IMMUNOGLOBULINS IN PERIODONTAL DISEASE

Periodontal disease is the major cause of tooth loss in man. The initial histological picture of the inflamed gingiva is characteristic of local inflammatory reaction involving polymorphonuclear leukocytes, vasculitis and localized tissue loss. Subsequent clinical stages of periodontal disease (mild gingivitis) show histological evidence of the involvement of the immune response with initial accumulation of macrophages, and lymphocytes devoid of surface staining immunoglobulins (presumably T cells). As the disease progresses, a predominance of surface and cytoplasmic staining lymphocytes and plasma cells are seen (severe gingivitis and periodontitis). Whether the occurrence of the immunoglobulin positive lymphocytes and the concurrent loss of collagen and resorption of alveolar bone seen in periodontitis is indicative of a direct cause and effect relationship has been a controversy.^ The majority of investigations in the periodontal field have involved the use of peripheral blood lymphocytes or serum. Blastogenic responses of peripheral blood lymphocytes and serum antibody titers from periodontal patients to a variety of oral bacteria have not shown any correlation between response and the severity of disease. The need to study the local immune response in inflamed gingiva is apparent. Since there are no baseline studies on the functional capabilities of the lymphoid cells present in gingiva from periodontitis patients, an in depth study involving the role of the immunoglobulin positive lymphocytes was investigated.^ Inflamed gingiva from four clinically defined periodontal disease states (mild gingivitis, severe gingivitis, periodontitis and severe periodontitis) were placed in gingival organ cultures. Class specific immunoglobulins were quantitated in gingival organ culture supernatants using an indirect sandwich technique. A significant difference in mean levels of IgA and IgG was seen between mild gingivitis and periodontitis (P < .00l, P = .001), as well as in IgG levels between periodontitis and severe periodontitis (P = .001). The predominance of IgG in gingival organ culture supernatants and the statistically significant findings that the overall mean levels of IgG between mild gingivitis and periodontitis (P = .014) and between severe periodontitis and periodontitis (P = .001) suggested a possible indicator of periodontal disease. The presence of IgG in gingival organ culture supernatants was shown to be a product of actively secreting plasma cells. The incorporation of radiolabelled amino acids into IgG was noted over a seven-day period with a peak response at day 4-5. The inhibition of IgG synthesis by cyclohexamide confirmed the contention that IgG was a product of de novo synthesis and not serum derived.^ The specificity of immunoglobulins derived from gingival organ cultures were studied using a whole bacterial agglutination test. Oral bacteria frequently cultured from periodontal patients were assessed for their ability to be agglutinated by gingival organ culture supernatants. A positive correlation of antibody titer and severity of disease was seen with five strains of Actinomyces viscosus, two of Actinomyces naeslundii and one Actinomyces israelii. The agglutination of bacteria was shown to be due to the specific interaction of immunoglobulin and cell-wall antigen. ^

Molecular detection of Bartonella spp. in deer ked pupae, adult keds and moose blood in Finland

SUMMARY The deer ked (Lipoptena cervi) is a haematophagous ectoparasite of cervids that harbours haemotrophic Bartonella. A prerequisite for the vector competence of the deer ked is the vertical transmission of the pathogen from the mother to its progeny and transstadial transmission from pupa to winged adult. We screened 1154 pupae and 59 pools of winged adult deer keds from different areas in Finland for Bartonella DNA using PCR. Altogether 13 pupa samples and one winged adult deer ked were positive for the presence of Bartonella DNA. The amplified sequences were closely related to either B. schoenbuchensis or B. bovis. The same lineages were identified in eight blood samples collected from free-ranging moose. This is the first demonstration of Bartonella spp. DNA in a winged adult deer ked and, thus, evidence for potential transstadial transmission of Bartonella spp. in the species.

Strategic use of serology for the diagnosis of bovine tuberculosis after intradermal skin testing

Diagnostic tests based on cell-mediated immunity are used in programmes for eradication of bovine tuberculosis (Mycobacterium bovis). Serological assays could be applied as ancillary methods to detect infected animals. Our objective was to evaluate two serological techniques: M. bovis Ab Test (IDEXX, USA) and Enferplex™ TB assay (Enfer, Ireland) in animals tested simultaneously with the single and comparative intradermal tests and the interferon-gamma assay. This work was performed at two stages. First, a preliminary panel of samples collected prior to intradermal tests from tuberculosis-free (n=60) and M. bovis-infected herds (n=78) was assayed, obtaining high specificity: 100% (M. bovis Ab Test) and 98.3% (Enferplex TB assay) but low sensitivity (detection of M. bovis infected animals): 23.9% (M. bovis Ab Test) and 32.6% (Enferplex TB assay). Subsequently, the use of serological techniques was further studied in two herds with M. bovis infection (n=77) using samples collected prior to, and 72 h and 15 days after PPD inoculation. The highest level of detection of infected animals for serology was achieved at 15 days post-intradermal tests taking advantage of the anamnestic effect: 70.4% and 85.2% in herd A, and 66.7% and 83.3% in herd B, using M. bovis Ab Test and Enferplex TB assay, respectively. Quantitative results (average values obtained with M. bovis Ab Test ELISA and degree of positivity obtained with Enferplex TB assay) were higher in animals showing lesions compatible with tuberculosis. No significant differences were observed in the number of confirmed infected animals detected with either serological technique.

Direct visualization of the outer membrane of mycobacteria and corynebacteria in their native state.

The cell envelope of mycobacteria, which include the causative agents of tuberculosis and leprosy, is crucial for their success as pathogens. Despite a continued strong emphasis on identifying the multiple chemical components of this envelope, it has proven difficult to combine its components into a comprehensive structural model, primarily because the available ultrastructural data rely on conventional electron microscopy embedding and sectioning, which are known to induce artifacts. The existence of an outer membrane bilayer has long been postulated but has never been directly observed by electron microscopy of ultrathin sections. Here we have used cryo-electron microscopy of vitreous sections (CEMOVIS) to perform a detailed ultrastructural analysis of three species belonging to the Corynebacterineae suborder, namely, Mycobacterium bovis BCG, Mycobacterium smegmatis, and Corynebacterium glutamicum, in their native state. We provide new information that accurately describes the different layers of the mycobacterial cell envelope and challenges current models of the organization of its components. We show a direct visualization of an outer membrane, analogous to that found in gram-negative bacteria, in the three bacterial species examined. Furthermore, we demonstrate that mycolic acids, the hallmark of mycobacteria and related genera, are essential for the formation of this outer membrane. In addition, a granular layer and a low-density zone typifying the periplasmic space of gram-positive bacteria are apparent in CEMOVIS images of mycobacteria and corynebacteria. Based on our observations, a model of the organization of the lipids in the outer membrane is proposed. The architecture we describe should serve as a reference for future studies to relate the structure of the mycobacterial cell envelope to its function.

Impact of Acquired Pellicle Modification on Adhesion of Early Colonizers

New preventive approaches against dental erosion caused by acidic drinks and beverages include fortification of beverages with natural polymers. We have shown that the mixture of casein and mucin significantly improved the erosion-inhibiting properties of the human pellicle layer. This study aimed to investigate the effect of pellicle modification by casein, mucin and a casein-mucin mixture on the adhesion of early bacterial colonizers. Test specimens of human tooth enamel were prepared, covered with saliva and coated with 0.5% aqueous (aq.) casein, 0.27% aq. mucin or with 0.5% aq. casein-0.27% aq. mucin, after which the adhesion of Streptococcus gordonii, Streptococcus oralis, and Actinomyces odontolyticus was measured after incubation for 30 min and 2 h. log10 colony-forming units were compared by nonparametric tests. All three bacterial strains adhered in higher number to pellicle-coated enamel than to native enamel. The protein modifications of pellicle all decreased the counts of adhering bacteria up to 0.34 log10/mm2, the most efficient being the casein-mucin mixture. In addition to the recently shown erosion-reducing effect by casein-mucin, modification of the pellicle may inhibit bacterial adherence compared to untreated human pellicle.

Lactoferrin: An adjuvant to enhance the efficacy of the BCG vaccine

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is a disease with world wide consequences, affecting nearly a third of the world's population. The established vaccine for TB; an attenuated strain of Mycobacterium bovis Calmette Guerin (BCG), has existed virtually unchanged since 1921. Intensive research is focused on developing a TB vaccine that can surpass and improve the existing BCG vaccine. Lactoferrin, an iron binding protein found in mucosal secretions and granules of neutrophils was hypothesized to be an ideal adjuvant to enhance the efficacy of the BCG vaccine. Specifically, Lactoferrin enhanced the ratio of IL-12:IL-10 production from macrophages stimulated with LFS or infected with BCG, indicating the potential to affect T-cell development in vivo. Five different vaccination protocols were investigated for generation of host protective responses against MTB infection using Lactoferrin admixed to the BCG vaccine. Mice immunized and boosted at 2 weeks with BCG/Lactofefrin increased host protection against MTB infection by decreasing organ bacterial load and reducing lung histopathology. The observed postchallenge results paralleled with increasing production of IFN-γ, IL-2, TNF-α, and IL-12 from BCG stimulated splenocytes. In vitro studies examined possible mechanisms of Lactoferrin action on BCG infected macrophages and dendritic cells. Addition of Lactoferrin to BCG infected macrophages and dendritic cells increased stimulation of presensitized CD3+ and CD4+ T-cells. Analysis by fluorescent activated cell sorting (FACS) revealed an increase in surface expression of MHC I and decreased ratio of CD80/86 from BCG infected macrophages cultured with Lactoferrin. In contrast, Lactoferrin decreased surface expression of MHC I, MHC II, CD80, CD86, and CD40, but increased CD 11c, from BCG infected dendritic cells, indicating involvement of adhesion molecules. Overall, these studies indicate that Lactoferrin is a useful and effective adjuvant to improve efficacy of the BCG vaccine by enhancing generation of mycobacterial antigen specific T-cell responses through promotion of antigen presentation and T-cell stimulation.^

Development and Characterization of an in vitro Four-species Anaerobic Dental Biofilm Model

Dental caries is the most common chronic disease worldwide. It is characterized by the demineralization of tooth enamel caused by acid produced by cariogenic dental bacteria growing on tooth surfaces, termed bacterial biofilms. Cariogenesis is a complex biological process that is influence by multiple factors and is not attributed to a sole causative agent. Instead, caries is associated with multispecies microbial biofilm communities composed of some bacterial species that directly influence the development of a caries lesion and other species that are seemingly benign but must contribute to the community in an uncharacterized way. Clinical analysis of dental caries and its microbial populations is challenging due to many factors including low sensitivity of clinical measurement tools, variability in saliva chemistry, and variation in the microbiota. Our laboratory has developed an in vitro anaerobic biofilm model for dental carries to facilitate both clinical and basic research-based analyses of the multispecies dynamics and individual factors that contribute to cariogenicity. The rational for development of this system was to improve upon the current models that lack key elements. This model places an emphasis on physiological relevance and ease of maintenance and reproducibility. The uniqueness of the model is based on integrating four critical elements: 1) a biofilm community composed of four distinct and representative species typically associated with dental caries, 2) a semi-defined synthetic growth medium designed to mimic saliva, 3) physiologically relevant biofilm growth substrates, and 4) a novel biofilm reactor device designed to facilitate the maintenance and analysis. Specifically, human tooth sections or hydroxyapatite discs embedded into poly(methyl methacrylate) (PMMA) discs are incubated for an initial 24 hr in a static inverted removable substrate (SIRS) biofilm reactor at 37°C under anaerobic conditions in artificial saliva (CAMM) without sucrose in the presence of 1 X 106 cells/ml of each Actinomyces odontolyticus, Fusobacterium nucleatum, Streptococcus mutans, and Veillonella dispar. During days 2 and 3 the samples are maintained continually in CAMM with various exposures to 0.2% sucrose; all of the discs are transferred into fresh medium every 24 hr. To validate that this model is an appropriate in vitro representation of a caries-associated multispecies biofilm, research aims were designed to test the following overarching hypothesis: an in vitro anaerobic biofilm composed of four species (S. mutans, V. dispar, A. odontolyticus, and F. nucleatum) will form a stable biofilm with a community profile that changes in response to environmental conditions and exhibits a cariogenic potential. For these experiments the biofilms as described above were exposed on days 2 and 3 to either CAMM lacking sucrose (no sucrose), CAMM with 0.2% sucrose (constant sucrose), or were transferred twice a day for 1 hr each time into 0.2% sucrose (intermittent sucrose). Four types of analysis were performed: 1) fluorescence microscopy of biofilms stained with Syto 9 and hexidium idodine to determine the biofilm architecture, 2) quantitative PCR (qPCR) to determine the cell number of each species per cm2, 3) vertical scanning interferometry (VSI) to determine the cariogenic potential of the biofilms, and 4) tomographic pH imaging using radiometric fluorescence microscopy after exposure to pH sensitive nanoparticles to measure the micro-environmental pH. The qualitative and quantitative results reveal the expected dynamics of the community profile when exposed to different sucrose conditions and the cariogenic potential of this in vitro four-species anaerobic biofilm model, thus confirming its usefulness for future analysis of primary and secondary dental caries.

Alojamiento de terneros

A lo largo de las próximas páginas se van a abordar una serie de aspectos relacionados con los alojamientos para terneros. Las posibilidades de diseño son diversas y en la elección de la solución más adecuada pesan un buen número de factores condicionantes que hay que considerar detenidamente. Quizá sean los terneros el grupo de animales de una explotación bovina al que se le ha prestado una menor atención, en razón, quizá, de no tratarse de una fase productiva, en el sentido monetario del término. Es evidente que terneros criados en ambientes poco favorables puede que no lleguen nunca a expresar todo su potencial genético, bien se trate de producción de leche o de producción de carne. Aún más, los datos recogidos en muchísimas granjas nos dicen que se mueren demasiados terneros, con las pérdidas económicas que ello supone. Una de las principales razones de los altos índices de mortalidad es el inadecuado alojamiento en el que se coloca a los terneros durante esta fase crítica que son sus 2 -3 primeros meses de vida. Estudiaremos, por tanto, las necesidades ambientales de estos animales así como las condiciones básicas que deben cumplir los locales donde se alojan. Para finalizar esta introducción, no podemos olvidar que los alojamientos, por sí solos, no garantizan el éxito de una explotación, sino que es fundamental garantizar cada uno de los demás pilares de la producción animal: alimentación, manejo, higiene, sanidad y calidad genética de los propios animales.

Calidad del forraje y del heno

Podríamos definir la calidad del forraje y/o del heno como “el potencial que éstos tienen de producir una respuesta deseada en el animal”. Dicho de otra forma, el grado en que cubre las necesidades del animal que lo consume. Se analizan pues, en este capítulo, aquellos factores que condicionan la calidad del forraje verde original así como los que determinan la calidad del heno que se obtiene y que no han sido expuestos en capítulos anteriores. Asimismo, y de forma previa, se citan algunos de los parámetros de calidad que van a condicionar la respuesta del ganado, tanto en lo que a su nivel de ingestión voluntaria se refiere como a la respuesta productiva que se obtiene, función, entre otras cosas, del valor nutritivo del heno ingerido.

El proceso de henificación

En este capítulo se describe el proceso de henificación y, por tanto, todos los pasos que llevan a que el forraje segado pierda la cantidad de agua suficiente para poder ser conservado con las menores pérdidas nutritivas posibles. Asimismo, se analizan las causas de las pérdidas de valor alimenticio que el forraje experimenta desde el estado verde original. Durante el proceso que conduce a la formación del heno se experimentan una serie de modificaciones en la composición química del forraje original, lo que conlleva cambios en el valor nutritivo y de la digestibilidad del mismo. Estas pérdidas, que son consecuencia del nivel de humedad del forraje en desecación, hacen necesario acelerar dicho proceso de secado. Por ello se comentan diversos métodos para lograrlo como el acondicionado del forraje y la desecación química, método éste poco utilizado.

Condiciones ambientales y bienestar. La ventilación

En este breve primer capítulo de la primera monografía sobre conservación de forrajes se quiere poner de manifiesto la gran importancia que en la actualidad tienen los forrajes conservados desde el punto de vista económico, así como desde el punto de vista del manejo de la alimentación del ganado. Asimismo, los forrajes juegan un importante papel en el mantenimiento del suelo agrícola, permitiendo una gran diversidad de rotaciones de cultivo. De alguna forma, el cul- tivo de forrajes contribuye notablemente a lo que se ha venido en denominar “agricultura sostenible”, al permitir conservar una buena estructura del suelo, mejorar la infiltración del agua, evitar la erosión y controlar las plagas de forma natural. No cabe duda que, al igual que todos los alimentos fibrosos que no pueden ser aprovechados directamente por el hombre, gracias a los forrajes cultivados transformamos una enorme cantidad de recursos vegetales en alimentos de origen animal, lo que evidencia la importancia de estos cultivos en la alimentación humana.

Condiciones ambientales y bienestar. La ventilación

Probablemente las condiciones ambientales sean una de las principales asignaturas pendientes en los alojamientos de vacuno de leche. La concurrencia en gran parte de nuestro país de inviernos fríos y veranos muy calurosos añaden una dificultad más a la ya compleja tarea de diseñar una ventilación correcta. Los cambios bruscos de tiempo tampoco ayudan. Conscientes de la dificultad de la tarea, hemos querido ofrecer en este capítulo unas sencillas recomendaciones y aportar algunas sugerencias al hilo de lo que se está haciendo en otros países con sistemas de producción similares al nuestro. De esta forma, resaltamos la importancia de evitar las corrientes de aire pero proporcionar una amplia superficie de entrada y de salida de aire. Para que ello pueda ser posible, incluso en invierno, sugerimos la instalación de cortavientos. Finalmente, queremos resaltar la necesidad de que la renovación de aire llegue a todos los rincones de los alojamientos sin que se presenten zonas ciegas, y de evitar una humedad relativa excesiva, motivo de estrés térmico, de patologías respiratorias y de deterioro acelerado de los edificios.

Comederos

El alimento sólido es un ingrediente básico en la producción de leche, de ahí que dediquemos un capítulo al diseño y al manejo de la zona de alimentación. El comedero debe permitir una distribución adecuada de la ración, proporcionar espacio suficiente a las vacas para que éstas puedan consumir la cantidad que necesitan, estar limpio y libre de residuos de comidas anteriores y ser fácil de limpiar. La ingestión de alimentos se ve afectada por una serie de factores ambientales y de manejo. Sobre los primeros (clima y entorno) no se puede actuar o resulta caro y difícil, pero sobre las segundas (diseño y dimensionamiento adecuado, espacio disponible, etc.) sí podemos influir. De esta forma, un comedero correctamente diseñado da lugar a un acercamiento del animal más frecuente, más duradero y a una mayor ingestión. Asimismo, evitará numerosas lesiones debidas a una presión excesiva de las vacas sobre él. También nos referiremos a su mantenimiento y limpieza, para lo que es esencial una buena elección de materiales.

Alojamientos y bienestar. Aspectos principales

El bienestar de los animales no se restringe a una alimentación y manejo adecuados, sino que empieza en el propio diseño y concepción de la explotación en su conjunto. Cuestiones como la orientación de los edificios, la disposición entre ellos, su emplazamiento en la parcela, etc., cobran gran relevancia cuando pensamos en las patologías que se derivan de una mala calidad del aire por una falta de renovación del mismo. Debemos pensar también en el comportamiento natural de las vacas a la hora de diseñar el interior de los alojamientos y evitar soluciones que provocan malestar, incomodidad o lesiones al animal y que, en muchas ocasiones, son difíciles y/o costosas de corregir. En modelos intensivos de producción de leche, el suelo habitual de hormigón se ha convertido en una de las principales causas de problemas podales de las vacas y en origen de no pocos accidentes. Conocer cómo debe ser el suelo sobre el que caminan los animales es un asunto vital para el bienestar económico de la explotación.