Detection and quantification of Anaplasma marginale DNA in blood samples of cattle by real-time PCR

A TaqMan-based real-time PCR assay was developed for the diagnosis of Anaplasma marginale infection of cattle. The established assay was proven to be highly specific, since no cross-reactions were observed with other Anaplasma species of ruminants, including the closely related Anaplasma centrale, or other haemoparasites of ruminants (Anaplasma bovis, Anaplasma ovis, Anaplasma phagocytophilum, Babesia bovis, Babesia bigemina, Theileria annulata and Theileria buffeli). The detection limit was equal to that of nested (n)PCR (10(1) copies of standard DNA and 3 x 10(1) infected erythrocytes ml(-1) of blood). The assay was also reproducible, as shown by satisfactory low intra-assay and inter-assay coefficients of variation. Fifty-four blood samples of ruminants (cattle, n = 51; sheep, n = 2; goats, n = 1), that had been tested previously by reverse line blot (RLB) hybridisation, were subjected to an nPCR assay and the newly established real-time PCR assay. By using real-time PCR, A. marginale DNA was detected in 39/51 bovine samples, with DNA titres ranging from 3.60 x 10(3) to 5.70 x 10(8) copies ml(-1) of blood, whereas sheep and goat samples tested negative. The concordance with nPCR was 100%, whereas a unique sample that had tested negative by RLB gave positive results by nPCR and real-time PCR. The established assay could overcome the limitations of existing diagnostic methods, allowing for simultaneous detection and quantification of the A. marginale DNA in bovine blood, that is essential to support the clinical diagnosis, to assess the carrier status of the animals and to evaluate the efficacy of vaccines and antirickettsial drugs.     

Feline vaccine‐associated sarcomagenesis: Is there an inflammation‐independent role for aluminium?

Aluminium has been found in feline vaccine‐associated sarcomas. In this study, we investigated the potential for aluminium to contribute directly to tumourigenesis. Our results indicated that an aluminium hydroxide adjuvant preparation was cytotoxic and mutagenic in human‐Chinese hamster ovary (CHO) hybrid cells in vitro. Moreover, CHO cells deficient in DNA double strand break (DSB), but not single‐strand break (SSB), repair, were particularly sensitive to aluminium exposure compared with repair proficient cells, suggesting that aluminium is associated with DSBs. In contrast to CHO cells, primary feline skin fibroblasts were resistant to the cytotoxic effects of aluminium compounds and exposure to an aluminium chloride salt promoted cell growth and cell cycle progression at concentrations much less than those measured in particular feline rabies vaccines. These findings suggest that aluminium exposure may contribute, theoretically, to both initiation and promotion of tumours in the absence of an inflammatory response.     

Integrating meteorological factors for better understanding of the urban form-air quality relationship

Landscape Ecology - Understanding the urban form-air quality relationship is essential to mitigate intra-urban air pollution and to improve urban ecology. However, few studies considered urban form...     

Studies on the various sugar-urea and sugar-starch-urea relationships with reference to the fermentation in the rumen and the digestibility of nutrients in fattening cattle]

Recommendations are given saying that sugar-to-crude fibre rations of not more than 1.7:1 and sugar-to-urea ratios of 12:1 should be used in the fattening of cattle fed urea-supplemented rations of roughages in which 40% to 50% of the crude protein content of the ration had been replaced by urea. The amount of sugar per kg live-weight should not exceed 4 g. Higher quantities reduce the digestibility of crude fibre and, additionally, decrease the NH3 level and the pH in the rumen. At the same time the amount of volatile fatty acids is increased while the content of ruminal acetic acid decreases. Variations in the sugar-to-starch ratio had no statistically significant influence on the nitrogen balance whereas it was found that increasing quantities of sugar significantly decreased the digestibility of crude fibre. The proportional content of sugar in the readily metabolisable carbohydrates contained in the ration should not exceed 20% to 50%.     

Infectious bovine keratoconjunctivitis: seasonal variation in cultural, biochemical and immunoreactive properties of Moraxella bovis isolated from the eyes of cattle

The eyes of 20 young cattle were examined over an 18 month period in which 12 members of the group contracted infectious bovine keratoconjunctivitis (IBK). On each of 23 occasions cultural, biochemical and immunoreactive properties of up to 6 isolates of Moraxella bovis derived from each eye were determined. Relationships between the clinical response of eyes, phenotypic properties of M. bovis and annual variations in the level of solar ultraviolet radiation of 280 to 320 nm wavelength were examined. M. bovis was isolated from all IBK-affected and some unaffected eyes less than one month after the maximum annual level of the mean weekly UV radiation (2,840 mWh.m-2 X nm-1) was recorded. A high proportion of M. bovis from IBK lesions were simultaneously active in haemolysis, agar corrosion, gelatin liquefaction and litmus milk peptonisation. Some of these characteristics showed marked dissociation despite consistent reactivity in the fluorescent antibody test, which had a sensitivity and specificity of 95%. Fall in the mean weekly level of UV radiation below 1,438 mWh X m-2 X nm-1 in autumn was accompanied by healing of ulcers, persistent scar formation and a decline in the number of M. bovis isolated from affected eyes. A slower decline in the number of M. bovis isolated from apparently healthy eyes occurred in the winter and occasional fresh IBK lesions occurred.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adaptation of the Sensititre broth microdilution technique to antimicrobial susceptibility testing of Mycoplasma gallisepticum.

A technique is described for determining the antimicrobial susceptibility of Mycoplasma gallisepticum, using the Sensititre broth microdilution system. Fourteen M. gallisepticum field isolates and one reference isolate (R-strain) were tested in duplicate against seven antimicrobials. Isolates were susceptible to oxytetracycline, furaltadone, and lincomycin/spectinomycin, but not to amoxycillin and apramycin. Susceptibility to erythromycin and tylosin varied. These data are in agreement with those reported by other workers using more traditional methods, but this adaptation of the broth microdilution technique eliminates any variation attributable to the time-consuming preparation of antimicrobial dilutions associated with these methods.     

In vitro activity of danofloxacin, tylosin and oxytetracycline against mycoplasmas of veterinary importance

The activities of danofloxacin, a novel fluoroquinolone, and two other antimicrobials were determined in vitro against field isolates of seven Mycoplasma species of veterinary importance isolated from cattle, swine and poultry in five European countries. The minimum inhibitory concentrations (MIC) of danofloxacin, tylosin and oxytetracycline were determined against a total of 68 isolates. Danofloxacin showed excellent activity against isolates of all Mycoplasma species (range 0·008 to 0·5 μg ml⁻¹), but in some isolates there was evidence of reduced sensitivity to tylosin (range 0·008 to 2·0 μg ml⁻¹) and oxytetracycline (range 0·008 to over 16·0 μg ml⁻¹). Danofloxacin was more active than other antimicrobials against, M hyopneumoniae, M dispar and M bovigenitalium, and showed activity similar to that of tylosin against M bovis and M gallisepticum. Tylosin was the most active against M synoviae and M hyosynoviae. Generally, oxytetracycline showed the poorest activity, but was superior to tylosin against M bovigenitalium. A second (final) MIC reading was taken for all isolates 14 or 21 days after the initial reading, and MIC values rose during that time. However, the increase seen in danofloxacin values (typically one to two dilutions) was less than that seen for tylosin and oxytetracycline. It is concluded that danofloxacin is highly active in vitro against all of the Mycoplasma species tested, and thus shows great potential for the treatment of respiratory and other infections caused by Mycoplasma species in cattle, pigs and poultry.     

Genetic characterization by fluorescent AFLP of Pseudomonas savastanoi pv. savastanoi strains isolated from different host species

The genetic diversity of 71 Pseudomonas savastanoi pv. savastanoi strains isolated from different host species and from diverse geographical regions was determined by fluorescent amplified fragment length polymorphism (f-AFLP) analysis. The study was carried out using three different selective primer combinations. Strains of P. syringae pv. syringae , P. syringae pv. phaseolicola , P. syringae pv. glycinea , P. syringae pv. tagetis and P. amygdali were also included as outgroups. Based on cluster analysis of f-AFLP data, all P. savastanoi pv. savastanoi strains showed a high degree of similarity, grouping in a cluster and forming a taxon clearly separate from outgroup strains. AFLP analyses failed to support placing strains of P. savastanoi pv. savastanoi , P. syringae pv. phaseolicola and P. syringae pv. glycinea in the same species. Strains of P. savastanoi pv. savastanoi formed subclusters that correlated with the host species. Strains identified within these subclusters were related to the geographical region where the strains were isolated. Strains of P. savastanoi pv. savastanoi from olive were divided into two subclusters. Strains from oleander were differentiated from those from ash and were divided into two additional subclusters, distinct from olive strains. Three strains isolated from jasmine showed a high level of similarity among them but, at a lower Dice similarity coefficient, were linked to a subcluster including olive strains. Finally, two strains isolated from privet were similar to strains from olive and were included in the same subcluster.