Identification of pathogenic Leptospira strains in tissues of a premature foal by use of polymerase chain reaction analysis.

Studies were carried out to determine the cause of death in a prematurely born Thoroughbred foal that died 24 hours after birth. Necropsy revealed gross lesions suggestive of septicemia. A commercial Leptospira polymerase chain reaction (PCR) assay designed to specifically amplify the hemolysis-associated protein 1 (hap1) gene present only in pathogenic Leptospira strains detected the presence of Leptospira DNA in various tissues of the foal. Histologic examination of lung, liver, kidney, and myocardium revealed numerous spirochetes in Warthin-Starry-stained tissue sections. Results of PCR analysis and histologic examination suggested a leptospiral infection in the newborn foal. At the moment of death, the infection coexisted with a streptococcal-associated aspiration bronchopneumonia and postpartum septicemia. These findings indicate that the PCR assay based on the amplification of the hap1 gene represents a useful tool for specific detection of pathogenic leptospira in field samples taken from horses.     

Development and validation of a radioimmunoassay for thyrotropin in cattle.

In mammals, thyrotropin, or thyroid-stimulating hormone (TSH), assay is used for the diagnosis of primary hypothyroidism. Hypothyroidism is the most common type of thyroid disorder in cattle. The aim of this study was to develop and validate, under physiologic and pathologic conditions, a radioimmunoassay (RIA) for bovine TSH (bTSH). Double RIA was performed with purified bTSH and specific bovine antiserum. Laboratory validation included research of minimal detection limit, accuracy, and reproducibility. The physiologic validation included a thyrotropin-releasing hormone (TRH) challenge performed on euthyroid cows and a follow-up of bTSH concentration over a 24-hour period. Furthermore, bTSH concentration was assayed in a large population of healthy dairy and beef cows to define reference interval. The pathologic validation was made by assaying bTSH and thyroid hormones on healthy and goitrous newborn calves. The minimum detection limit (MDL) for bTSH assay was 1.3 microU/ml. The recovery was 101% to 106%. The intra- and interassay coefficients of variation (CVs) ranged from 5% to 11% and 11% to 15%, respectively. The RIA covered the whole range of physiologic bTSH values, as shown by bTSH values induced by TRH-challenge. A pulsatile secretion of bTSH was observed, accompanied by a diurnal variation with lower night values than day values. Reference intervals of bTSH ranged from 1.3 to 13.0 microU/ml for beef and dairy breeds. Finally, bTSH easily discriminated goitrous newborn calves from healthy ones, leading to the definition of a cutoff value of 35 microU/ml. The bTSH assay positively reacted to physiologic and pathologic conditions. The accuracy and precision of the RIA were satisfying.     

Serologic and molecular characterization of tickborne pathogens in lions (Panthera leo) from the Fasano Safari Park, Italy.

Lions (Panthera leo) are an endangered species threatened by illegal hunting, habitat loss, and infectious diseases. Little is known about the tick-borne pathogens that infect lions and could contribute to population declines. The objective of this study was to characterize Rickettsia spp., Anaplasma phagocytophilum, and Coxiella burnetii infections in 10 lions from the Fasano Safari Park in Italy by serology, polymerase chain reaction, and sequence analysis. Although animals did not show clinical signs of tick-borne diseases, evidence of infection with C. burnetii, spotted fever group Rickettsia sp., and A. phagocytophilum were found in 50%, 20%, and 10% of the lions, respectively. One of the lions tested positive for all three pathogens. This study is the first report of molecular evidence of infection with C. burnetii, Rickettsia sp., and A. phagocytophilum in lions and provides evidence that these felids become infected and serve as hosts for tick-transmitted bacteria.     

Evaluation of the API 50CH and API ZYM systems for rapid characterization of Clavibacter michiganensis subsp. sepedonicus, causal agent of potato ring rot

API 50CH and API ZYM systems were used to characterize fifty-three strains of Clavibacter michiganensis subsp. sepedonicus from different geographic locations and several reference strains of the same and different species, including other potato pathogens. Clavibacter michiganensis subsp. sepedonicus strains displayed a high level of homogeneity, both in carbohydrate utilization and in enzymatic activity. Using API 50CH and API ZYM it was possible to differentiate C. michiganensis subsp. sepedonicus strains from the remaining taxa analysed in this study, which included representative strains of the other subspecies of C. michiganensis as well as other bacterial pathogens affecting potatoes. Therefore, these systems could be used as an effective method to characterize C. michiganensis subsp. sepedonicus. Such a procedure would constitute an alternative system to the conventional nutritional and physiological identification tests currently included in the official methods employed in the European Union to detect and identify this bacterium. The results obtained with the API systems agreed with the current taxonomic classification of C. michiganensis, clearly separating sepedonicus from the other subspecies belonging to this species.     

Comparison ofPotato Virus Y andPlum Pox Virus transmission by two aphid species in relation to their probing behavior

Two different aphid species,Myzus persicae (Sulzer) andHyalopterus pruni (Geoffroy) (Homoptera: Aphididae), were used to analyze their ability to transmit two different potyviruses,Potato virus Y (PVY) andPlum pox virus (PPV), to pepper (Capsicum annuum) andNicotiana benthamiana plants, respectively. In parallel experiments,M. persicae consistently transmitted both viruses with high efficiency, whereasH. pruni always failed to transmit either virus. This is in contrast to previous reports describingH. pruni as a vector of these potyviruses. Different aphid probing behavior among individual aphids of each species was obtained in electrical penetration graph (EPG) experiments performed on pepper plants. This suggested thatH. pruni did not transmit these potyviruses due to behavioral differences during probing that impeded virus acquisition and/or inoculation. It was found thatM. persicae usually makes its first probe within the first 2 min, whereasH. pruni individuals remained for more than 10 min on the plant before starting to probe. Furthermore,M. persicae individuals displayed their first intracellular puncture during the first minute of probing whereasH. pruni needed ∼ 15 min to penetrate the cell plasmalemma with their stylets. In addition, intracellular stylet punctures occurred very frequently forM. persicae but was a rare event, never exceeding a single one, forH. pruni. The relevance of these findings for the epidemiological spread of potyviruses by different aphid species is discussed. http://www.phytoparasitica.org posting May 14, 2006.     

Evaluation of cell wall preparations for proteomics: a new procedure for purifying cell walls from Arabidopsis hypocotyls

Background  

The ultimate goal of proteomic analysis of a cell compartment should be the exhaustive identification of resident proteins; excluding proteins from other cell compartments. Reaching such a goal closely depends on the reliability of the isolation procedure for the cell compartment of interest. Plant cell walls possess specific difficulties: (i) the lack of a surrounding membrane may result in the loss of cell wall proteins (CWP) during the isolation procedure, (ii) polysaccharide networks of cellulose, hemicelluloses and pectins form potential traps for contaminants such as intracellular proteins. Several reported procedures to isolate cell walls for proteomic analyses led to the isolation of a high proportion (more than 50%) of predicted intracellular proteins. Since isolated cell walls should hold secreted proteins, one can imagine alternative procedures to prepare cell walls containing a lower proportion of contaminant proteins.     

Response of black rot in cabbage to spatial distribution of inoculum

Disease progress of black rot in cabbage crop was studied over three years in field plots to compare the effects of uni-focal and multi-focal inoculum applied in equal amounts per plot. Disease progress (plant incidence and leaf incidence) was plotted over time, three dimensional maps were made, and disease aggregation was studied by means of geostatistics, black-black counts and Moran's I statistic. Black rot progress was primarily due to focus expansion. Secondary foci may appear at short distances from the initial focus but they usually merge with the expanding initial focus. Anisotropy occurred occasionally but was of minor importance. Disease proceeds faster in plots with multi-focal inoculation than in those with uni-focal inoculation. Probably, serious epidemics in Dutch cabbage fields originate from large numbers of foci.     

A comparison of the adrenal cortical response to ACTH stimulation in Angora and non-Angora goats

The adrenal cortex is believed to be implicated in the high incidence of abortion in the Angora goat. Stimulation testing with adrenocorticotrophic hormone (ACTH) was used to assess the adrenal cortical function in 5 Angora does from herds with a history of abortion and 5 non-Angora does. An acute test involving a single intramuscular (i.m.) injection of 0.25 mg of synthetic ACTH was given during anoestrus, at mid-oestrus, on day 90 and on day 120 of gestation. Blood samples were collected from the jugular vein at 30 min intervals for 1 h before and 5 h after injection. Cortisol concentrations rose within 30 min and returned to baseline values within 3.5 h. Cortisol production was lower (p<0.01) in the pregnant state compared to the non-pregnant state in both groups. Production of cortisol was consistently lower (p<0.05) in the Angora does compared to the non-Angora does during anoestrus and pregnancy and marginally so at mid-oestrus. A chronic stimulation test involving once daily injections of 0.5 mg of a depot form of ACTH i.m. for 7 days commencing on day 90 of pregnancy was also conducted. Cortisol concentrations rose to reach a peak on the third day of treatment in both groups. The values then declined in the Angora does despite continued ACTH treatment, while those for the non-Angora does exhibited a second peak. During and following this treatment, two non-Angora does delivered live kids (day 95, day 120). Out of 7 Angora pregnancies, one Angora doe aborted two dead fetuses at day 116. No significant difference in the cortisol response in the acute test was detected between the animals that aborted and their respective cohorts, but the two non-Angora does that aborted had significantly lower cortisol concentrations during depot ACTH administration. Progesterone and oestradiol levels did not differ between Angora and non-Angora animals during pregnancy or on the test days. The results suggest that the steroidogenic response of the adrenal cortex to ACTH stimulation is significantly less in Angora does with a history of abortion than it is in non-Angora does and support the view that the Angora goat would make a more limited adrenal cortical response to a stressful occurrence during pregnancy.     

Potential errors in measurement of nonuniform sap flow using heat dissipation probes

The empirical calibration of Granier-type heat dissipation sap flow probes that relate temperature difference (DeltaT) to sap velocity (v) was reevaluated in stems of three tropical tree species. The original calibration was confirmed when the entire heated probe was in contact with conducting xylem, but mean v was underestimated when part of the probe was in contact with nonconducting xylem or bark. Analysis of the effects of nonuniform sap velocity profiles on heat dissipation estimates showed that errors increased as v and the proportion of the probe in nonconducting wood increased. If half of a 20-mm probe is in sapwood with a v of 0.15 mm s(-1) and the other half is in nonconducting wood, then mean v for the whole probe can be underestimated by as much as 50%. A correction was developed that can be used if the proportion of the probe in nonconducting wood is known. Even with the entire heated probe in contact with conducting xylem, v would be underestimated when radial velocity gradients are present. In this case, the error would be smaller except when velocity gradients are very steep, as can occur in species with ring-porous wood anatomy. Errors occur because the relationship between DeltaT and v is nonlinear. Mean DeltaT along the probe is therefore not a measure of mean v, and users of heat dissipation probes should not assume that v is integrated along the length of the probe. The same type of error can occur when DeltaT is averaged through time while v is changing, but the error is small unless there are sudden, step changes between zero and high sap velocity. It is recommended that relatively short probes (20 mm or less) be used and that probes longer than the depth of conducting sapwood be avoided. Multiple probes inserted to a range of depths should be used in situations where steep gradients in v are expected. If these conditions are met, heat dissipation probes remain useful and widely applicable for measuring sap flow in woody stems.