A field vaccine trial in Tanzania demonstrates partial protection against malignant catarrhal fever in cattle.

Malignant catarrhal fever (MCF) is a fatal lymphoproliferative disease of cattle that, in East Africa, results from transmission of the causative virus, alcelaphine herpesvirus 1 (AlHV-1), from wildebeest. A vaccine field trial involving an attenuated AlHV-1 virus vaccine was performed over two wildebeest calving seasons on the Simanjiro Plain of northern Tanzania. Each of the two phases of the field trial consisted of groups of 50 vaccinated and unvaccinated cattle, which were subsequently exposed to AlHV-1 challenge by herding toward wildebeest. Vaccination resulted in the induction of virus-specific and virus-neutralizing antibodies. Some cattle in the unvaccinated groups also developed virus-specific antibody responses but only after the start of the challenge phase of the trial. PCR of DNA from blood samples detected AlHV-1 infection in both groups of cattle but the frequency of infection was significantly lower in the vaccinated groups. Some infected animals showed clinical signs suggestive of MCF but few animals went on to develop fatal MCF, with similar numbers in vaccinated and unvaccinated groups. This study demonstrated a baseline level of MCF-seropositivity among cattle in northern Tanzania of 1% and showed that AlHV-1 virus-neutralizing antibodies could be induced in Tanzanian zebu shorthorn cross cattle by our attenuated vaccine, a correlate of protection in previous experimental trials. The vaccine reduced infection rates by 56% in cattle exposed to wildebeest but protection from fatal MCF could not be determined due to the low number of fatal cases.

Conservation and variation of the parapoxvirus GM-CSF-inhibitory factor (GIF) proteins.

The GIF protein of orf virus (ORFV) binds and inhibits the ovine cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2). An equivalent protein has so far not been found in any of the other poxvirus genera and we therefore investigated whether it was conserved in the parapoxviruses. The corresponding genes from both the bovine-specific pseudocowpox virus (PCPV) and bovine papular stomatitis virus (BPSV) were cloned and sequenced. The predicted amino acid sequences of the PCPV and BPSV proteins shared 88 and 37 % identity, respectively, with the ORFV protein. Both retained the six cysteine residues and the WSXWS-like motif that are required for biological activity of the ORFV protein. However, an analysis of the biological activity of the two recombinant proteins revealed that, whilst the PCPV GIF protein bound to both ovine and bovine GM-CSF and IL-2 with very similar binding affinities to the ORFV GIF protein, no GM-CSF- or IL-2-binding activity was found for the BPSV protein.

Glycosylation, disulfide bond formation, and the presence of a WSXWS-like motif in the orf virus GIF protein are critical for maintaining the integrity of Binding to ovine granulocyte-macrophage colony-stimulating factor and interleukin-2.

Orf virus (ORFV), the type species of the family Parapoxviridae, encodes a protein (GIF) that binds and inhibits the ovine cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2). There is no obvious sequence homology between the ORFV protein and any known mammalian GM-CSF- or IL-2-binding proteins. We demonstrate here that many of the biochemical properties of mammalian GM-CSF receptors that are required for efficient binding of GM-CSF are also critical to the GIF protein for binding to ovine GM-CSF (ovGM-CSF). Site-directed mutagenesis of the GIF protein demonstrated, first, the importance of disulfide bonds, and second, that a sequence motif (WDPWV), related to the WSXWS motif of the type 1 cytokine receptor superfamily, was necessary for biological activity. Finally, glycosylation of the GIF protein was also critical for binding to GM-CSF.

Orf virus encodes a novel secreted protein inhibitor of granulocyte-macrophage colony-stimulating factor and interleukin-2.

The parapoxvirus orf virus encodes a novel soluble protein inhibitor of ovine granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2). The GM-CSF- and IL-2-inhibitory factor (GIF) gene was expressed as an intermediate-late viral gene in orf virus-infected cells. GIF formed homodimers and tetramers in solution, and it bound ovine GM-CSF with a K(d) of 369 pM and ovine IL-2 with a K(d) of 1.04 nM. GIF did not bind human GM-CSF or IL-2 in spite of the fact that orf virus is a human pathogen. GIF was detected in afferent lymph plasma draining the skin site of orf virus reinfection and was associated with reduced levels of lymph GM-CSF. GIF expression by orf virus indicates that GM-CSF and IL-2 are important in host antiviral immunity.

Analysis of pancreatic development using a cell lineage label.

We have devised a new culture system for in vitro culture of pancreatic buds from mouse embryos which enables the organ to grow as a flat branched structure suitable for wholemount immunostaining. This system has been used to analyze pancreatic development. We have also used the ROSA-26 gene trap mouse strain as a source of tissue which expresses lacZ in a stable manner, in all cell types, during in vitro culture. Combinations of lacZ epithelium and unlabeled mesenchyme show that both exocrine and endocrine cells arise from the epithelium, and smooth muscle cells from the mesenchyme. Although previously suspected, this is the first formal proof that both exocrine and endocrine cells are of endodermal origin. Combinations of lacZ epithelium with unlabeled stomach mesenchyme give similar results and show that stomach mesenchyme has the same trophic effect as pancreatic mesenchyme. When a lacZ and an unlabeled epithelium are combined with an unlabeled mesenchyme, both acini and islets in the resulting culture can be of mixed cell composition. This shows that neither of the chief structural units of the pancreas is formed by clonal growth from a single cell.

Chloroplast division in higher plants requires members of two functionally divergent gene families with homology to bacterial ftsZ.

The division of plastids is critical for viability in photosynthetic eukaryotes, but the mechanisms associated with this process are still poorly understood. We previously identified a nuclear gene from Arabidopsis encoding a chloroplast-localized homolog of the bacterial cell division protein FtsZ, an essential cytoskeletal component of the prokaryotic cell division apparatus. Here, we report the identification of a second nuclear-encoded FtsZ-type protein from Arabidopsis that does not contain a chloroplast targeting sequence or other obvious sorting signals and is not imported into isolated chloroplasts, which strongly suggests that it is localized in the cytosol. We further demonstrate using antisense technology that inhibiting expression of either Arabidopsis FtsZ gene (AtFtsZ1-1 or AtFtsZ2-1) in transgenic plants reduces the number of chloroplasts in mature leaf cells from 100 to one, indicating that both genes are essential for division of higher plant chloroplasts but that each plays a distinct role in the process. Analysis of currently available plant FtsZ sequences further suggests that two functionally divergent FtsZ gene families encoding differentially localized products participate in chloroplast division. Our results provide evidence that both chloroplastic and cytosolic forms of FtsZ are involved in chloroplast division in higher plants and imply that important differences exist between chloroplasts and prokaryotes with regard to the roles played by FtsZ proteins in the division process.

Susceptibility testing of Klebsiella spp.--an international collaborative study in quality assessment.

In order to compare the prevalence of antibiotic resistance in different geographical areas, it is necessary to ensure agreement between laboratories on the assignment of strains to 'susceptible' and 'resistant' categories. An international quality assessment was performed to investigate the performance of susceptibility testing of Klebsiella spp. Ninety-five strains of klebsiellae were selected from clinical isolates at the London Hospital Medical College (LHMC). These included strains with a diversity of susceptibility profiles to amoxycillin/clavulanate, piperacillin, ceftazidime, cefuroxime, ciprofloxacin, gentamicin and trimethoprim. The strains were sent to 13 participating laboratories in Europe and the USA and laboratories were asked to test the susceptibility of these strains to these antibiotics by their usual methods. They were also asked to provide details of the method used to test susceptibility. Several different standard recommended testing methods were used. Reporting of susceptibilities was generally accurate, but a number of anomalies were noted. Discrepancies of reporting between the LHMC and the participating laboratories was more marked for resistant strains, particularly in the detection of resistance to cefuroxime and ciprofloxacin, as well as the assignment of susceptibility and resistance to piperacillin and amoxycillin/clavulanate. Some discrepancies could be attributed to the use of different breakpoints, leading to differing assignment of susceptibility. Methodological variations including disc content, inoculum and failure to measure and interpret zone sizes consistently also led to anomalies. This quality assessment programme has helped to identify problems in susceptibility testing which should be investigated further.

Why "sounds are judged longer than lights": application of a model of the internal clock in humans.

Three experiments, using temporal generalization and verbal estimation methods, studied judgements of durations of auditory (500-Hz tone) and visual (14-cm blue square) stimuli. With both methods, auditory stimuli were judged longer, and less variable, than visual ones. The verbal estimation experiments used stimuli from 77 to 1183 msec in length, and the slope of the function relating mean estimate to real length differed between modalities (but the intercept did not), consistent with the idea that a pacemaker generating duration representations ran faster for auditory than for visual stimuli. The different variability of auditory and visual stimuli was attributed to differential variability in the operation of a switch of a pacemaker-accumulator clock, and experimental data suggested that such switch effects were separable from changes in pacemaker speed. Overall, the work showed how a clock model consistent with scalar timing theory, the leading account of animal timing, can address an issue derived from the classical literature on human time perception.

Resistance to Meloidogyne incognita Race 3 and Rotylenchulus reniformis in Wild Accessions of Gossypium hirsutum and G. barbadense from Mexico.

Forty-six accessions of G. hirsutum and two of G. barbadense were examined for resistance to Meloidogyne incognita race 3 and Rotylenchulus reniformis in environmental growth chamber experiments, with the objective of finding new sources of resistance. Only the G. barbadense accessions, TX-1347 and TX-1348, supported significantly less reproduction by R. reniformis than the susceptible control, Deltapine 16 (USDA accession SA-1186). However, they were highly susceptible to M. incognita race 3. The G. hirsutum accessions TX-1174, TX-1440, TX-2076, TX-2079, and TX-2107 had levels of resistance to M. incognita race 3 as great as or greater than those of Clevewilt 6 and Wild Mexican Jack Jones, which are the primary sources of resistance to M. incognita race 3 in the most resistant breeding lines. No accession was as resistant as the highly resistant line Auburn 623 RNR (SA-1492). Resistant accessions were from the Mexican coastal states of Campeche, Quintana Roo, Tabasco, Veracruz, and Yucatan. Populations of R. reniformis from Alabama, Mississippi, Louisiana, and Texas, and of M. incognita race 3 from Mississippi, Texas, and California, had similar reproductive rates on resistant genotypes. Thus, new sources of resistance to M. incognita race 3 but not to R. reniformis were identified in wild accessions of G. hirsutum from southern Mexico.

Susceptibility testing of Haemophilus influenzae--an international collaborative study in quality assessment.

In order to compare the prevalence of antibiotic resistance in different geographical areas, it is necessary to ensure that agreement is achieved between laboratories on the assignment of strains to 'susceptible' and 'resistant' categories. An international quality assessment study, involving 15 laboratories in eight countries, was performed to investigate the standard of performance of the susceptibility testing of Haemophilus influenzae. One hundred and fifty strains of H. influenzae were distributed from the London Hospital Medical College (LHMC) to all laboratories who were asked to test the susceptibility of the strains to ampicillin, chloramphenicol, tetracycline, trimethoprim, cephalosporins and ciprofloxacin. Laboratories were also asked to provide the details of methodology to test the susceptibility. Significant discrepancy between the LHMC and the participating laboratories appeared in the detection of resistance to ampicillin (especially beta-lactamase-negative strains resistant to ampicillin) as well as the assignment of susceptibility and resistance to chloramphenicol, tetracycline and trimethoprim. Often these reflected the use of inappropriate breakpoints which led to erroneous assignment of susceptibility. Other variations including disc content, medium and supplement, inoculum as well as failure to measure zone sizes properly also led to some repeating anomalies.

Speeding up an internal clock in humans? Effects of click trains on subjective duration.

Four experiments investigated the effect of trains of clicks (usually 5 s long and at 5 or 25 Hz) on subjective duration in humans, as previous research had suggested that such a manipulation would speed up the pacemaker of an internal clock by increasing participants' arousal. The four experiments used temporal generalization, pair comparison of duration, verbal estimation, and production of short durations. In all cases, preceding the durations to be judged by clicks changed their subjective length in a manner broadly consistent with the idea that pacemaker speed was increased, by an average of about 10%.

The cytokine response of afferent lymph following orf virus reinfection of sheep.

Abstract The in vivo dynamics of differentiated cells and interleukin (IL)-lß, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon (IFN)-γ titres in afferent lymph were compared following orf virus reinfection and inactivated virus injection of previously infected sheep. The biphasic lymphoblast and cytokine response in the lymph to virus reinfection is consistent with a response initially to orf virus as recall antigen followed by a response to viral replication. CD4 T cells increased in output over other cell types in the lymph in both groups. A rapid immune/inflammatory response was detected in lymph plasma as an increase in cytokine titres within 24 h of virus reinfection or injection. Lymph cells producing IL-1ß and IL-8 appeared prior to those producing GM-CSF in both groups. In spite of variations in the concentration of individual cytokines in lymph following reinfection, both the size of the orf lesion and the time to resolve were similar in all cases. Résumé- Les dynamiques in vivo des cellules différenciées et des taux d'IL-1ß, de GM-CSF et d'interferon γ dans la lymphe afférente furent comparés chez des moutons antéricurement infectés après une réinfection par le virus de l'ecthyma contagieux et après injection d'un virus inactivé. La réponse biphasique lymphoblastique et des cytokines à la réinfection virale est compatibles avec une réponse primaire au virus de l'ecthyma contagieux comme antigène mémoire suivie par une réponse secondaire à la réplication virale. Dans les 2 groupes, le nombre de TCD4 est plus élevé que les autres populations cellulaires mises en évidence dans la lymphe. Une réponse de type immune/inflammatoire est révélée dans le plasma par une élévation des taux de cytokines dans les 24 heures qui suivent la réinfection virale ou l'injection de virus inactivé. Les lymphocytes producteurs dTL-1ß et d'IL-8 apparaissent avant les lymphocytes producteurs de GM-CSF dans les deux groupes. En dépit des variations de concentration des cytokines individuelles dans le lymphe après reinfection, à la fois la taille des lesions d'ecthyma et les délais de guérison sont identiques dans tous les cas. [Haig, D., Deane, D., Percival, A., Myatt, N., Thomson, J., Inglis, L., Rothel, J., Heng-Fong Seow, Wood, P., Miller, H. R. P., Reid, H. W. The cytokine response of afferent lymph following orf virus reinfection of sheep (Effet sur les cytokines de la lymphe afferente d'une reinfection par les virus de l'ecthyma contagieux chez le mouton.) Veterinary Dermatology 1996; 7: 11-20.] Resumen Se comparó la actividad de células diferenciadas y los titulos de interleuquina (IL)-1ß, IL-8, factor de estimulación de colonias de granulocitos y macrófagos (GM-CSF) e interferon (IFN)-y entre reinfecciones por el virus del ectima contagioso e inyección de virus inactivado de ovinos previamente infectados. La respuesta a la reinfección en forma bifásica linfoblástica y de citoquinas en la linfa está de acuerdo con una respuesta inicial al virus del ectima por estimulación antigénica, seguida por una respuesta a la replicación viral. Las células T CD4 aumentaron respecto a otros tipos celulares en la linfa de ambos grupos. Se detectó una respuesta inmune/inflamatoria rápida en el linfa-plasma en forma de aumento de los titulos de citoquinas dentro de las 24 h de reinfección o inyección del virus. Las células linfáticas productoras de IL-1ß e IL-8 aparecicron antes que las productoras de GM-CSF en ambos grupos. A pesar de las variaciones en la concentración de citoquinas individuates en la linfa después de la reinfección, tanto el tamaño de la lesión por el virus del ectima contagioso como el tiempo de resolución fueron similares en todos los casos. [Haig, D., Deane, D., Percival, A., Myatt, N., Thomson, J., Inglis, L., Rothel, J., Heng-Fong Seow, Wood, P., Miller, H. R. P., Reid, H. W. The cytokine response of afferent lymph following orf virus reinfection of sheep (Produccion de citoquinas en el ganglio linfatico afferente tras la reinfección por el virus del ectima contagioso.) Veterinary Dermatology 1996; 7: 11-20.] Zusammenfassung- Es wurde die in-vivo-Dynamik der Titer differenzierter Zellen und Interleukin (1L)-1ß, IL-8, Granulozytenmakrophagenkolonien-stimulierender Faktor (GM-CSF) und Interferon (IFN)-γ in afferenter Lymphe nach einer Reinfektion mit ORF-Virus und einer Injektion inaktivierten Virusmaterials von früher infizierten Schafen verglichen. Die biphasische Lymphoblasten- und Zytokin-Reaktion in der Lymphe auf die Virusreinfektion stimmte mit einer initialen Reaktion gegenüber ORF-Virus überein, da der Wiederabruf von Antigen von einer Reaktion auf die Virusreplikation gefolgt wird. CD4-T-Zellen vermehrten sich im Output stärker als andere Zelltypen in der Lymphe beider Gruppen. Es wurde eine rasche immunologische/entzündliche Reaktion im Lymphplasma in Form eines Anstieges von Zytokin-Titern innerhalb von 24 Stunden nach Virusreinfektion oder -injektion festgestellt. Lymphatische Zellen, die IL-1ß und IL-8 produzieren, erschienen vor dem Auftreten von solchen, die GM-CSF produzieren, in beiden Gruppen. Trotz des Schwankens der Konzentration der individuellen Zytokine in der Lymphe nach Reinfektion, waren sowohl die Größe der ORF-Veränderungen und die Zeit der Heilung ähnlich in alien Fällen. [Haig, D., Deane, D., Percival, A., Myatt, N., Thomson, J., Inglis, L., Rothel, J., Heng-Fong Seow, Wood, P., Miller, H. R. P., Reid, H. W. The cytokine response of afferent lymph following orf virus reinfection of sheep (Die ZytokinReaktion afferenter Lymphe nach einer Reinfektion mit orf-virus beim schaf.) Veterinary Dermatology 1996; 7: 11-20.].

The survival and growth of ovine afferent lymph dendritic cells in culture depends on tumour necrosis factor-alpha and is enhanced by granulocyte-macrophage colony-stimulating factor but inhibited by interferon-gamma.

An in vitro culture system is described which allows an analysis of the signals responsible for the survival, growth and functional maturation of afferent lymph dendritic cells (ALDC), a subpopulation of migrating dermal dendritic cells involved in antigen carriage and presentation to T-cells. Purified ALDC survived and grew for up to 30 days in lymph node conditioned medium and survived 14 days in recombinant ovine (rov) TNF-alpha whereas none were detected after 24 h in rov GM-CSF, rov IFN-gamma or rh M-CSF. However, when rov GM-CSF was added to cultures along with rov TNF-alpha, increased numbers of ALDC compared with input numbers (growth) were recorded on Days 14 and 21. In contrast, when 50-200 units ml-1 of rov IFN-gamma were added to cultures of ALDC along with TNF-alpha or rov TNF-alpha plus rov GM-CSF, cell survival and growth was inhibited. Antibody blocking studies confirmed the cytokine specificity of these effects. ALDC cultured in rov TNF-alpha or rov TNF-alpha plus rov GM-CSF retained MHC Class-II and ov CD-1 antigen expression and accessory function for autologous ov CD-4 T-cell proliferation, although at reduced levels compared with freshly isolated cells. Neither fresh nor cultured ALDC expressed coagulation factor XIIIa.

Haemopoietic cell responses in the blood and bone marrow of sheep infected with the abomasal nematode Telodorsagia circumcincta.

The generation of bone marrow and blood haemopoietic progenitor colony-forming cells (CFCs) in sheep given primary or challenge infections with the nematode parasite Telodorsagia circumcincta is described. Ten days after a primary infection, the frequency of early multipotential-CFCs, eosinophil-CFCs, macrophage-CFCs and mast cell or basophil-CFCs was greater than in controls. These frequencies then declined to pre-infection levels by day 21. Blood CFCs (mainly macrophage-CFCs and eosinophil-CFCs) also increased after infection, indicating a migration of CFCs, presumably to the site of infection. Ten days after challenge infection there was less marked myelopoiesis than in the primary infection on day 10, though both eosinophil-CFCs and mast cell or basophil-CFCs were significantly above control values. Blood CFC output (mainly macrophage-CFCs and eosinophil-CFCs) reached a peak 2-6 days after challenge, evidence of rapid recruitment to the site of infection. Telodorsagia circumcincta infection is therefore associated with an increase in myelopoiesis, particularly for the cell types characteristic of the local inflammatory response to abomasal nematodes. There was no correlation between any of the haemopoietic cell responses measured and worm burdens in individual animals after either primary or challenge infection.

Chicken skeletal muscle ryanodine receptor isoforms: ion channel properties.

To define the roles of the alpha- and beta-ryanodine receptor (RyR) (sarcoplasmic reticulum Ca2+ release channel) isoforms expressed in chicken skeletal muscles, we investigated the ion channel properties of these proteins in lipid bilayers. alpha- and beta RyRs embody Ca2+ channels with similar conductances (792, 453, and 118 pS for K+, Cs+ and Ca2+) and selectivities (PCa2+/PK+ = 7.4), but the two channels have different gating properties. alpha RyR channels switch between two gating modes, which differ in the extent they are activated by Ca2+ and ATP, and inactivated by Ca2+. Either mode can be assumed in a spontaneous and stable manner. In a low activity mode, alpha RyR channels exhibit brief openings (tau o = 0.14 ms) and are minimally activated by Ca2+ in the absence of ATP. In a high activity mode, openings are longer (tau o1-3 = 0.17, 0.51, and 1.27 ms), and the channels are activated by Ca2+ in the absence of ATP and are in general less sensitive to the inactivating effects of Ca2+. beta RyR channel openings are longer (tau 01-3 = 0.34, 1.56, and 3.31 ms) than those of alpha RyR channels in either mode. beta RyR channels are activated to a greater relative extent by Ca2+ than ATP and are inactivated by millimolar Ca2+ in the absence, but not the presence, of ATP. Both alpha- and beta RyR channels are activated by caffeine, inhibited by Mg2+ and ruthenium red, inactivated by voltage (cytoplasmic side positive), and modified to a long-lived substate by ryanodine, but only alpha RyR channels are activated by perchlorate anions. The differences in gating and responses to channel modifiers may give the alpha- and beta RyRs distinct roles in muscle activation.