation of mpl (adhesion) and rodA (rod shape), indicating L. monocytogenes adaptation to shear stress. This study provides fundamental information on the multi-species biofilm formation by L. monocytogenes under stress.
Spatiotemporal mapping of spontaneous smooth muscle motility in capacious organs: the ex vivo urinary bladder and in vivo gravid uterus of the rabbit: a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University, Palmerston North, New Zealand
(Massey University, 2023-08-20) Hulls, Corrin
The temporal and spatial dynamics of propagating myogenic contractions in the wall of the resting ex vivo urinary bladder and in vivo gravid uterus of the rabbit were characterised by spatiotemporal maps of area strain rate, of linear strain rate and contractile patch analysis, and related to cyclic variation in intravesical pressure (pves) in the bladder, and electrophysiological recordings in the gravid uterus. In the urinary bladder, patches of propagating contractions (PPCs) enlarged and involuted with a frequency in near synchrony with peaks in intravesical pressure. Maximum area percentage of the anterior surface of the bladder undergoing contraction and the sizes of individual PPCs also coincided with the peak in pves. Moreover, pves varied cyclically with total area of contraction and with the indices of the size and aggregation of PPCs, indicating that PPCs grew and involuted by a combination of peripheral enlargement or shrinkage and by coalescence or fission with other PPCs, their areas being maximal at or around the peak in pves. Bladder PPCs originated and propagated within temporary patch domains (TPDs) and comprised groups of near synchronous cyclic individual contractions (PICs). The TPDs were located principally along the vertical axis of the anterior surface of the bladder, either to the left or the right of midline and changed in location from one side to side and from side to tip or base. The sites of origin of PICs within PPCs were inconsistent, consecutive contractions often propagating in opposite directions along linear maps of strain rate. Similar patterns of movement of PPCs within TPDs of the same form occurred in areas of the anterior bladder wall that had been stripped of mucosa. The synchronisation and extended propagation of PICs within PPCs and the concurrent variation in pves of the bladder were sometimes lost or diminished, uncoordinated PICs then occurred, propagated shorter distances, and had little effect on pves. There was no evidence that any influence of bladder shape on stress influenced the principal direction of propagation of either PCCs or PICs or the disposition of TPDs. The disposition and dynamics of PPCs and their component individual myogenic contractions in the wall of the resting ex vivo tetrodotoxinized urinary bladder of the rabbit were characterised by spatiotemporal maps and related to cyclic variation in pves before and after the administration of carbachol, isoprenaline, carbenoxolone, and the RhoA-inhibitor Y-27632. The results confirm that the bladder wall can exhibit two contractile states that are of similar frequencies to those of the two types of electrophysiological discharge described by previous workers. In the first of these, large low frequency cyclic PPCs predominate. In the second, small irregular, higher frequency PICs predominate. Comparison of the effects of the drugs on the timing and disposition of contraction suggested indicated that the local spatial spread of contractions in PPCs was governed largely by myocytes, whilst the propagation, frequency, and duration of PPCs was likely governed via gap junctions between interstitial cells of cajal- intermuscular (ICC-IM) and myocytes. Spontaneous and oxytocin-induced contractile activity was quantified in the bicornuate uteri of pregnant rabbits maintained in situ, using data from electrophysiological recordings and spatiotemporal maps, and compared statistically. Spontaneous contractions occurred over a range of frequencies in gravid animals at 18-21 and at 28 days of gestation and propagated both radially and longitudinally over the uterine wall overlying each foetus. Patches of contractions were randomly distributed over the entire surface of the cornua and were pleomorphic in shape. No spatial coordination was evident between longitudinal and circular muscle layers nor temporal coordination that could indicate the activity of a localised pacemaker. The density and duration of contractions decreased, and their frequency increased with the length of gestation in the non-labouring uterus. Increasing intravenous doses of oxytocin had no effect on the mean frequencies, or the mean durations of contractions in rabbits of 18-21 days gestation but caused frequencies to decrease and durations to increase in rabbits of 28 days gestation, from greater spatial and temporal clustering of individual contractions. This was accompanied by an increase in the distance of propagation, the mean size of the patches of contraction, the area of the largest patch of contraction and the overall density of patches. Together these results suggest that progressive smooth muscle hypertrophy and displacement with increasing gestation is accompanied by a decrease in smooth muscle connectivity causing an increase in wall compliance and that oxytocin restores connectivity and decreases compliance, promoting volumetric expulsion rather than direct propulsion of the foetus by peristalsis. The latter effects were reversed by the β2 adrenergic receptor agonist salbutamol thus reducing area of contraction, duration, and distance of propagation. The characteristics of smooth muscle contraction that are associated with the maintenance of tone during accommodation appear to be to be similar in the capacious organs discussed. Hence, it appears there is patchy rather than uniform local revision of the state of tonal contraction over the surfaces of the various capacious organs during accommodation that can undergo neural modulation. There appears to be less similarity in regard to the mechanisms that secure the voiding contractions in these capacious structures. Whilst the actuation of the necessary shorter term increase in tone may be ultimately dependent upon a mechanosensitive myogenic reflex, the relative contributions of autonomic, hormonal, mechanical and voluntary reflexes that reset the threshold of this reflex and aid in the relatively rapid expulsion of the contents, appear to vary between organs.
Understanding host and microbial metabolites in functional gut disorders : a thesis presented in particle fulfilment of the requirements for the degree of Doctor of Philosophy in Nutritional Science at Massey University, Palmerston North, New Zealand
(Massey University, 2021) James, Crystal Shanalee
Interactions between diet, host, and the gut microbiome can result in beneficial or detrimental effects on human health. Functional gut disorders (FGDs) are an example of the negative effects of these interactions. However, an understanding of the mechanisms behind FGDs remains unknown. Metabolomics is a powerful tool to understand possible mechanisms. It was hypothesised that key metabolite groups in faecal samples would differ between or within FGD subtypes and healthy controls reflective of mechanistic perturbations. This PhD aimed to characterise the metabolite profile of FGD individuals (functional constipation (FC), IBS-constipation (IBS-C), functional diarrhoea (FD), IBS-diarrhoea (IBS-D), IBS-mixed (IBS-M)) and healthy controls. The concentration of faecal bile acids and plasma amino acids were quantified to ascertain changes in known metabolites associated with FGDs. The faecal metabolome was characterised to potentially identify wider perturbations, and this was then integrated with dietary intake, plasma metabolome abundance and faecal microbiota composition for a systems biology analysis. Constipation (FC + IBS-C) and diarrhoea (FD + IBS-D) were combined to determine differences between healthy controls and disease states. Faecal bile acid concentrations differed between all FGD participants and healthy controls. In the combined analysis of the diarrhoea (FD+IBS-D) and constipation (FC+IBS-C) groups, the diarrhoea group had a higher concentration of bile acids than the constipation group or healthy control group. The concentration of plasma amino acids did not differ between FGD participants and healthy controls. Furthermore, analysis of key amino acid groups showed that only the concentration of branched chain amino acids were different between all subtypes and healthy controls. Characterisation of faecal polar, semi-polar and lipid metabolites showed a differential relative abundance of some polar and semi-polar metabolites (e.g., riboflavin, nicotinic acid) between diarrhoea and healthy control groups, and constipation and healthy control groups. Substantial changes in the abundance of some lipids (e.g., ceramides, triglycerides) were evident between constipation and healthy control groups, and diarrhoea and healthy control groups. Integration of the faecal metabolome with other datasets (faecal microbiome, plasma metabolome, dietary intake) showed the faecal metabolome and microbiome separated healthy controls from constipation or diarrhoea. Additionally, differential positive and negative correlations were observed between faecal lipids (triglycerides and diglycerides) and microbial species (Firmicutes and Bacteroidetes). This PhD thesis presents novel insights into the metabolite signature characterising FGD participants and healthy controls and provides directions for future research.
Spatiotemporal mapping of the motility of the ex vivo rabbit caecum : a thesis presented in partial fulfilment of the requirements for the degree of Masters of Physiology in Digestive Biomechanics (Physical Process of Digestion) at Massey University, Turitea, New Zealand
(Massey University, 2015) Hulls, Corrin
This work sought to determine the contractile factors influencing the coordination of inflow and out flow from the caecum, and the mixing and mass transfer within. Specifically, the work was focussed on the ileocaecal junction in the domestic rabbit (Oryctolagus cuniculus). The salient questions to answer were; 1. What are the contractile movements in the body of the caecum and associated structures of the rabbit caecum? 2. How are contractile movements coordinated at the body of the rabbit caecum and how does this affect the pattern of motility? The following two main experimental works of this thesis were all conducted using live gut rabbit caecum preparations maintained ex vivo. Spatiotemporal mapping and electromyography was used to visualize and quantify contractile activity and coordination in the caecum. 1. High definition radial, strain rate and intensity spatiotemporal mapping was used to quantify contractile movements of the body and associated structures of the rabbit caecum. 2. Coordination between contractile events at different sites in the basal portion of the rabbit caecum and its associated structures were identified by electrophysiological recordings with simultaneous one dimensional, and a novel two dimensional, spatiotemporal mapping technique. The following are the main findings and implications of the work. 1. The body of the caecum exhibited two patterns of motility that appeared autonomous, i.e. occurred independently of any contractile activity at the inlet or outlet. Firstly, a pattern termed ladder activity consisted of orderly sequential contractions in the spiral turns in the corpus ceci. Secondly, less localised, rapidly propagating synchronous contractions that were termed mass peristalsis. 2. Movements of the ileum and sacculus rotundus occurred at the same frequency and were broadly coordinated. Further, the findings suggest that the action of the sacculus rotundus may result from its distension with chyme by ileal peristalsis and that the subsequent propagation of contraction along the basal wall of the caecum toward the colon may be augmented by this local distension. 3. The caecum and proximal colon/ampulla coli act reflexly to augment colonic outflow. When the caecum is distended and mass peristalsis is instituted, the action of the latter overrides the inherent rhythm and direction of haustral propagation in the adjacent portion of the proximal colon but not in the terminal ileum. In conclusion, coordination, mixing and mass transfer in the rabbit caecum is a very complex, dynamic and largely autonomous process. Further, spatiotemporal mapping techniques enabled the identification and visualization of previously unknown contractile movements within the rabbit caecum.
Factors influencing mixing and mass transfer in the small intestine : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Digestive Biomechanics (Physical Process of Digestion) at Massey University, Turitea, New Zealand
(Massey University, 2015) Lim, Ian Yuen Feung
This work sought to determine the factors influencing mixing and mass transfer in the small intestine. Specifically, the work was focussed on the gut periphery (i.e. perivillous region) of the terminal ileum in the brushtail possum (Trichusurus vulpecula). The salient questions to answer were; 1. What are the microrheological properties and disposition of mucus in the perivillous space? 2. What are the disposition and movements of the mucosa and the associated villi during postprandial gut motility patterns of pendular contractions? 3. Are villi rigid structures during physiological levels of lumen flow? The following three main experimental works of this thesis were all conducted using live gut wall samples maintained ex vivo. In addition, computational models were developed incorporating the novel findings detailed in this thesis to assist in visualizing mixing and mass transfer in the perivillous space. 1. The properties of the perivillous fluid environment were assessed by multiple-particle-tracking of the Brownian motion of fluorescent microbeads on gut samples. 2. The movements and disposition of the mucosal surface and associated villi during pendular contractions were observed for whole lengths of everted gut samples. 3. Flow velocities in the perivillous space of gut samples were determined by microparticle-image-velocimetery of microbeads. The movement of villi in response to physiological levels of lumen flow were quantified by image analysis. The following are the main findings and implications of the work. 1. The perivillous fluid environment consisted of discrete viscoelastic bodies dispersed within a watery Newtonian phase. Such characteristics of the fluid environment were thought to be conducive for mixing and mass transfer, and likened to the processes of gel filtration. 2. Gut pendular contractions generated transient mucosal microfolds, which resulted in the formation of periodic congregation and separation of villous tips. Such a mechanism was predicted (using computational simulations) to augment mixing and mass transfer of nutrients at the gut periphery. 3. Villi were rigid structures, which were more prone to pivot than to bend, while intervillous fluid was predicted to be quasi-static during physiological levels of lumen flow. Such a feature of villi supports a perivillous mixing and mass transfer mechanism driven by mucosal microfolding In conclusion, mixing and mass transfer in the perivillous space are governed by more complex dynamics than previously assumed and by factors previously unknown.
A study of the transport of spermatozoa in the reproductive tract of the ewe after artificial insemination : a thesis presented in partial fulfilment of the requirements for the degree of Master of Agricultural Science at Massey University
(Massey University, 1975) Giles, Kerin Harle
Two preliminary experiments were conducted with artificially inseminated ewes to examine the considerable variation in counts of spermatozoa recovered in flushings from the reproductive tract. In the second trial, road transport stress immediately after insemination caused a reduction in the number of spermatozoa reaching the Fallopian tubes. In the third trial, sperm transport after insemination with fresh, undiluted semen at two sites in the tract at predetermined times relative to synchronised ovulation (progestagen-HCG treatment) was investigated. Deposition of semen at the uterine end of the cervix or within the uterus using a specially adapted inseminating pippette, resulted in the recovery of higher numbers of spermatozoa from the uterus and Fallopian tubes 4 hours after insemination, than did insemination at the external cervical os. Twenty-four hours after insemination similar numbers of spermatozoa were found in all segments for both sites of insemination. Deposition of semen before or after the expected time of ovulation, or at both times, had little effect on the numbers of spermatozoa recovered. More ova with accessory spermatozoa were recovered from oviducts containing high numbers of sperm. The transport of spermatozoa after deposition of fresh or frozen semen into the uterus by surgical and non-surgical methods, was studied in a fourth experiment. The number of spermatozoa recovered from the uterus and isthmus was similar for both types of semen, but only 'fresh' spermatozoa deposited at surgery, reached the ampulla in significant numbers. 'Frozen' sperm deposited by both methods, and fresh sperm deposited non-surgically, did not reach the ampulla within 4 hours of insemination. Impaired transport of 'frozen' spermatozoa to the site of fertilization appeared to be a result of reduced longevity of these cells within the female tract, and nay involve factors such as breakage of spermatozoa in the uterus, and delayed transport in the oviducts. The improvement of sperm transport in the ewe through the development of non-surgical, intrauterine inseminations and using either freshly extended or frozen semen, are discussed.
Studies of radiopaque markers in cats and dogs : a thesis presented in partial fulfilment of the requirements for the degree of Master of Veterinary Science at Massey University
(Massey University, 2000) Bruce, Sara J
Adverse reactions to food are exceedingly common reasons for the presentation of cats and dogs to veterinarians. Of those cases, a relatively small number involve a truly immune-mediated reaction to the food substance. However, differentiating those that are from the more common food intolerances is usually difficult and often impossible. In addition, certain individuals with a true food hypersensitivity are difficult to manage with conventional diets. The identification and availability of nutritionally complete commercially prepared diets with a protein component that is truly novel to the patient under investigation is often the stumbling block to successful diagnosis and management of food hypersensitivity. The recent development of protein hydrolysate based pet foods for dogs and cats provides an exciting tool for more reliable diagnosis and management or food hypersensitivity in those species. Chapter 1 of this thesis describes the immunological and pathophysiological basis of food hypersensitivity and presents some of the key areas of recent research that have lead to a deeper, if still incomplete understanding of the aetiological mechanisms responsible. The development and maintenance of oral tolerance is discussed including the key roles that the resident antigen presenting cells in the mucosa play. From that description follows a presentation of some of the current hypotheses regarding mechanisms by which oral tolerance is lost or not established. These include the action of mucosal adjuvants, parasitism, IgA deficiency and alterations in mucosal permeability. Building on this discussion is an examination of the methods currently available to veterinarians for the diagnosis of food hypersensitivity, their clinical usefulness and limitations. The importance of obtaining a complete and accurate dietary history is emphasized. The difficulties in doing so and the consequences of not doing so are discussed. As stated, the recent development of diets in which the protein content has been hydrolysed provides a new tool for the veterinarian. Some of the practical aspects behind producing hydrolysate diets are presented and the theoretical basis, especially the importance of the molecular weight of remaining polypeptide fragments, is emphasized. Finally recommendations as to their use and the role that they may play in the future are discussed. Chapter 2 describes the initial assessment of 2 candidate hydrolysates, one made from fish and the other from chicken proteins. The method used for this initial experiment was high-performance size-exclusion liquid chromatography. The investigation revealed the chicken hydrolysate to have the more favourable molecular weight profile of the two. The finding that 92.9% of the hydrolysate was of a molecular weight less than 5kDa is supportive of its potential value in a hypoallergenic diet. The molecular weight profile was then compared with a selection of those published in the human medical literature. Cow's milk hydrolysates have been widely available and used for the past 2-3 decades. The chicken hydrolysate appeared to compare very favourably to a number of extensively hydrolysed human infant formulae that have been demonstrated experimentally and clinically to be truly hypoallergenic. Despite the extensive use of molecular weight profiles to compare hydrolysates, they remain unreliable as predictors of allergenicity. Chapter 3 describes the experimental evaluation of the antigenicity of the chicken hydrolysate. Following successful immunization of dogs to the intact parent protein, an IgG inhibition ELISA was developed using sera from the immunized dogs. It was demonstrated that the hydrolysate retained some ability to bind IgG but that at equal levels of antibody binding, the concentration of the hydrolysate solution required was 66 times greater than that of the intact parent protein. It is likely that this represents a clinically highly significant reduction in antigenicity. Of the limitations of the inhibition ELISA study, perhaps the greatest from the perspective of hydrolysate diet analysis is its inability to differentiate the molecular weight of the IgG-binding fragments. This is important since if they are less than 6-10kDa, they are unlikely to participate in IgE-mediated allergic reactions. Chapter 4 describes the experiment chosen to determine the size of the remaining IgG-binding fragments, namely Western blotting. It was established that the major antigenic fraction remaining in the parent protein following SDS-PAGE separation was a c.69kDa protein consistent with chicken serum albumin. It was demonstrated by both the Western blotting and the HP-SEC that this antigen was absent from the hydrolysate. The actual size of the few remaining binding fragments in the hydrolysate was not, however, clearly elucidated. It was concluded that the chicken hydrolysate assessed during this thesis is a promising candidate for inclusion as the peptide component of a diet for the diagnosis and management of food hypersensitivity in dogs and cats. In addition, the diet has theoretical promise for the prevention of food hypersensitivity during periods of mucosal inflammation such as idiopathic inflammatory bowel disease and acute gastroenteritis. Ultimately, clinical trials are required to conclusively demonstrate the value of the hydrolysate in the diagnosis and management of these disorders.
Stimulation of oesophageal motility in the cat : a thesis presented in partial fulfilment of the requirements for the degree of Master of Agricultural Science in Physiology at Massey University of Manawatu in the Department of Veterinary Biology
(Massey University, 1966) Wheeler, John Spencer
Literature concerned with reports or stimuli which cause or modify oesophageal activity have been reviewed and has been associated with the evidence obtained in the experiments undertaken. The preparations of cats which have been used in these experiments were:- Anaesthetised cats. Chloralose 70 mgm/kgm injected as a solution in saline (70°C), or an organic solvent (dimethyl sulphoxide). Decerebrated cats. Decerebrated cats with the spinal cord sectioned at the sixth cervical vertebra. Decerebrated cats with the spinal cord sectioned at the third cervical vertebra. Cats with the central nervous system totally destroyed by pithing. Oesophageal motility has been recorded by isometric and isotonic systems. In all cases oesophageal contractions were detected by balloons 3-5 cc volume. Contractions of the terminal oesophagus in response to distension of this region have been obtained in the absence of an extrinsic innervation. These results, and those obtained in decerebrate preparations, indicated the existence of both local and inter-oesophageal reactions. These intrinsic reactions were found to be reflexly modified by stimuli arising in other parts of the preparations, particularly the alimentary tract. The reflexes appeared to be mediated certainly through vagal and possibly through sympathetic afferents to centres in the medulla oblongata or pons. Vagal efferent pathways have been shown to affect the oesophagus, and indications of a sympathetic efferent innervation of the oesophagus have been obtained. Stimuli which facilitated the local responses of the terminal oesophagus to distension were:- Greater degrees of distension of this region of the oesophagus. (Very high levels of distension tended to be inhibitory.) Acidification of the mucosa of the terminal regions of the oesophagus. Acidification of the isolated stomach to a pH of about 2.0. Stimuli which inhibited the response of the terainal oesophagus to distension were:- Distension of a more cranial region of the oesophagus. High degrees of distension of the oesophagus at the point from which the recording was taken. Distension of the stomach. Noxious stimuli arising from the operative procedures. These facilitatory and inhibitory stimuli were found to summate. The oesophageal response observed was a reflection of the nett afferent discharge. Reactions of the oesophagus to cholinergic and adrenergic drugs and blocking agents such as atropine, hexamethonium and nicotine have been studied. The results are discussed in relation to the function of the oesophagus in the intact animal and in relation to how co-ordinated responses are obtained in swallowing and other circumstances.

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