‘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.     

Five field trials on the efficacy of a bovine respiratory syncytial virus vaccine

Five field trials evaluated whether immunization of beef cattle prior to weaning, at weaning, or immediately upon arrival at the feedlot with a commercial bovine respiratory syncytial virus (BRSV) vaccine would reduce subsequent treatment for respiratory disease.

Bovine respiratory syncytial virus vaccination was associated with a significant (p<0.05) reduction in treatment rate in one of three groups of calves immunized prior to weaning (−12%) and in calves immunized upon arrival at the feedlot (−4%).

There was no significant (p>0.05) effect of the BRSV vaccine on treatment rate in calves immunized at weaning, in calves immunized upon arrival at the Saskatoon bull test station, or in yearlings immunized upon arrival at the feedlot.

Although the trend in these field trials was to a sparing effect of the BRSV vaccine, the small reduction in treatment rate may not justify the cost of the vaccination program.

     

Control of Fusarium wilt in carnation grown on rockwool by Pseudomonas sp. strain WCS417r and by Fe-EDDHA

In carnations grown on rockwool disease incidence of fusarium wilt caused byFusarium oxysporum f.sp.dianthi (Fod) was reduced when Fe-EDDHA instead of Fe-DTPA was used as iron source in the nutrient solution. Addition ofPseudomonas sp. strain WSC417r intensified this reduction in the cultivar Pallas, moderately resistant to Fusarium, but not in the susceptible cultivar Lena. Treatment of plants with Fe-EDDHA instead of Fe-DTPA as iron source resulted in higher numbers and percentages on the roots, ofin vitro antagonistic fluorescent pseudomonads. However, differences were only significant at 56 days after planting for cv. Lena and at 14 and 28 days after planting for cv. Palas. Both chelators, at different concentrations, had no effect on root colonization by eitherPseudomonas sp. strain WCS417r orFod strain WCS816. However, when coinoculated, reduced numbers of propagules ofFusarium were found at concentrations of Fe-EDDHA lower than 10^–5 M.Higher concentrations of the siderophore fusarine produced byFod strain WCS816 were demonstrated when Fe-EDDHA instead of Fe-DTPA was used as iron source in culture media. At equal concentrations, no such differences were found in the amount of siderophore produced by WCS417r. Germ tube length ofFod was less with Fe-EDDHA than with Fe-DTPA. The reduction of germ tube length was stronger when the purified siderophore of WCS417r was added in excess to the culture media with Fe-EDDHA than those with Fe-DTPA. Therefore, the observed reduction of germ tube growth can not completely be explained by iron deprivation. It appeared that EDDHA exhibited a toxic effect for conidia ofFod strain WCS816 as well.we conclude that the observed disease reduction by Fe-EDDHA is a consequence of a limitation of iron availability forFod. This limitation is possibly intensified by the increase in number or percentage of antagonistic fluorescent pseudomonads that strongly compete for iron. The additional effect after bacterization withPseudomonas strain WCS417r in Fe-EDDHA treated carnations of cv. Pallas is likely to be due, at least partly, to a direct competition for iron between the siderophores ofFod strain WCS816 and ofPesudomonas sp. strain WCS417r.Samenvatting Verwelkingsziekte in anjers op steenwol, veroorzaakt doorFusarium oxysporum f. sp.dianthi (Fod), werd gereduceerd indien het ijzer-chelaat Fe-EDDHA in plaats van Fe-DTPA werd toegevoegd aan de nutriëntenvloeistof. Bacterisatie metPseudomonas sp. stam WCS417r had een additioneel effect bij de matig resistence cultivar Pallas maar niet bij de vatbare cultivar Lena. Toevoeging van Fe-EDDHA in plaats van Fe-DTPA aan planten als ijzerbron resulteerde op de wortels in hogere aantallen en percentages fluorescerende pseudomonaden, diein vitro antagonistisch waren ten opzichte vanFod. De verschillen waren echter alleen significant 56 dagen na planten voor de cultivar Lena en 14 en 28 dagen na planten voor de cultivar Pallas. Beide chelaten vertoonden bij verschillende concentraties geen effect op de kolonisatie van de wortel door beide microorganismen. Echter, wanneer beide micro-organismen gezamelijk werden toegevoegd nam de wortelkolonisatie doorFod stam WCS816 af bij concentraties lager dan 10^–5 M Fe-EDDHA. Er werd meer van het siderofoor fusarine doorFod stam WCS816 geproduceerd bij concentraties lager dan 10^–4 M Fe indien Fe-EDDHA in plaats van Fe-DTPA als ijzerbron aan het cultuurmedium was toegevoegd. Er werd geen effect van beide chelaten gevonden op de siderofoorproduktie door WCS417r. Indien een overmaat van het gezuiverde siderofoor van WCS417r werd toegevoegd aan Fe-EDDHA werden een sterkere afname van de kiembuislengte gevonden dan toevoeging aan Fe-DTPA. De reductie van de kiembuislengte bleek niet volledig verklaard te kunnen worden door een afname van de ijzerbeschikbaarheid. Het chelaat EDDHA heeft ook een toxisch effect op conidiën van fusarium.Wij concluderen, dat de waargenomen reductie van de verwelkingziekte door Fe-EDDHA een gevolg is van de afname van de ijzerbeschikbaarheid voorFod. Dit wordt waarschijnlijk versterkt door de ontwikkeling van een antagonistische, fluorescerendePseudomonas-populatie die sterk concurreren om ijzer. Het additioneel effect dat door bacterisatie metPseudomonas sp. WCS417r van de met Fe-EDDHA behandelde matig resistante anjers (Pallas) werd verkregen is voor een deel het gevolg van een directe concurrentie om ijzer tussen de sideroforen vanFod stam WCS816 en vanPseudomonas sp. stam WCS417r.     

Reproductive performance of nili-ravi buffaloes after a single injection of GnRH earlypost partum

Summary Sixty dairy buffaloes (second to fourth lactation) from a large buffalo farm were used to compare the effects of single intramuscular injections of 100 µg gondadotrophin releasing hormone (GnRH), 250 µg GnRH or saline given on day 14post partum. The buffaloes had calved at the end of the breeding season (December). Milk samples for progesterone determination were taken at the time of injection and then three times a week either until first insemination or until around day 90post partum. GnRH given at 14 dayspost partum resulted in quicker completion of uterine involution, earlier resumption of ovarian activity, shorter intervals between calving and conception and a better first service conception rate in non-suckled dairy buffaloes. Differences between the results obtained by a GnRH dose level of 100 µg and 250 µg were non-significant. In the post-treatment period cases of prolonged luteal activity were common in all groups of buffaloes. Therefore the sequential administration of GnRH and prostaglandin is suggested for the management of post-partum reproductive activity in problem herds.

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Desempeno Reproductivo De Bufalos Nili-Ravi Despues De Una Sola Inyeccion De GnRH Temprano Despues Del Post-Parto

Resumen Sesenta búfalos lecheros (segunda a cuarta lactancia) pertenecientes a un hato grande fueron utilizados para comparar los efectos de inyecciones intramusculares de 100 µg de GnRH, 250 µg y de solución salina, aplicadas 14 días después del parto. Los búfalos habian parido al final de la estación de pariciones (diciembre). Se tomaron muestras de leche para medir progesterona, en el momento de la inyección y después tres veces por semana hasta la primera inseminación, o hasta cerca de los 90 días post-parto. La GnRH administrada 14 días post-parto, dio como resultado una rápida involución uterina, más rápido retorno a la actividad ovárica, más cortos intervalos entre partos y concepción, y un mejor índice servicio-concepción en búfalos lecheros que no estaban amamantando. Los resultados de dosis de 100 µg y 250 µg no fueron significativos. En el período post-tratamiento, los casos de actividad luteal prolongada fueron comunes en todos los grupos de búfalos. Se sugiere entonces, la administración secuencial de GnRH y prostaglandina, parar el manejo de la actividad reproductiva post-parto en hatos problema.

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Performances De Reproduction Du Buffle Nili-Ravi Apres Une Injection Unique PrecocePost Partum De Gonadotrophine (GnRH)

Résumé Soixante bufflesses laitières (de la seconde à la quatrième lactation), appartenant à une grande ferme d'élevage de buffles, ont été utilisées pour comparer les effets d'une injection unique intramusculaire de 100 µg de gonadotrophine, ou de 250 µg du même produit ou d'une solution de sérum physiologique. Les bufflesses avaient mis bas à la fin de la saison de reproduction soit en décembre. Des prélèvements de lait ont été effectués pour déterminer le taux de progestérone au moment de l'injection d'une part, puis trois fois par semaine d'autre part jusqu'à la première insémination ou aux alentours d'une période de 90 jours après le part. La gonadotrophine, libérée sous forme d'hormone (GnRH), injectée 14 jours après la mise bas a eu pour effet un retour complet plus rapide de l'involution utérine et des intervalles plus courts entre la mise bas et la conception ainsi qu'un meilleur taux de conception à la première monte chez les femelles laitières non allaitantes. Les différences entre les résultats obtenus par une dose de 100/µg ou de 250/µg de gonadotrophine (GnRH) n'étaient pas significatives. Pendant la période post-thérapeutique, des cas d'activité lutéale prolongée ont été courants dans tous les groupes de bufflesses. En conséquence, l'administration séquentielle de GnRH et de prostaglandine est conseillée pour contrôler l'activité post-partum de reproduction dans les troupeaux qui présentent des problèmes de cet ordre.

     

Comparisons of frequencies and egg shell characteristics of broken and intact eggs within diverse populations of chickens

1. Differences were found among eight populations of White Plymouth Rock pullets in the frequency of broken eggs.

2. The shells of such eggs were thinner than those of intact eggs in seven of the populations.

3. The number of defective eggs, the specific gravity of the eggs, and the percentage hen‐day egg production of normal eggs were significantly correlated within populations with the incidence of broken eggs.

4. No significant correlations were found within populations between the incidence of egg breakage and either egg weight, body weight, or shank length.     

Subclinical pneumonia associated with an experimental adenovirus infection in the domestic fowl and the effect of concurrent infectious laryngotracheitis virus.

A CELO-type adenovirus (AV) isolated from fowls with respiratory disease was inoculated experimentally into the tracheas of young birds. No symptoms referable to respiratory infection were evident. Post mortem examination between days 2 and 5 after inoculation revealed pneumonia involving up to 30 per cent of the surface of the lungs. Histologically, a focal to diffuse interstitial lymphocytic infiltration and bronchiolar degeneration were present. Concurrent infections with a mild strain of infectious laryngotracheitis virus (ILT) failed to enhance the pathogenicity of either the AV or ILT infections.     

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