A bead-based suspension array for the multiplexed detection of begomoviruses and their whitefly vectors.

Bead-based suspension array systems enable simultaneous fluorescence-based identification of multiple nucleic acid targets in a single reaction. This study describes the development of a novel approach to plant virus and vector diagnostics, a multiplexed 7-plex array that comprises a hierarchical set of assays for the simultaneous detection of begomoviruses and Bemisia tabaci, from both plant and whitefly samples. The multiplexed array incorporates genus, species and strain-specific assays, offering a unique approach for identifying both known and unknown viruses and B. tabaci species. When tested against a large panel of sequence-characterized begomovirus and whitefly samples, the array was shown to be 100% specific to the homologous target. Additionally, the multiplexed array was highly sensitive, efficiently and concurrently determining both virus and whitefly identity from single viruliferous whitefly samples. The detection limit for one assay within the multiplexed array that specifically detects Tomato yellow leaf curl virus-Israel (TYLCV-IL) was quantified as 200fg of TYLCV-IL DNA, directly equivalent to that of TYLCV-specific qPCR. Highly reproducible results were obtained over multiple tests. The flexible multiplexed array described in this study has great potential for use in plant quarantine, biosecurity and disease management programs worldwide.

Nonpathogenic Isolates of the Citrus Black Spot Fungus, Guignardia citricarpa, Identified as a Cosmopolitan Endophyte of Woody Plants, G. mangiferae (Phyllosticta capitalensis).

ABSTRACT The population structure of Guignardia citricarpa sensu lato (anamorph: Phyllosticta citricarpa), a fungus of which strains pathogenic to citrus are subject to phytosanitary legislation in the European Union and the United States, was investigated. Internal transcribed spacer sequences revealed two phylogenetically distinct groups in G. citricarpa. This distinction was supported by amplified fragment length polymorphism analysis that also supported the exclusion of two isolates that had apparently been misclassified as G. citricarpa. On cherry decoction agar, but not on other media, growth rates of group I isolates were lower than those of group II isolates. Conidial dimensions were similar, but group I isolates formed conidia with barely visible mucoid sheaths, whereas those of group II formed conidia with thick sheaths. Cultures of isolates belonging to group I produced rare infertile perithecia, whereas fertile perithecia were formed by most isolates of group II. Colonies of isolates belonging to group I were less dark than those of group II, with a wider translucent outer zone and a lobate rather than entire margin. On oatmeal agar, exclusively group I isolates formed a yellow pigment. Group I harbored strains from citrus fruits with classical black spot lesions (1 to 10 mm in diameter) usually containing pycnidia. Group II harbored endophytic strains from a wide range of host species, as well as strains from symptomless citrus fruits or fruits with minute spots (<2-mm diameter) without pycnidia. These observations support the historic distinction between slowly growing pathogenic isolates and morphologically similar fast-growing, nonpathogenic isolates of G. citricarpa. The latter proved to belong to G. mangiferae (P. capitalensis), a ubiquitous endophyte of woody plants with numerous probable synonyms including G. endophyllicola, G. psidii, P. anacardiacearum, and P. theacearum. G. mangiferae occurs in the European Union and the United States on many host species including citrus, and does not cause symptoms of citrus black spot, justifying its exclusion from quarantine measures.

Molecular Characterization of Natural Hybrids of Phytophthora nicotianae and P. cactorum.

ABSTRACT Hybrid isolates of Phytophthora nicotianae x P. cactorum from five different hosts (Cyclamen, Lavandula, Lewisia, Primula, and Spathiphyllum spp.) were identified by their atypical morphology and their well-defined heterozygous isozyme patterns. The hybrid nature of these isolates was tested by restriction fragment length polymorphism analysis of the internal transcribed spacer (ITS) region of rDNA, generating fragments typical for both P. nicotianae and P. cactorum. In hybrid isolates, polymerase chain reactions (PCR) with primers derived from unique parts of the ITS region (ITS-PCR) of both species yielded a combination of unique amplicons typical of both parental species. Eleven hybrid isolates, three isolates of each parental species and two atypical isolates from Rhododendron and Idesia spp. close to P. cactorum, were analyzed for amplified fragment length polymorphisms (AFLP). Consistent differences in AFLP patterns existed among the hybrid isolates, strongly indicating that these hybrids have arisen from independent hybridization events between P. nicotianae and P. cactorum. The two atypical isolates morphologically resembling P. cactorum were identical to the latter species in ITS-restriction fragment length polymorphism and response to the specific PCR primers but were intermediate between P. nicotianae x P. cactorum and P. cactorum in isozyme profiles and AFLP patterns. Since the introduction of hydroponic systems in greenhouses in the Netherlands, outbreaks of Phytophthora diseases are occurring in previously unaffected host species. This may be due to interspecific hybridization events resulting in novel pathogenic behavior.

Spirometric variables recorded after lobeline administration in healthy Friesian and Belgian white and blue calves: normal values and effects of somatic growth.

The purposes of the present study were: (1) to develop original equations to predict spirometric variables (SV) in healthy Friesian and Belgian White and Blue (BWB) calves < 1 year of age; and (2) to determine the effects of somatic growth on SV. Sixty-seven Friesian and 500 BWB calves were investigated. For each calf, the following SV were calculated: (1) the average minute volume derived using all the ventilatory cycles recorded during the 15 s of maximal ventilatory changes induced by lobeline administration (0.25 mg/kg, i.v.) (15-s LMV); (2) the maximal tidal volume, the maximal peak expiratory flow and the maximal peak inspiratory flow recorded from single breaths after lobeline administration (MVTL, MPEFLand MPIFL, respectively); (3) the ventilatory reserve (VRL= 15-s LMV - VEr; VEr= minute volume at rest). Mass specific (s) values were also calculated. All SV changed linearly with somatic growth in both Friesian and BWB calves. Since the rise in SV was more related to body weight than the age of calves, equations for reference values of SV always had body weight as the only independent variable. In the youngest calves, s SV were lower in the BWB breed. In the oldest calves (i.e. 1 year of age), s MPEFLand s MPIFLremained small in BWB calves whereas s MVTL, s 15-s LMV and s VRLwere almost equal in both breeds. These results could be related to the lower resistance to respiratory disorders in BWB calves < 1 year of age than in Friesian calves < 1 year of age.

Role of the cholinergic pathway in mediating platelet-activating factor-induced pulmonary dysfunctions in unsedated calves.

The purpose of this study was to investigate the possible role of the cholinergic pathway in mediating platelet-activating factor (PAF)-induced pulmonary dysfunctions in unsedated calves. In a placebo group, PAF infusion challenge induced significant dysfunctions in the pattern of breathing [a significant increase in respiratory rate (RR) and a significant decrease in tidal volume (VT)], the mechanics of breathing [a significant increase in total lung resistance (RL) and a significant decrease in dynamic lung compliance (CLdyn)] and gas exchange, whereas in atropine pre-treated calves, PAF infusion challenge induced a significant increase in RR, VT and HR and a significant decrease in CLdyn. The RL increase was prevented by atropine pre-treatment. On the basis of our findings, we suggest that, in cattle, the PAF-induced pattern of breathing dysfunctions and the diffuse bronchoconstriction and microvascular leakage of small airways are not mediated through the cholinergic pathway. By contrast, our data suggest that PAF-induced bronchoconstriction of upper airways is at least partly mediated through muscarinic receptors.

Ketoprofen and phenylbutazone attenuation of PAF-induced lung inflammation in calves.

The purposes of this study were: (1) to investigate which arachidonic acid metabolites contributed to platelet-activating factor (PAF) induced pulmonary dysfunction; and (2) to compare the effect of two non-steroidal anti-inflammatory drugs, phenylbutazone and ketoprofen in a model of PAF-induced reversible lung inflammation in six calves. In placebo and phenylbutazone groups, PAF infusion induced significant dysfunctions in the pattern of breathing, mechanics of breathing and gas exchange. These dysfunctions were prevented by ketoprofen pretreatment, except for the mechanics of breathing which was moderately but significantly altered by the PAF challenge. In all calves, leukotriene (LT) B4 plasma concentrations did not significantly increase above baseline values at any time. Prostaglandin (PG) E2 plasma concentrations showed a minor significant increase in phenylbutazone pretreated calves (55.8 +/- 25.8 pg/mL from 36.7 +/- 16.13 pg/mL). Thromboxane (TX) B2 plasma concentration was significantly increased during PAF challenge in placebo- and phenylbutazone-pretreated groups, but not in ketoprofen-pretreated calves (1580.0 +/- 1370 from 42.7 +/- 10.7 pg/mL; 2340 +/- 477 from 63 +/- 32 pg/mL; and 36.5 +/- 4.12 from 39.3 +/- 12.0 pg/mL, respectively). These data suggest that TXA2 is an important cyclooxygenase metabolite of arachidonic acid produced in response to PAF and that ketoprofen (intramuscular injection, 3 mg/kg) is more effective than phenylbutazone (intramuscular injection, 10 mg/kg) in preventing respiratory dysfunctions induced by the PAF challenge 30 min after drug administration. Ketoprofen did not suppress totally the PAF-induced changes in mechanics of breathing, which suggests that PAF or a secondary release of mediators could have a direct action on airway smooth muscle.

Effect of platelet-activating factor on bovine pulmonary function.

The objectives of this study were to determine whether the infusion of platelet-activating factor would modify bovine pulmonary function, heart rate and platelet count and whether any such modifications could be antagonised by the prior intravenous injection of a specific antagonist (WEB 2086). In saline-pretreated calves, the respiratory rate, total lung resistance (RL) and maximal changes in transpulmonary pressure (delta Ptp) were significantly increased whereas lung dynamic compliance (CLdyn), tidal volume (VT), platelet count and heart rate were significantly decreased. The changes in RL, CLdyn, VT and delta Ptp were abolished by pretreatment with WEB 2086, whereas respiratory rate remained significantly increased to 125 per cent of the baseline value, but less than in the saline-pretreated calves in which it was 250 per cent of the baseline value.

Effect of serotonergic blockade on calf pulmonary function after the intravenous administration of 3-methylindole.

This study was designed to investigate whether 3-methylindole (3-Mi), a tryptamine analogue, could cause pulmonary injury in calves other than by cytotoxicity. Injection of 3-Mi resulted in a marked increase of respiratory rate, decrease of tidal volume and increase in minute ventilation. Pulmonary mechanics values were also profoundly affected, lung dynamic compliance being reduced to approximately one-third of its baseline value and total pulmonary resistance being increased two-fold. Arterial oxygen partial pressure was dramatically reduced. Successive challenges with 3-Mi after physiological saline pretreatment resulted in quantitatively identical alterations of pulmonary function values. Conversely, all these ventilatory, mechanical and gas exchange changes were abolished by pretreatment with serotonergic antagonists. It was concluded that intravenous administration of 3-Mi to healthy calves induced immediate and reversible bronchoconstriction which affected both central and peripheral airways. Because the effect was abolished by pretreatment with antiserotonin drugs, it is suggested that 3-Mi acts either directly by stimulating serotonergic receptors or indirectly through the release of serotonin from platelets. Current concepts of the physiopathological cascade underlying the toxicity of 3-Mi should, therefore, be re-evaluated in the light of this pharmacological mechanism.

Immuno-epidemiology of malaria: a study in a tribal area of West Irian.

An investigation of malariometric indices in relation to immunoglobulin levels, rheumatoid factors, and antithyroglobulins was carried out on 78 members of the Arfak tribe near Manokwari in Western New Guinea, in the course of a WHO assessment of malaria control activities in that region. The population investigated had been exposed to a period of epidemic malaria, as indicated by the small differences in malariometric indices between consecutive age groups. Typically high spleen sizes were recorded, as found generally among Papuans in similar situations. Falciparum malaria was most prevalent, almost equal to cases of vivax and malariae malaria together. IgM levels were very high, while those of IgG, IgA and IgD were not elevated. Total serum protein was rather low. No correlation between malariometric indices, autoantibodies, and immunoglobulin levels could be found. In particular there was no correlation between IgM levels and spleen indices, such as has been found in many other surveys. It is suggested that splenomegaly may show no correlation with the IgM level in Papuan populations without previous selection.