BoLA class I polymorphism and in vitro immune response to M. bovis antigens

SUMMARY: From a sample of 119 Friesian calves, serologically typed for BoLA class I, 47 subjects were chosen expressing 9 different MHC types (A6, A6.9, A10, A11, A14, A15, A30, W16, M103) with the same age and reared in the same farm conditions. The animals were s.c. injected with a water in oil suspension of killed M. bovis and the treatment was repeated two days later. Before the treatment and 21 days later, calves were bled and on PBM (peripheral blood mononuclear leucocytes) were performed the following tests: 1. Lymphocyte Stimulation with bovine and avian PPDs (Purified protein derivative of Mycobacterium bovis and Mycobacterium avium, respectively). 2. Phagocytic activity towards M. bovis. 3. Class II molecules expression on cell surface. 4. Percentage of leucocyte populations and subpopulations. In the in vitro Lymphocyte Stimulation test, all the animals and classes were responders. Animals bearing A10 BoLA class I presented c.p.m. (counts per minute) and index values higher than the other cattle; these values were significantly positively related both to bovine and avian PPDs (P < .01). By variance analysis A14 BoLA type showed a slight positive significant correlation with more efficient phagocytic activity. BoLA class I type did not seem to significantly affect percentage of class II positive cells and leucocyte percentages on PBM. ZUSAMMENFASSUNG: Der BoLA Klasse I Polymorphismus und in vitro immunologische Antwort gegen die Antigene von M. bovis Aus einer Stichprobe von 119 für BoLA Klasse I serologisch typisierten Friesian K?lber, wurden 47 Subjekte ausgew?hlt, die 9 verschiedene MHC Typen ausdrückten (A6, A6.9, A10, A11, A14, A15, A30, W16, M103). Alle waren gleich alt und in gleichen Haltungsbedingungen. Die Tiere wurden mit einer Wasser-in-Ol Suspension abget?teter M. bovis subkutan injiziert und die Behandlung nach zwei Tagen wiederholt. Vor und 21 Tage nach Behandlung wurden die folgenden Tests ausgeführt: 1. Lymphozyten-Stimulationstest mit bovinen und Geflügel PPDs. 2. Phagozyten Aktivit?t gegen M. bovis. 3. Zeil-Oberfl?chen, Expression der Klasse II Moleküle. 4. Anteile der Lymphozyten Populationen und Subpopulationen. Im in vitro Lymphozyten-Stimulationstest waren alle Tiere und Klassen responder. Tiere mit A10 BoLA I zeigten h?here c.p.m. und Indexwerte als die anderen; diese Werte waren in signifikant positiver Beziehung mit der PPD von M. bovis und auch mit M. avium (P < .01). BoLA Typ A14 zeigte leicht signifikant positive Korrelation mit wirksamerer Phagozyten Aktivit?t. BoLA Klasse I Typ scheint nicht den Prozentsatz der positiven Zellen der Klasse II und der Leukozyten der PBM signifikant zu beeinflussen. RESUMEN: Polimorfismo de BoLA clase I y immunidad a los antigenos del M. bovis Se escojeron 47 novillos dentro de un grupo de 119 animales que segun analisis previamente hecha tenian BoLA de clase I. Estos 47 novillos fueron escojidos de manera que tuvieran 9 distintos tipos de MHC (A6, A6.9, A10, A11, A14, A15, A30, W16, M103), la misma edad, las mismas condiciones de cria. Estos animales fueron inoculados subcutaneo con M. bovis matados en una suspension oleosa y la misma inoculacion fue repetida una secunda vez despues de dos dias. Por cada animal se tomaron muestras de sangre antes y 21 dias despues de la inoculacion de arriba. Las muestras de sangre fueron pruebaoas con: 1. Stimulacion Lymhocitaria con PPD bovina y avicola. 2. Actividad phagocitaria a M. bovis. 3. Expresion sobre la superficie celular de moleculas de clase II. 4. Porcentaje de poblaciones y de subpob-laciones de leucocitos. Todos los animales y todos los tipos de MHC dieron respuestas positivas en las pruebas de Stimulacion Lymphocitaria. Los animales que tenian la BoLA A10 presentaron valores de c.p.m. y indices mas altos de los demas animales. Estos valores se encontraron significativamente y positivamente relacionados sea a la PPD bovina que a la PPD avicola. Por medio de la analisis de varianzas se encontro que el tipo BoLA A14 muestraba una correlacion significativa y algo positiva con una mejor actividad fagocitaria. Los tipos de clase BoLA I no parecieron que influenzaran de manera appreciable el porcentaje de positividad por la clase II y el porcentaje de leucocitos en la sangre PBM.     

‘Plantibodies’: a flexible approach to design resistance against pathogens

Engineering resistance against various diseases and pests is hampered by the lack of suitable genes. To overcome this problem we started a research program aimed at obtaining resistance by transfecting plants with genes encoding monoclonal antibodies against pathogen specific proteins. The idea is that monoclonal antibodies will inhibit the biological activity of molecules that are essential for the pathogenesis. Potato cyst nematodes are chosen as a model and it is thought that monoclonal antibodies are able to block the function of the saliva proteins of this parasite. These proteins are, among others, responsible for the induction of multinucleate transfer cells upon which the nematode feeds. It is well documented that the ability of antibodies to bind molecules is sufficient to inactivate the function of an antigen and in view of the potential of animals to synthesize antibodies to almost any molecular structure, this strategy should be feasible for a wide range of diseases and pests.Antibodies have several desirable features with regard to protein engineering. The antibody (IgG) is a Y-shaped molecule, in which the domains forming the tips of the arms bind to antigen and those forming the stem are responsible for triggering effector functions (Fc fragments) that eliminate the antigen from the animal. Domains carrying the antigen-binding loops (Fv and Fab fragments) can be used separately from the Fc fragments without loss of affinity. The antigen-binding domains can also be endowed with new properties by fusing them to toxins or enzymes. Antibody engineering is also facilitated by the Polymerase Chain Reaction (PCR). A systematic comparison of the nucleotide sequence of more than 100 antibodies revealed that not only the 3′-ends, but also the 5′-ends of the antibody genes are relatively conserved. We were able to design a small set of primers with restriction sites for forced cloning, which allowed the amplification of genes encoding antibodies specific for the saliva proteins ofGlobodera rostochiensis. Complete heavy and light chain genes as well as single chain Fv fragments (scFv), in which the variable parts of the light (V_L) and heavy chain (V_H) are linked by a peptide, will be transferred to potato plants. A major challenge will be to establish a correct expression of the antibody genes with regard to three dimensional folding, assembly and intracellular location.     

Effects of Latent Infection of Stock Plants and Abundance of Thrips on the Occurrence of <Emphasis Type="Italic">Tomato spotted wilt virus </Emphasis>in Chrysanthemum Fields

DAS-ELISA proved to be reliable enough to detect a latent infection by Tomato spotted wilt virus (TSWV) in asymptomatic stock plants of chrysanthemum. A high density of Frankliniella occidentalis, the predominant vector, in the presence of latently infected stock plants resulted in a high incidence of disease in the chrysanthemum production field. The incidence of disease was low when the vector thrips were not abundant in spite of the presence of latently infected stock plants. These results suggest that an infestation of the vector thrips causes severe secondary spread of TSWV originating from latently infected stock plants in chrysanthemum production fields. Received 27 July 2001/ Accepted in revised form 27 November 2001     

Indole-3-acetic acid biosynthesis in <Emphasis Type="Italic">Aciculosporium take</Emphasis>, a causal agent of witches' broom of bamboo

 Aciculosporium take (Ascomycota; Clavicipitaceae) is a causal agent of witches' broom of bamboo plants. The symptoms of this disease are believed to be induced by plant hormones, particularly auxins. Indole-3-acetic acid (IAA) was identified in cultures of this fungus in an l-tryptophan-supplemented liquid medium. IAA production was confirmed on 30 isolates of A. take from various hosts and locations at levels up to 1 mg/l. The biosynthetic pathway of IAA in A. take culture was examined by analyzing intermediate products and by feeding experiments. The results showed that the indole-3-pyruvic acid pathway (l-tryptophan → indole-3-pyruvic acid → indole acetaldehyde → IAA) was the dominant pathway in A. take. Received: June 3, 2002 / Accepted: July 25, 2002     

Inconspicuous restraint of rice seedling growth by root-infecting fungi in soil of a rice paddy field

 Rice seedling growth, estimated by plant height and root development and discoloration, was better in pasteurized soil than in unpasteurized soil obtained from a flooded rice field. Rice seedlings also grew better in sterilized soil modified by adding roots harvested from the pasteurized soil than in soil modified by adding roots harvested from the unpasteurized soil. The results demonstrate that seedling growth in the rice field soil was inhibited by soil microorganisms, even though no typical symptoms such as seedling blight or damping-off appeared. Pythium aristosporum is suggested to be involved in the inhibition. Thus, it appears that inconspicuous restraint of rice seedling growth could occur in soils of rice paddy fields. Received: May 20, 2002 / Accepted: October 16, 2002 Acknowledgments The authors thank Dr. T. Ichitani, former professor at Osaka Prefectural University, for providing an isolate of Pythium aristosporum for comparison, and Mr. Mitsuaki Sato of Akita Prefectural College of Agriculture for technical assistance.     

Functional heterogeneity in resources within landscapes and herbivore population dynamics

Large mammalian herbivores are notorious for their propensity towards population irruptions and crashes, yet many herbivore populations remain relatively stable. I explore how resource heterogeneity within landscapes dampens population instability, using a metaphysiological modelling approach considering patch state distributions. Resource heterogeneity is functionally stabilizing through spreading consumption away from preferred resources before these become critically depleted. Lower-quality resources act as a buffer against starvation during critical periods of the seasonal cycle. Enriching resource quality is destabilizing, even if patch diversity is maintained, because food quantity then becomes the limitation. The potential consequences of landscape fragmentation are explored using the Serengeti ecosystem, characterised by broadscale resource gradients, as a hypothetical example. Further insights provided by the model are illustrated with specific examples concerning the effects of patch scales and waterpoint distribution. A metaphysiological modelling approach enables the basic consequences of landscape heterogeneity to be distinguished from further effects that may arise from specific patch scales and configurations, without the distracting detail of spatially explicit models.     

Validation of the specificity and sensitivity of species-specific primers that provide a reliable molecular diagnostic for <Emphasis Type="Italic">Xiphinema diversicaudatum</Emphasis>, <Emphasis Type="Italic">X. index</Emphasis> and <Emphasis Type="Italic">X. vuittenezi</Emphasis>

Xiphinema diversicaudatum and X. index are vector nematode species of economic importance in viticulture regions as they can transmit Arabis Mosaic, Grapevine Fanleaf and Strawberry Latent Ringspot viruses to grapevine. Wang et al. (2003) designed species-specific diagnostic primers from ribosomal genes for both these vector species as well as a vector and a non-vector species X. italiae and X. vuittenezi, respectively. Our study aimed to confirm the specificity and determine the sensitivity and reliability of the primers for the two vector species, X. diversicaudatumand X. indexwhen challenged with closely related longidorid species and general nematode communities typical of vineyard soil. With one exception, no PCR product was observed when the primers were tested against six Longidorus, one Paralongidorus and one Xiphinema non-target species. Occasionally (three out of eight replicate PCR reactions) a weak PCR product was noted when primers for X. index were tested with L. elongatus. Furthermore, when challenged with a range of non-target nematode species comprising the nematode community typical of viticulture soil, no PCR product was amplified. An experimental dilution series of extracted DNA rigorously demonstrated that DNA from an equivalent single specimen of the target virus-vector species, X. diversicaudatum and/or X. index, could be detected amongst 1000 equivalent non-targetX. vuittenezi. Also, extracted DNA from an equivalent single target specimen was detected when added to DNA extracted from the overall soil nematode community. The primers were assessed further by using serial mixtures of actual nematodes rather than extracted DNA to simulate field soil. Using this method, a single target nematode could be detected amongst 200 non-target specimens. Given their specificity, sensitivity and reliability, it appears that these diagnostic primers will be of great benefit to phytosanitary/quarantine services related to the viticulture industry.