Microbial ecology of the equine hindgut during oligofructose-induced laminitis

Alimentary carbohydrate overload is a significant cause of laminitis in horses and is correlated with drastic shifts in the composition of hindgut microbiota. Equine hindgut streptococcal species (EHSS), predominantly Streptococcus lutetiensis, have been shown to be the most common microorganisms culturable from the equine caecum prior to the onset of laminitis. However, the inherent biases of culture-based methods are estimated to preclude up to 70% of the normal caecal microbiota. The objective of this study was to evaluate bacterial population shifts occurring in the equine caecum throughout the course of oligofructose-induced laminitis using several culture-independent techniques and to correlate these with caecal lactate, volatile fatty acid and degrees of polymerization 3-7 fructo-oligosaccharide concentrations. Our data conclusively show that of the total microbiota present in the equine hindgut, the EHSS S. lutetiensis is the predominant microorganism that proliferates prior to the onset of laminitis, utilizing oligofructose to produce large quantities of lactate. Population shifts in lactobacilli and Escherichia coli subpopulations occur secondarily to the EHSS population shifts, thus confirming that lactobacilli and coliforms have no role in laminitis. A large, curved, Gram-negative rod previously observed during the early phases of laminitis induction was most closely related to the Anaerovibrio genus and most likely represents a new, yet to be cultured, genus and species. Correlation of fluorescence in situ hybridization and quantitative real-time PCR results provide evidence supporting the hypothesis that laminitis is associated with the death en masse and rapid cell lysis of EHSS. If EHSS are lysed, liberated cellular components may initiate laminitis.

Fluorescence in situ hybridization analysis of hindgut bacteria associated with the development of equine laminitis

In the article 'Fluorescence in situ hybridization analysis of hindgut bacteria associated with the development of equine laminitis' (Milinovich et al., 2007), it is found that with reference to Horse 1, the histological signs of laminitis were first observed at 12 h post-oligofructose administration, and not 30 h as was indicated in the Results section under the subheading 'Induction of Laminitis' and in Fig. 1.

Bacterial Community Structure in the Hindgut of Wild and Captive Dugongs (Dugong dugon)

Dugongs (Dugong dugon) are marine mammals that obtain nutrients through hindgut fermentation of seagrass, however, the microbes responsible have not been identified. This study used denaturing gradient gel electrophoresis (DGGE) and 454-pyrosequencing to profile hindgut bacterial communities in wild dugongs. Faecal samples obtained from 32 wild dugongs representing four size/maturity classes, and two captive dugongs fed on cos lettuce were screened using DGGE. Partial 16S rRNA gene profiles of hindgut bacteria from wild dugong calves and juveniles were grouped together and were different to those in subadults and adults. Marked differences between hindgut bacterial communities of wild and captive dugongs were also observed, except for a single captive whose profile resembled wild adults following an unsuccessful reintroduction to the wild. Pyrosequencing of hindgut communities in two wild dugongs confirmed the stability of bacterial populations, and Firmicutes (average 75.6% of Operational Taxonomic Units [OTUs]) and Bacteroidetes (19.9% of OTUs) dominated. Dominant genera were Roseburia, Clostridium, and Bacteroides. Hindgut microbial composition and diversity in wild dugongs is affected by ontogeny and probably diet. In captive dugongs, the absence of the dominant bacterial DNA bands identified in wild dugongs is probably dependent upon prevailing diet and other captive conditions such as the use of antibiotics. This study represents a first step in the characterisation of a novel microbial ecosystem-the marine hindgut of Sirenia.

Variation in the hindgut microbial communities of the Florida manatee, Trichechus manatus latirostris over winter in Crystal River, Florida

The Florida manatee, Trichechus manatus latirostris, is a hindgut-fermenting herbivore. In winter, manatees migrate to warm water overwintering sites where they undergo dietary shifts and may suffer from cold-induced stress. Given these seasonally induced changes in diet, the present study aimed to examine variation in the hindgut bacterial communities of wild manatees overwintering at Crystal River, west Florida. Faeces were sampled from 36 manatees of known sex and body size in early winter when manatees were newly arrived and then in mid-winter and late winter when diet had probably changed and environmental stress may have increased. Concentrations of faecal cortisol metabolite, an indicator of a stress response, were measured by enzyme immunoassay. Using 454-pyrosequencing, 2027 bacterial operational taxonomic units were identified in manatee faeces following amplicon pyrosequencing of the 16S rRNA gene V3/V4 region. Classified sequences were assigned to eight previously described bacterial phyla; only 0.36% of sequences could not be classified to phylum level. Five core phyla were identified in all samples. The majority (96.8%) of sequences were classified as Firmicutes (77.3 ± 11.1% of total sequences) or Bacteroidetes (19.5 ± 10.6%). Alpha-diversity measures trended towards higher diversity of hindgut microbiota in manatees in mid-winter compared to early and late winter. Beta-diversity measures, analysed through permanova, also indicated significant differences in bacterial communities based on the season.

Morphological study of the hindgut in larvae of Anticarsia gemmatalis Hübner(Lepidoptera: Noctuidae)

A lagarta da soja (Anticarsia gemmatalis Hübner) tem grande interesse econômico, pois afeta significativamente a cultura da soja em todo o mundo. Este trabalho descreve a morfologia do intestino posterior de larvas de A. gemmatalis, com ênfase nos seus aspectos histológicos. O intestino posterior é constituído por regiões morfologicamente distintas, identificadas como piloro, íleo, cólon e reto. Independente da região, a parede do intestino posterior é constituída por fina cutícula, epitélio simples e camada muscular. A íntima cuticular apresenta espículas no anel intersticial posterior, entre o intestino médio e o posterior, e na região posterior do piloro. A musculatura do reto é formada por camada única de largas fibras circulares, diferindo das demais regiões do intestino posterior que apresentam duas camadas de fibras musculares. As extremidades distais dos túbulos de Malpighi atravessam as paredes do reto, constituindo o sistema criptonefridial característico de Lepidoptera.

TNM staging of midgut and hindgut (neuro) endocrine tumors: a consensus proposal including a grading system

Criteria for the staging and grading of neuroendocrine tumors (NETs) of midgut and hindgut origin were established at the second Consensus Conference in Frascati (Rome) organized by the European Neuroendocrine Tumor Society (ENETS). The proposed tumor-node-metastasis (TNM) classifications are based on the recently published ENETS Guidelines for the Diagnosis and Treatment of gastroenteropancreatic NETs and follow our previous proposal for foregut tumors. The new TNM classifications for NETs of the ileum, appendix, colon, and rectum, and the grading system were designed, discussed, and consensually approved by all conference participants. These proposals need to be validated and are meant to help clinicians in the stratification, treatment and follow-up of patients.

Changes in equine hindgut bacterial populations during oligofructose-induced laminitis

In the horse, carbohydrate overload is thought to play an integral role in the onset of laminitis by drastically altering the profile of bacterial populations in the hindgut. The objectives of this study were to develop and validate microbial ecology methods to monitor changes in bacterial populations throughout the course of experimentally induced laminitis and to identify the predominant oligofructose-utilizing organisms. Laminitis was induced in five horses by administration of oligofructose. Faecal specimens were collected at 8 h intervals from 72 h before to 72 h after the administration of oligofructose. Hindgut microbiota able to utilize oligofructose were enumerated throughout the course of the experiment using habitat-simulating medium. Isolates were collected and representatives identified by 16S rRNA gene sequencing. The majority of these isolates collected belonged to the genus Streptococcus, 91% of which were identified as being most closely related to Streptococcus infantarius ssp. coli. Furthermore, S. infantarius ssp. coli was the predominant oligofructose-utilizing organism isolated before the onset of lameness. Fluorescence in situ hybridization probes developed to specifically target the isolated Streptococcus spp. demonstrated marked population increases between 8 and 16 h post oligofructose administration. This was followed by a rapid population decline which corresponded with a sharp decline in faecal pH and subsequently lameness at 24-32 h post oligofructose administration. This research suggests that streptococci within the Streptococcus bovis/equinus complex may be involved in the series of events which precede the onset of laminitis in the horse.

Cell migration and proliferation on homogeneous and non-homogeneous domains : modelling on the scale of individuals and populations

Cell migration is a behaviour critical to many key biological effects, including wound healing, cancerous cell invasion and morphogenesis, the development of an organism from an embryo. However, given that each of these situations is distinctly different and cells are extremely complicated biological objects, interest lies in more basic experiments which seek to remove conflating factors and present a less complex environment within which cell migration can be experimentally examined. These include in vitro studies like the scratch assay or circle migration assay, and ex vivo studies like the colonisation of the hindgut by neural crest cells. The reduced complexity of these experiments also makes them much more enticing as problems to mathematically model, like done here. The primary goal of the mathematical models used in this thesis is to shed light on which cellular behaviours work to generate the travelling waves of invasion observed in these experiments, and to explore how variations in these behaviours can potentially predict differences in this invasive pattern which are experimentally observed when cell types or chemical environment are changed. Relevant literature has already identified the difficulty of distinguishing between these behaviours when using traditional mathematical biology techniques operating on a macroscopic scale, and so here a sophisticated individual-cell-level model, an extension of the Cellular Potts Model (CPM), is been constructed and used to model a scratch assay experiment. This model includes a novel mechanism for dealing with cell proliferations that allowed for the differing properties of quiescent and proliferative cells to be implemented into their behaviour. This model is considered both for its predictive power and used to make comparisons with the travelling waves which result in more traditional macroscopic simulations. These comparisons demonstrate a surprising amount of agreement between the two modelling frameworks, and suggest further novel modifications to the CPM that would allow it to better model cell migration. Considerations of the model’s behaviour are used to argue that the dominant effect governing cell migration (random motility or signal-driven taxis) likely depends on the sort of invasion demonstrated by cells, as easily seen by microscopic photography. Additionally, a scratch assay simulated on a non-homogeneous domain consisting of a ’fast’ and ’slow’ region is also used to further differentiate between these different potential cell motility behaviours. A heterogeneous domain is a novel situation which has not been considered mathematically in this context, nor has it been constructed experimentally to the best of the candidate’s knowledge. Thus this problem serves as a thought experiment used to test the conclusions arising from the simulations on homogeneous domains, and to suggest what might be observed should this non-homogeneous assay situation be experimentally realised. Non-intuitive cell invasion patterns are predicted for diffusely-invading cells which respond to a cell-consumed signal or nutrient, contrasted with rather expected behaviour in the case of random-motility-driven invasion. The potential experimental observation of these behaviours is demonstrated by the individual-cell-level model used in this thesis, which does agree with the PDE model in predicting these unexpected invasion patterns. In the interest of examining such a case of a non-homogeneous domain experimentally, some brief suggestion is made as to how this could be achieved.

Fractional models for the migration of biological cells in complex spatial domains

Travelling wave phenomena are observed in many biological applications. Mathematical theory of standard reaction-diffusion problems shows that simple partial differential equations exhibit travelling wave solutions with constant wavespeed and such models are used to describe, for example, waves of chemical concentrations, electrical signals, cell migration, waves of epidemics and population dynamics. However, as in the study of cell motion in complex spatial geometries, experimental data are often not consistent with constant wavespeed. Non-local spatial models have successfully been used to model anomalous diffusion and spatial heterogeneity in different physical contexts. In this paper, we develop a fractional model based on the Fisher-Kolmogoroff equation and analyse it for its wavespeed properties, attempting to relate the numerical results obtained from our simulations to experimental data describing enteric neural crest-derived cells migrating along the intact gut of mouse embryos. The model proposed essentially combines fractional and standard diffusion in different regions of the spatial domain and qualitatively reproduces the behaviour of neural crest-derived cells observed in the caecum and the hindgut of mouse embryos during in vivo experiments.