The Use of Green Fluorescent Protein-Tagged Recombinant Viruses to Test Lettuce mosaic virus Resistance in Lettuce

ABSTRACT Seed certification and the use of cultivars containing one of two, probably allelic, recessive genes, mo1(1) and mo1(2), are the principal control methods for Lettuce mosaic virus (LMV) in lettuce. Although for a few LMV isolates, mo1(2) confers resistance with most isolates, the genes mo1(1) or mo1(2) confer a tolerance, and virus accumulation is readily detected in mo1-carrying plants. This phenotype complicates evaluation of the resistance status, in particular for mo1(1), for which there are no viral strains against which a true resistance is expressed. Two green fluorescent protein (GFP)-tagged viruses were constructed, derived from a non-resistance breaking isolate (LMV-0) and from a resistance-breaking isolate (LMV-E). An evaluation of 101 cultivars of known status was carried out with these recombinant viruses. Using the LMV-0-derived recombinant, identification of mo1-carrying cultivars was simple because, contrary to its wild-type parent, systemic movement of LMV-0-GFP was abolished in resistant plants. This assay detected four cases of misidentification of resistance status. In all these cases, further tests confirmed that the prior resistance status information was incorrect, so that a 100% correlation was observed between LMV-0-GFP behavior and the mo1 resistance status. Similarly, the LMV-E-derived recombinant allowed the identification of mo1(2) lettuce lines because its systemic movement was restricted in mo1(2) lines but not in susceptible or in mo1(1) lines. The tagged viruses were able to systemically invade another host, pea, irrespective of its resistance status against another member of the genus Potyvirus, Pea seed-borne mosaic virus. The use of these recombinant viruses could therefore greatly facilitate LMV resistance evaluation and speed up lettuce breeding programs.     

Repetitive Applications of the Biocontrol Agent Pseudomonas putida 06909-rif/nal and Effects on Populations of Phytophthora parasitica in Citrus Orchards

ABSTRACT Pseudomonas putida 06909-rif/nal was applied repetitively during the irrigation season in two citrus orchards over 3 years. In a mature (50-yearold) commercial citrus orchard covering 2.02 ha, weekly applications of Pseudomonas putida 06909-rif/nal with an in-field fermentor resulted in soil populations that fluctuated between 2.83 log CFU + 1 per g of soil and 4.35 log CFU + 1 per g of soil. Resulting rhizosphere populations of Phytophthora parasitica were significantly reduced in 1999 but not 1997 or 1998. In a newly planted citrus orchard, yearly applications of Pseudomonas putida 06909-rif/nal at the beginning of the irrigation season resulted in high soil populations of Pseudomonas putida 06909-rif/nal that declined rapidly and never reduced the rhizosphere populations of Phytophthora parasitica. When Pseudomonas putida 06909-rif/nal was applied weekly, soil populations increased throughout the 1997 and 1998 irrigation seasons, reaching a maximum in 1998 and remained high throughout the 1999 irrigation season. Rhizosphere populations of Phytophthora parasitica were significantly reduced in 1998. Yearly applications of the fungicide metalaxyl and the nematicide phenamiphos reduced rhizosphere populations of Phytophthora parasitica in 1997 but not in 1998 or 1999. Pseudomonas putida 06909-rif/nal was uniformly distributed throughout the soil profile to a depth of 75 cm in both yearly and weekly applications. When applied through low-volume minisprinklers, Pseudomonas putida 06909-rif/nal was found in aerosols up to 3 m away.     

Role of inflammatory mediators in priming, activation, and deformability of bovine neutrophils

OBJECTIVE: To determine the capacity of inflammatory mediators tumor necrosis factor-alpha (TNF-alpha), interleukin-8 (IL-8), platelet-activating factor (PAF), lipopolysaccharide (LPS), and leukotoxin to prime, activate, or alter deformability of adult bovine neutrophils. SAMPLE POPULATION: Blood collected from 5 healthy adult Holstein cows. PROCEDURE: Isolated neutrophils or whole blood was incubated with TNF-alpha, IL-8, PAF, LPS, or leukotoxin, and neutrophil chemiluminescence, degranulation, deformability, shape change, CD11b expression, and size distribution was measured. RESULTS: Incubation with TNF-alpha, IL-8, PAF, and LPS primed neutrophils for oxygen radical release but caused minimal oxygen radical release by themselves. None of the inflammatory mediators induced degranulation. Incubation with TNF-alpha and PAF resulted in a decrease in neutrophil deformability and induced shape change in neutrophils. Incubation with PAF consistently resulted in an increase in neutrophil size as measured by use of flow cytometry. Only IL-8 caused an increase in expression of CD11b by neutrophils. CONCLUSIONS AND CLINICAL RELEVANCE: Inflammatory mediators tested had minimal effects on neutrophil oxygen radical production or degranulation but did prime neutrophils for oxygen radical production. Incubation with PAF and TNF-alpha caused a decrease in neutrophil deformability and altered neutrophil shape and size. Results of our study indicate that PAF- and TNF-alpha-induced changes in neutrophil deformability and size may cause integrin- and selectin-independent trapping of neutrophils in the lungs of cattle with pneumonic pasteurellosis.     

Use of canine red blood cell with high concentrations of potassium, reduced glutathione, and free amino acid as host cells for in vitro cultivation of Babesia gibsoni

OBJECTIVE: To determine the usefulness of canine RBC with high concentrations of potassium, reduced glutathione (GSH), and amino acid(i.e., HK cells) for in vitro cultivation of Babesia gibsoni. ANIMALS: RBC were obtained from 3 dogs that had inherited HK cells and from 3 genetically unaffected dogs that, therefore, had RBC with lower potassium (LK) concentrations (i.e., LK cells). PROCEDURES: First, B. gibsoni were cultivated using HK or LK cells in alpha-modification of Eagle medium, consisting of Earle salts with glutamine and without ribosides, deoxyribosides, and sodium bicarbonate under a humidified atmosphere containing 5% CO2 at 37 C. Second, parasites were cultivated with LK- or HK-cell lysates. Finally, HK cells were separated into 3 fractions (bottom, middle, top layers) by density gradient centrifugation, and B. gibsoni were cultivated with each of the HK-cell fractions. In addition, the concentrations of free amino acids and reduced glutathione (GSH) in each HK-cell fraction were measured. RESULTS: B. gibsoni preferentially multiplied in HK-cell cultures rather than in LK-cell cultures. Furthermore, the addition of HK-cell lysate to the culture medium resulted in enhanced multiplication of the parasites. Higher multiplication of the parasites was observed in HK cells from the top layer, compared with HK cells from the middle and bottom layers. The HK cells from the top layer had higher concentrations of glutamate, aspartate, and GSH, compared with HK cells from the middle and bottom layer. CONCLUSIONS: Canine HK cells are useful host cells for in vitro cultivation of B. gibsoni, and the high concentrations of glutamate, aspartate, and GSH may result in enhancement of multiplication of the parasites in HK cells.     

Infection of turkeys with Ornithobacterium rhinotracheale and Mycoplasma synoviae

Within 1 mo, two separate outbreaks of respiratory disease occurred in two flocks on the multiage market turkey farm in Slovenia. More severe dinical signs and higher mortality were observed in male birds. Ornithobacterium rhinotracheale (ORT) was isolated in pure culture from tracheas of the affected birds in both outbreaks. Commercial enzyme-linked immunosorbent assay test showed the presence of antibodies to ORT in sera of birds from both clinically affected flocks and also in two flocks of younger birds without clinical sings. Immunoblotting with ORT culture isolated during the outbreak as an antigen confirmed the presence of antibodies to ORT in sera of turkeys of all four flocks examined. In addition, three different serologic assays also detected antibodies to Mycoplasma synoviae (MS) in three out of four flocks. The concomitant infection with MS did not show an obvious effect on mortality rates nor on the antibody response against ORT. Younger birds appeared to be less susceptible to ORT pathogenicity because in those flocks the infection was subclinical.     

Effects of injection position and transponder size on the performances of passive injectable transponders used for the electronic identification of cattle

A total of 686 Tiris half-duplex passive injectable transponders (PIT) of two sizes (23 and 32 mm) were randomly injected s.c. in three positions, armpit, ear scutulum, and upper lip, in 343 fattening calves (1 to 3 mo old). Injections were performed by two trained and two untrained operators. Losses and breakages on the farm were recorded at wk 1, 3, 7, 11, and 15 in restrained animals using two types of hand-held transceivers with a stick antenna. Dynamic reading efficiency (DRE) in animals running through a raceway was also evaluated at wk 1 and 3 and monthly until slaughter, using a stationary transceiver working at 137 dB x microV x m(-1) at 3 m. The total number of PIT that fell or broke in the slaughtering line, the location method, and the recovery time were also recorded. Results on the farm showed low breakages on average (0.4%) and differences (P < 0.05) in losses according to position (armpit, 1.7%; ear, 5.2%; and lip, 14.0%). An interaction (P < 0.05) between position x size was observed, and losses were greatest using a 32-mm PIT in the lip. The DRE was affected (P < 0.05) by PIT position and size, and values were greater for the 32-mm PIT in all positions (armpit: 99.9 +/- 0.1 vs 95.8 +/- 4.9%; ear: 93.8 +/- 2.2 vs 81.9 +/- 4.6%; lip: 66.8 +/- 4.9 vs 53.4 +/- 4.7%, respectively, for 32 vs 23 mm). Recovery of PIT in the abattoir was on average 96.7, 96.7, and 99.2% for armpit, ear, and lip, respectively (P > 0.05). Most of the PIT injected in the armpit were recovered by sight or palpation, but 31.9% were recovered after cutting the muscles around the area and 10.7% were recovered on the internal side of the hide, which jeopardized carcass identification. Recovery of PIT injected in the ear was 23.4% in the hide and 76.6% in the auricular muscles of the head. The easiest recovery was in the lip, 8.9% of PIT were located in the hide and 91.1% in the head. Recovery time was affected (P < 0.05) by position: the quickest was lip (27 +/- 2 s), followed by ear (52 +/- 5 s) and armpit (78 +/- 7 s). In conclusion, taking into account retention and reading performances, injection of a 32-mm PIT into the armpit showed the best results on the farm, but a careful and longer recovery was needed in the abattoir. Improvement of recovery methodology and time would be necessary in order to recommend injection of PIT in the armpit instead of in the ear for cattle tracking or monitoring.     

GM1 gangliosidosis in shiba dogs

A six-month-old shiba dog with a one-month history of progressive motor dysfunction showed clinical signs of a cerebellar disorder, including ataxia, dysmetria and intention tremor of the head. Histopathological and ultrastructural studies revealed distended neurons packed with membranous cytoplasmic bodies throughout the central nervous system. The activities of lysosomal acid beta-galactosidase in its leucocytes and liver were less than 2 per cent of the control levels, and the compound accumulated in the brain was identified as GM1 ganglioside. A sibling which died immediately after birth was shown to have a beta-galactosidase deficiency in the brain and visceral organs. A family study revealed that the sire and dam of the probands were heterozygotes with approximately half of the normal level of beta-galactosidase activity, suggesting an autosomal recessive pattern of inheritance.     

Chiral inversion of R(-) fenoprofen and ketoprofen enantiomers in cats

The chiral inversion process is a characteristic metabolic pathway for different aryl-2-propionic acids or profens. Important variations have been observed between these individual compounds as well as between animal species. In this study, R(-) fenoprofen [R(-)FPF] and R(-) ketoprofen [R(-) KTF] were used to investigate their comparative stereoconversion in cats. After intravenous (i.v.) administration of R(-) FPF, the percentage of chiral inversion was 93.20+/-13.70%. A highly significant correlation (r: 0.978) was observed between the clearance of R(-) FPF and the chiral inversion process. After i.v. administration of R(-) KTF, the percentage of inversion was only 36.73+/-2.8%. No correlation between the clearance of R(-) KTF and this process was observed. R(-) FPF was metabolized by the pathways of thioesterification - chiral inversion processes. For R(-) KTF, the competitive metabolic pathways, glucuronidation and hydroxylation may be involved. However, these metabolic steps are saturable or less functional in cats. Moreover, the thioesterification of R(-) KTF in in vitro studies has been shown to be important in carnivores. The lack of correlation between clearance and chiral inversion process of R(-) KTF may be finally explained by deviation of thioesterification to other metabolic pathways of lipids and/or aminoacid conjugation, particulary glicine derivatives.