Lygus Hahn (Hemiptera: Miridae) are serious pests of economically important field, fruit, vegetable, and greenhouse crops in Canada. The release of European Peristenus Förster (Hymenoptera: Braconidae) in the USA has resulted in significant suppression of this pest and has renewed interest in the release of European Peristenus spp. in Canada. Prior to the release of exotic Peristenus spp., ecological host range studies need to be conducted to define their habitat and host associations. <p>These associations can be difficult to study using conventional methods. Morphological similarity of related parasitoids prevents species-level identification by dissection. Host rearing is time-consuming and can result in high levels of host and parasitoid mortality. To facilitate identification of immature Peristenus spp. in their hosts, a multiplex PCR assay was developed. This assay provided a specific and sensitive tool to screen individual insects for three parasitoid species simultaneously. <p>To validate the utility of the multiplex PCR assay in ecological host range studies, parasitism and parasitoid species composition obtained using conventional and molecular techniques were compared. Molecular methods compared favorably with conventional methods; however, more complete species composition information was available with the multiplex assay. To improve the quality of risk assessment studies and extract the most accurate ecological host range data, molecular methods were used to evaluate host-parasitoid associations in mirid populations collected in two ecoregions. Several new host-parasitoid associations were recorded for <i>P. digoneutis</i> and <i>P. relictus</i>, but parasitism of non-target mirids was low. <p>Parasitism of the target host collected from different plant species was evaluated to help clarify Peristenus host-plant associations. Despite the investigation of three different host plant species, no difference was observed in the parasitism level or parasitoid species composition in <i>L. rugulipennis</i>. The post-release utility of the multiplex assay was investigated in Canada, where Lygus parasitoids may have dispersed following release in the USA. To confirm establishment, samples were analyzed using the multiplex PCR assay, and P. digoneutis was detected for the first time in southern Ontario.

The use of reduced oxygen-packaging techniques has resulted in anaerobic bacteria emerging as a problem for the brewing industry over the last twenty-five years. The genus Pectinatus, consisting of the species<i>P. cerevisiiphilus</i> and<i> P. frisingensis</i>, is a concern for producers of unpasteurized beer. As a result, there is an ongoing need to both understand this genus and develop rapid detection methodologies to combat its presence in the brewery. The objectives of this study were to sequence and characterize the flagellin genes from both Pectinatus species and evaluate the genes and proteins from a taxonomic and detection-suitability standpoint. <p>A combination of micro-protein sequencing, polymerase chain reaction (PCR) and Bubble-PCR was used to completely sequence one flagellin gene from each Pectinatus species. This knowledge was then utilized to sequence the flagellin gene from four additional Pectinatus isolates, two from each species. To confirm the identity of the flagellin genes, one flagellin gene from each species was cloned, expressed and detected with Pectinatus-specific antibodies. A discrepancy between of the predicted protein size and the actual protein size led to tests for glycosylation, a post-translational modification. Taxonomic analyses, based on the flagellin genes, were conducted at both the superkingdom and genus levels. Finally, genus- and species-specific PCR primer sets were designed and tested for the specific detection of Pectinatus in the brewery. <p>Cloning and expression data confirmed the identity of the sequenced genes as Pectinatus flagellin genes. Glycosylation was positively confirmed to be a post-translational modification for five of the six strains tested. Phylogenetic analysis revealed that both of the Pectinatus species grouped with the phylum Firmicutes (low G+C, Gram-positive bacteria) and that there was more diversity at the species level within the <i>P. frisingensis</i> flagellin gene than the <i>P. cerevisiiphilus</i> flagellin gene. As a final point, the detection of most Pectinatus isolates was achieved with the preliminary PCR primer sets designed, however, some non-Pectinatus beer spoilage organisms, primarily wort spoilage organisms, were also detected. Both the basic science and the applied results generated from this study will aid the brewing industry in its ongoing battle to control Pectinatus contamination.

The North American elk or wapiti is an indigenous species to Canada. Understanding of the reproductive physiology of elk is limited, as little research has been conducted in this field as compared to domestic farmed species. In order to make available the tools to study reproductive physiology of the elk this thesis describes the cloning and expression of elk pituitary glycoprotein hormone cDNAs. The common gonadotropin a-subunit, and FSH, LH and TSH b-subunit elk cDNAs were amplified by reverse transcription and polymerase chain reaction (RT-PCR). There was a high degree of nucleotide similarity between the elk a and b subunits when compared with reported sequences from other species. The cDNAs for the pituitary glycoprotein hormone genes were used as probes to investigate seasonal expression in the female elk pituitary gland. Steady state levels of the common a-subunit mRNA was observed regardless of the reproductive season, but a significant increase in expression occurred during the breeding season. The FSH and LH b-subunit genes were expressed at low levels in pituitary glands of animals during presumed anestrous and pregnancy, but levels considerably increased during estrus. In contrast, levels of TSH b-subunit mRNA were similar regardless of the reproductive status. The FSH cDNAs were also transfected into a Chinese hamster ovary (CHO) mammalian expression system, aimed at the production of recombinant elk FSH. Transfected CHO cell lines were screened for expression of a- and FSH b-subunit mRNA by Northern blot. Activity of FSH was equivalent to ~100 mIU/ml of recombinant human FSH (Gonal-FTM), identified by FSH receptor signaling in an in vitro cell based assay. In conclusion, this work represents an advance towards understanding the molecular basis of seasonal reproduction in elk. This information and the availability of elk recombinant FSH will be useful for the application of advanced reproductive technologies required for the rapid expansion of healthy, disease resistant, and genetically superior animals, which are important for domestic production and wildlife management.

Las micobacterias se pueden detectar por examen microscópico, por demostración de su ADN o rRNA específico tras amplificación del ácido nucleico correspondiente o por cultivo. Para aislarlas por cultivo, hace falta tratar las muestras con flora comensal (esputos, orinas, etc.) con diferentes sustancias, proceso llamado descontaminación.El objetivo de la Tesis ha consistido en evaluar: a) un nuevo método de descontaminación de muestras clínicas con C18-carboxypropilbetaína (CB-18) y compararla con el método de Tacquet-Tison, b) evaluar un sistema de amplificación de ADN por Reacción en Cadena de la Ligasa (sistema LCxMTB de Abbott, USA) con otro de amplificación de rRNA (AMTDT de Gen-Probe, USA) para detectar Mycobacterium tuberculosis en 74 biopsias pleurales parafinadas y c) comparar un nuevo medio de cultivo líquido no radiométrico (MB/BacT) con otro radiométrico BACTEC 12B y el medio sólido de Löwenstein-Jensen (L-J) para aislar micobacterias.Para evaluar el nuevo método CB-18 se procesaron 1201 muestras y se compararon los resultados con los obtenidos por el método de Tacquet-Tison. No hubo diferencias estadísticamente significativas en la detección de micobacterias por tinción. Se aislaron un 14% más de micobacterias tras descontaminar con CB-18, diferencia estadísticamente significativa tanto para el total de micobacterias como para micobacterias ambientales. No hubo diferencias entre los métodos para aislar M.tuberculosis. Este bacilo se detectó 2 días antes tras descontaminar con Tacquet (13 d.) que tras CB-18 (15 d.) Hubo más contaminaciones en los medios de cultivo tras descontaminar con CB-18, predominando los estreptococos del grupo viridans, los estafilococos y las levaduras.Se analizaron 57 biopsias pleurales parafinadas (BPP) de pacientes con tuberculosis pleural y 17 BPP de pacientes sin tuberculosis. Las 70 BPP se desparafinaron con xileno, se digerieron con proteinasa K, se inactivaron a 95ºC, se centrifugaron a 13.000 rpm. El sobrenadante se usó para realizar ambas técnicas de amplificación según los protocolos recomendados por los fabricantes. La sensibilidad para detectar M.tuberculosis fue del 52,6% para el AMTDT y del 63,2% para el LCxMTB, diferencia no significativa. La especificidad de ambas técnicas fue del 100%. Utilizando ambas técnicas conjuntamente se alcanza una sensibilidad del 80,7%. La sensibilidad para diagnosticar tuberculosis pleural mediante la baciloscopia, el cultivo de esputos y de líquidos pleurales había sido del 73,7% y si se añaden los resultados obtenidos por las técnicas de amplificación se eleva hasta el 96,5%. Las reacciones de amplificación aportaron un 22,8% de diagnósticos adicionales de las tuberculosis pleurales.Para evaluar el nuevo sistema de cultivo de micobacterias MB/BacT se descontaminaron 600 muestras por el método de Tacquet-Tison. Se compararon los resultados con los otros sistemas de uso habitual. Se aislaron 79 cepas de M.tuberculosis y 27 cepas de micobacterias ambientales. Se aislaron el 92%, 91% y 94% de las cepas, respectivamente, en L-J, BACTEC 12B y MB/BacT. Al combinar dos de estos medios de cultivo se aislaron prácticamente todas las cepas. De las muestras baciloscopia-negativas se aislaron significativamente más M.tuberculosis en MB/BacT que en BACTEC 12B. Las micobacterias crecieron en una media de 11,7 días en BACTEC, 2,5 días más tarde en MB/BacT y 15 días después en L-J. Las contaminaciones fueron más frecuentes en el MB/BACT, debidas a bacterias gram-positivas, por lo que se recomienda la adición de 1 mcg/mL de vancomicina al MB/BACT.Se concluye que el método CB-18 es adecuado como sistema de descontaminación de muestras clínicas para aislar micobacterias, que ambos sistemas de amplificación tienen una sensibilidad intermedia para detectar M.tuberculosis en BPP y que MB/BacT es un medio de cultivo líquido, no radiométrico, menos laborioso y alternativa adecuada al BACTEC para el aislamiento de micobacterias de muestras clínicas. / Mycobacteria can be detected by means of a microscopic exam, by recognizing their specific DNA or rRNA after having amplified its nucleic acid or for culture. In order to isolate them for culture, samples with commensal flora (sputum, urine, etc.) have to be treated with different substances, in a process known as decontamination.The objective of this thesis was to evaluate: a) a new method of decontamination of clinical samples using C18-carboxypropylbetaine (CB-18) and compare it with the method of Tacquet-Tison, b) compare a system of amplification of DNA by ligase chain reaction (LCxMTB, Abbott, USA) with an amplification of rRNA (AMTDT, Gen-Probe, USA) to detect Mycobacterium tuberculosis in 74 paraffinized pleural biopsies and c) compare a new liquid non radiometric culture medium (MB/BacT) with the radiometric BACTEC 12B and with the solid medium Löwenstein-Jensen (L-J) to isolate mycobacteriaIn order to evaluate the new CB-18 method, 1201 samples were processed and its results were compared to those obtained using the method of Tacquet-Tison. There were no statistical differences in the detection of mycobacteria by Ziehl-Neelsen staining. A 14% more of mycobacteria were isolated after being decontaminated with CB-18, which represents a significant statistical difference not only for all kinds of mycobacteria but also for nontuberculous mycobacteria. No difference was noticed among the methods used for isolating M.tuberculosis. This species was detected 2 days before in samples decontaminated with Tacquet-Tison(13 d.) than in samples by CB-18 (15 d.). There were more contaminations in culture mediums decontaminated with CB-18 and there was a predominance of group viridans Streptococcus, Staphylococcus spp. and yeasts.Forty-seven paraffin-embedded pleural biopsies (PEB) from patients with plural tuberculosis and 17 PEB from patients without tuberculosis were analysed. They were deparaffinized with xylene, digested with proteinase K, inactivated at 95º and centrifuged at 13,000 rpm. The supernatant was used in both techniques of amplification according to the protocols recommended by the manufacturers. The sensitivities of the AMTDT and LCxMTB were 52.6% and 63.2%, respectively (not stadistically significant). The specificity of both techniques was 100%. A sensitivity of 80.7% was achieved by using both techniques together. In fact, the sensitivity when diagnosing pleural tuberculosis by means of Zeehl-Neelsen staining, the sputum culture and of pleural liquids had been of 73.7%. The overall diagnostic yield with both culture and amplification techniques was 96.5%, with amplification techniques adding 22.8% of the diagnosis.Six hundred samples were decontaminated by using the Tacquet-Tison method in order to evaluate the new culture system of mycobacteria (MB/BacT). Seventy-nine strains on M.tuberculosis and 27 of nontuberculous mycobacteria were isolated, 92%, 91% and 94% respectively in L-J, BACTEC 12-B and MB-BacT. By combining two of these mediums practically all of the strains were isolated. From the Ziehl-Neelsen negative samples more M.tuberculosis were isolated using MB/BacT than using BACTEC 12B. The average number of days to detection of a single mycobacterial isolates was 14.2 days for MB/BacT, 26.1 days for egg-based media and 11.7 days for BACTEC. The contaminations were more frequent in MB/BacT, owing to the gram-positive bacteria; that's why, it seems recommended to add 1 mcg/mL of vancomycin to the MB/BacT. We can therefore conclude firstly that the CB-18 method is appropriate as a system of decontamination of clinical samples in which mycobacteria are supposed to be found; secondly, that both systems of amplification have an intermediate sensitivity to detect M.tuberculosis in PEB and, finally, that the MB/BacT is a reliable, nonradiometric, less labor-intensive alternative to BACTEC 12B for recovery of mycobacteria from clinical samples.

El cáncer colorrectal representa una de las patologías neoplásicas más frecuentes en nuestro medio. La afectación de los ganglios linfáticos es el factor pronóstico más importante de esta enfermedad. Alrededor de un 30 % de pacientes tratados quirúrgicamente con intención curativa y sin afectación ganglionar, presentarán recurrencia de su enfermedad. Las metástasis no detectadas en el estudio histopatológico convencional pueden ser la explicación de estos fallos terapéuticos. Los objetivos de esta tesis son:1. Identificar micrometástasis en ganglios linfáticos de pacientes con cáncer colorrectal sometidos a cirugía resectiva con intención curativa, mediante técnicas de inmunohistoquimia y RT-PCR, determinando la existencia de citokeratina 20 por ambas técnicas. 2. Evaluar el estadiaje de los pacientes con cada una de las técnicas, la convencional con tinción de hematoxilina eosina, y el obtenido mediante las técnicas de inmunohistoquimia y RT-PCR3. Determinar la implicación clínica del re-estadiaje con las nuevas técnicas, respecto al periodo libre de enfermedad y a la tasa de recurrencias de la misma durante el seguimiento a los cinco años. Se incluyeron 100 pacientes intervenidos de forma electiva por cáncer colorrectal en el Servicio de Cirugía General del Hospital Universitario Mútua de Terrassa, en Terrassa, Barcelona. Se excluyeron los pacientes intervenidos de urgencias, aquellos que presentaron metástasis a distancia en el momento del diagnóstico y a los que no se practicó cirugía con criterio oncológico. Se han incluido dos pacientes control (sin patología colónica maligna) para estudio específico de micrometástasis en ganglio linfático mediante inmunohistoquimia y RT-PCR.En el apartado de método se describe la técnica de linfadenectomía ultilizada así como el procesamiento del material linfático para la obtención del estadio ganglionar por las tres técnicas antes mencionadas. El tratamiento complementario de los pacientes se decidió en base al estadiaje fijado por el estudio histopatológico convencional con hematoxilina-Eosina. Se describe la batería de pruebas y programación de visitas efectuadas durante el control postoperatorio de los pacientes estudiados.La histopatología convencional, no clasificó correctamente el 37,5% de los pacientes que posteriormente fallecieron a causa de su enfermedad y el 50% de los pacientes que presentaron recidiva durante el seguimiento. La utilización de técnicas específicas para la identificación de micrometástasis puso de manifiesto la existencia de células neoplásicas en ganglios linfáticos clasificados como libres de enfermedad por la histopatología convencional. Mediante inmunohistoquimia se produjo un re-estadiaje del 13% de los pacientes. Por RT-PCR se alcanzó una re-estadificación del 41% de los pacientes.La determinación de micrometástasis mediante inmunohistoquimia no orientó en la identificación de pacientes a riesgo de sufrir recidiva de su enfermedad o muerte por cáncer colorrectal. Sin embargo, la detección de micrometástasis mediante RT-PCR fue positiva en el 88% (14 pacientes) de los pacientes que posteriormente desarrollaron una recidiva y en el 100% de los pacientes que presentaron muerte a causa de la evolución de su enfermedad.Según los resultados derivados de este estudio, se podría considerar la detección de micrometástasis por RT-PCR, una buena herramienta para la identificación de pacientes afectos de cáncer colorrectal en estadio II con factores de riesgo de mala evolución de su enfermedad. / Colorectal cancer is one of the most frequent malignant pathologies in our media. Lymph node involvement is the most important prognostic factor of this illness. Around 30 % of patients submitted to surgical procedures with curative intention and without lymph node involvement, will have recurrence of their neoplasm. Metastatic illness no detected in the former hystopathological study could be the explanation of these therapeutic mistakes.The objectives of this thesis are:1. To identify lymph node micrometastases in colorrectal cancers from patients submitted to oncologic resections with curative intention, by immunohistochemical and RT-PCR technique, determining cytokeratin 20 by both techniques2. To evaluate the new stage obtained by each technique, the conventional hematoxylin-eosin staining and the staging obtained with the specific techniques: immunohistochemestry and RT-PCR3. To determine if re-staging with specific techniques has clinical relevance in relation to free illness period and to relapse index during a follow up of five years. One hundred patients submitted to elective surgery for colorectal cancer were included. The study was developed in the Colorrectal Unit from General Surgery Department, Hospital Universitario Mútua Terrassa, in Terrassa, Barcelona. Patients with non-elective surgery, those with metastases at the moment of diagnostic and patients with a non-oncological resection (R0) were excluded. Two control patients (without malignant colorectal pathology) were included for specific study of micrometastases in lymph node by mean immunohistochemestry and RT-PCR. The methodology for lymphadenectomy is described as well as the hole process to get staging by the three techniques. Complementary treatment with chemotherapy was establish by hematoxylin-eosin hystopathological study. The follow up control during the study was describedHystopathological study didn't classify 37,5% of patients who lately will die because colorectal cancer progression during follow up and 50% of patients with recurrence o the illness.The use of specific techniques for micrometastases identification showed malignant cells in lymph nodes classify as free of illness by hematoxylin-eosin. Immunohistochemestry reclassified 13% of patients and RT-PCR 41% of patients.Micrometastases detection by immunohistochemestry didn't help top identify those patients with high risk of recurrence or who died because of colorrectal cancer. However, micrometastases detection by RT-PCR was positive in 88% (14 patients) of patients who will develop recurrence and in 100% of patients who will die because colorrectal cancer.The results of these study suggests that micrometastases detection by RT-PCR technique, could be in the future a promising method to identify patients with hystopathological stage II colorectal cancer with risk factors of poor prognosis.

Los programas de control de calidad microbiológica cada vez se aplican más a lo largo de la producción de la cadena alimentaria, siendo uno de ellos el programa de higienización. El propósito de los procedimientos de limpieza y desinfección es la destrucción de los microorganismos alterantes y de los patógenos de las superficies y del medio ambiente, para reducir el riesgo de la contaminación del alimento y evitando así la infección para los consumidores. Por lo tanto, para evaluar un programa de limpieza y desinfección es necesario muestrear las superficies y establecer los niveles residuales de contaminación. Existen diversos métodos para evaluar la contaminación microbiana de las superficies. Sin embargo, no hay un consenso para que un método estándar sea aceptado, debido a las ventajas y desventajas que presentan. Aún así, para la industria alimentaria, cada vez es más importante disponer de sistemas, rápidos y confiables para detectar la contaminación microbiana o incluso la presencia o ausencia de patógenos. En este estudio se usaron diferentes metodologías de control higiénico para vigilar y verificar la contaminación microbiana de superficies alimentarias. Para ello, se eligieron dos instalaciones diferentes con la finalidad de comparar las diferencias en base a los protocolos de limpieza-desinfección. Se evaluaron superficies en una industria alimentos deshidratados con un programa de higienización completo y en un supermercado con un tipo de higienización similar al empleado en el ámbito doméstico.En ambas instalaciones, los mayores recuentos se obtuvieron a partir de los discos, de acero inoxidable, adheridos a las superficies durante una semana y analizados con DEM. La DEM fue la metología más restrictiva. En la industria de alimentos deshidratados, las metodologías que seguidamente detectaron mayores recuentos fueron: la siembra del disco y el análisis de bioluminiscencia para la detección de ATP; mientras que en el supermercado fueron: el método de hisopado convencional y el análisis de bioluminiscencia para la detección de ATP. Conjuntamente, en ambas instalaciones se establecieron límites críticos estandarizados para cada metodología evaluada. En el supermercado, la presencia de patógenos como Salmonella spp., L. monocytogenes y S. aureus (coagulasa positiva) fue negativa y el recuento de enterobacterias obtenido fue muy bajo (<1 UFC/cm2).Además, se adaptó la PCR en tiempo real como método de control de patógenos (Salmonella Typhimurium y L. monocytogenes) en superficies de acero inoxidable en condiciones experimentales. La sensibilidad y eficacia de los kits iQCheck Salmonella e iQCheck Listeria monocytogenes fue demostrada utilizando caldo de soja triptona con 6 g/l de extracto de levadura (TSBYE) y Demi-Fraser (DFraser), respectivamente. Ambos medios de pre-enriquecimiento adicionados con Tween 80 como dispersante bacteriano. La sensibilidad de la PCR en tiempo real fue comparada con el método VIDAS a partir de biofilms y con el cultivo convencional (ISO) a partir de inóculos pequeños. La sensibilidad de la PCR en tiempo real fue mayor en ambos casos. La metodología propuesta podría ser una alternativa válida al método de cultivo convencional (ISO). / Microbiological quality control programs are becoming more common throughout all the stages of food production; sanitation is included among these programs. The purpose of cleaning and disinfection procedures is the destruction of spoilage microbes and pathogens from surfaces and the environment, in order to minimize the risk of contamination of food and thus avoiding infection for consumers. In order to assess a program of cleaning and disinfection it is necessary to sample surfaces to detect residual levels of contamination. There are lots of methods to enumerate microbial surface contamination. Nevertheless, there is no consensus on a standard method, because all of them have advantages and disadvantages. Still, for the food industry, the availability of reliable and rapid test systems to determine microbial contamination and the presence or absence of food-borne pathogens is becoming increasingly important. In this study, different hygienic control methodologies were used to assess microbiological contamination of food surfaces. Two different installations were selected to compare the differences based on the protocols of cleaning and disinfection. The evaluation of the surfaces of installations was performed in a dehydrated food manufacturing industry which had a complete sanitation programme and in a supermarket which only used basic sanitation, comparable to household cleaning.For both installations, the highest cell counts were obtained from the stainless steel coupons adhered to the surfaces for one week and analyzed with DEM. This was the most restrictive methodology. Direct spread of stainless steel coupons and bioluminescence assay for ATP determination were the methodologies that then showed higher cell counts in the food industry tested. Whereas, the conventional swab method and bioluminescence assay for ATP determination were the ones detecting higher cell counts at the supermarket. In both locations, standardized critical limits were established for each methodology. In the supermarket, the presence of pathogens such as Salmonella spp., L. monocytogenes and S. aureus (coagulase positive) was negative and Enterobacteriaceae counts was very low (<1 UFC/cm2).Real-time PCR assay was adapted to control food-borne pathogens such as Salmonella Typhimurium and L. monocytogenes on stainless steel surfaces in laboratory conditions. The sensitivity and efficiency of iQCheck Salmonella and iQCheck Listeria monocytogenes kits was demonstrated using triptic soy broth supplemented with 6 g/l of yeast extract (TSBYE) and Demi-Fraser (DFraser), respectively. Both pre-enrichment broths contained Tween 80 as dispersing agent for bacteria. The sensitivity of real-time PCR was tested using biofilms with VIDAS and traditional cultivation method (ISO) with small inocula levels. The sensitivity of real-time PCR was higher in both cases. The tested methodology could be a valid alternative to the conventional cultivation method (ISO).

Los objetivos de la presente tesis son los siguientes:- Determinar la sensibilidad en la detección del ARNm de la tirosinasa mediante la reacción en cadena de la polimerasa con transcripción reversa (RT-PCR) como marcador de células metastásicas de melanoma de úvea, en sangre periférica a partir de diluciones de varias líneas celulares de dicha tumoración. - Valorar la proporción de pacientes diagnosticados de melanoma de úvea, en los que mediante la misma técnica se detectan micrometástasis en sangre periférica.- Validar la RT-PCR para el rastreo metastásico en los pacientes afectos de melanoma de úvea respecto de los métodos estándar. (Radiografía de tórax, enzimas hepáticos y ecografía hepática).- Relacionar los factores clínicos y anatomo-patológicos pronósticos conocidos en el melanoma de úvea, con el resultado de la RT-PCR.

ABSTRACT Two experiments were conducted to determine the effects of various forage to concentrate ratios on the rumen microbial ecosystem and rumen fermentation parameters using culture-independent methods. In the first experiment, cattle were fed either a high concentrate (HC) or a high concentrate without forage (HCNF) diet. Comparison of rumen fermentation parameters between these two diets showed that duration of time spent below pH 5.2 and rumen osmolality were higher for HCNF. Calculations using Simpson’s index showed a greater diversity of dominant species for HCNF than in HC based on 16S rRNA PCR-DGGE. Real-time real-time PCR showed populations of Fibrobacter succinogenes (P=0.01) were lower in HCNF than HC diets. Ruminococcus spp., F. succinogenes and Selenomonas ruminantium were present at higher (P≤0.05) concentrations in solid than in liquid digesta in both diets. The second experiment compared cattle as they adapted from a strictly forage to a concentrate diet, after which they were subject to an acidotic challenge and a recovery period (Forage, Mixed Forage, High Grain, Acidosis and Recovery). A total of 153,621 high-quality bacterial sequences were obtained from biopsied rumen epithelium, and 407,373 sequences from the solid and liquid phases of rumen contents. Only 14 epithelial genera representing >1.0% of the epimural population differed (P ≤ 0.05) among dietary treatments. However, clustering showed a closer relation in bacterial profiles for the Forage and Mixed Forage diets as compared to the High Grain, Acidosis and Recovery diets. Several epithelial identified genera including Atopobium, Desulfocurvus, Fervidicola, Lactobacillus and Olsenella increased as a result of acidosis. However, any changes in bacterial populations during the acidosis challenge were not sustained during the recovery period. This indicates a high level of stability within the rumen epimural community. An epithelial core microbiome was determined which explained 21% of the enumerable rumen population across all treatment samples. Cluster analysis of the solid and liquid phase rumen bacterial showed that these populations differed (P ≤ 0.10) between forage and grain-based diets. Rumen core microbiome analysis found 32 OTU’s representing 10 distinct bacterial taxa in whole rumen contents for all dietary treatments. Heifers that developed clinical acidosis vs the subclinical acidosis showed increases in the genera Acetitomaculum, Lactobacillus, Prevotella, and Streptococcus. Variation in microbial taxa as an effect of both treatment and animal was evident in the solid and liquid fractions of the rumen digesta. However, impacts of a dietary treatment were transient and despite an acidotic challenge, rumen microbiota were able to recover within a week of perturbation. The bacterial populations in the rumen are highly diverse as indicated by DGGE analysis and showed clear distinction between not only dietary treatments, individual animals, but also between epithelial, liquid and solid associated populations on the same diet. Molecular techniques provide an increased understanding of the impact of dietary change on the nature of rumen bacterial populations and conclusions derived using these techniques may not match those previously derived using traditional laboratory culturing techniques.

No description available.

Starter cultures of different bacteria strains like lactic acid producing bacteria, Staphylococcus and Kocuria are used when making salami. Starter cultures give the sausage specific flavours and improve the quality and ripening of the final product. Probiotic strains can also be added during the production of salami. Studies have shown that probiotics are good for health and are therefore added to food, such as fermented sausages. In order to work as a probiotic strain, the bacteria have to survive during the production process, storage and through the whole human gastrointestinal tract. The aim of this study was to trace the probiotic strains Lactobacillus casei and Lactobacillus paracasei in salami samples to see if they had survived the production process. Methods used were DNA extraction, PCR, colony PCR and gel electrophoresis. Out of 100 samples in duplicate run in PCR, probiotics were found in only 3 of them. To see if screening of probiotics directly from plates was possible, a colony PCR was done. Colony PCR was made on colonies of two different strains of Lactobacillus casei, Lactobacillus paracasei and Lactobacillus sakei. From each bacteria strain, 5 colonies were analysed. Result showed that colony PCR, to screen for probiotic is a possible method.