In vitro effect of classical swine fever virus on a porcine aortic endothelial cell line

The effect of classical swine fever (CSF) virus on some phenotypic and functional features of an established porcine aortic endothelial cell (AOC) line was investigated. AOC cells show most of the characteristics of primary endothelial cells, avoiding the alterations and senescence that these cells undergo after a few passages in culture. AOC cells were susceptible to CSF virus infection to a high degree, reaching 90% of CSF virus positive cells after 24 h of infection; however as with other porcine susceptible cells, no cytopathic effect could be observed. In these conditions none of the surface molecules studied, including SLA-II MHC antigens, adhesion or co-stimulatory molecules, were altered by virus infection after 24 or 48 h. Functionally CSF virus infection induced a decrease in the pro-coagulant activity of the AOC cells, determined by the increase in the clot formation time shown by the lysates of these cells. This contrasts with the increase observed in the expression of mRNA corresponding to IL-1 alpha and IL-6, two proinflammatory and pro-coagulant cytokines, in CSF virus-infected AOC cells.     

Horizontal Maedi-Visna virus (MVV) infection in adult dairy-sheep raised under varying MVV-infection pressures investigated by ELISA and PCR

A three year long experimental study was carried out to investigate horizontal MVV-infection by PCR and ELISA, in 191 one year-old latxa dairy-sheep raised in two separate groups under low and high MVV-infection pressure, respectively. Sheep originated from a previous MVV-transmission study in lambs and seroprevalence among one year-old sheep in both groups was 15% approximately. The high infection-pressure group (H-group) consisted of 147 replacement ewes that joined a milk-producing, housed dairy-flock with 42-66% MVV-seroprevalence and the low infection-pressure group (L-group) were castrated males raised in a separate shed. In contrast to results obtained when infection was investigated in lambs, the overall degree of agreement between ELISA and PCR results was very good and there was some indication that it increased further as sheep became older. MVV-prevalence did not change in the L-group and increased to 57% in three year-old sheep in the H-group (p<0.001). Random effects logistic regression confirmed seroconversion was significantly higher in the H-group compared to the L-group and was highest during the year after the sheep were introduced in the dairy flock and did not increase with age as in previous studies using less sensitive antibody assays. The evidence that horizontal transmission can be very low in spite of prolonged close contact between infected and non-infected sheep is valuable for MVV-control purposes. Furthermore it highlights the need to investigate virus excretion dynamics in infected animals and animal to animal transmission to improve our overall understanding of horizontal MVV transmission in MVV endemic populations.     

Residue levels and effectiveness of pyrimethanil vs imazalil when using heated postharvest dip treatments for control of Penicillium decay on citrus fruit

The influence of fungicide concentration and treatment temperature on residue levels of pyrimethanil (PYR) in comparison with the commonly used fungicide imazalil (IMZ) was investigated in orange fruits following postharvest dip treatments. The dissipation rate of PYR residues was recorded as a function of storage conditions. The fungicide efficacy against green and blue molds caused by Penicillium digitatum and Penicillium italicum, respectively, was evaluated on different citrus varieties following the fungicide application at 20 or 50 degrees C. Residue levels of PYR in Salustiana oranges were significantly correlated with the fungicide dosage, but residue concentrations were notably higher (ca. 13-19-fold) after treatment at 50 degrees C as compared to treatments at 20 degrees C. After treatment at temperatures ranging from 20 to 60 degrees C, PYR and IMZ residues in Salustiana oranges were significantly correlated with dip temperatures. Dissipation rates of PYR during storage were negligible in both Salustiana and Tarocco oranges. Results obtained on wounded, noninoculated Miho satsumas revealed that when treatments were performed at 50 degrees C, PYR or IMZ concentrations needed to achieve the complete control of decay were 8- and 16-fold less than by treatment at 20 degrees C. When fruits were inoculated with either P. digitatum or P. italicum, the application of 400 mg L(-1) PYR at 20 degrees C or 100 mg L(-1) PYR at 50 degrees C similarly reduced green and blue mold development. These results were corroborated by storage trials on Marsh grapefruits and Tarocco oranges. The lowest concentration of PYR required to achieve almost total protection of the fruit against decay accounted for 100 mg L(-1) at 50 degrees C and 400 mg L(-1) at 20 degrees C, respectively. Treatments did not affect fruit external appearance, flavor, and taste. It is concluded that postharvest PYR treatment represents an effective option to control green and blue mold in citrus fruit and that integration of fungicide applications and hot water dips may reduce the possibility of selecting fungicide-resistant populations of the pathogen, as a consequence of increased effectiveness of the treatment.     

Glyceraldehyde bridging between flavanols and malvidin-3-glucoside in model solutions

Hydroxyl radicals (.OH) seem to have an important role in the oxidation of wine constituents and the production of important electrophilic aldehydes and ketones. In this experiment, glyceraldehyde, a .OH oxidation product of glycerol, recently described in wine, reacts with (+)-catechin, (-)-epicatechin, and malvidin-3-glucoside (Mv3gl), in model solutions, yielding new condensed phenolic compounds. The adduct compounds formed were separated by means of reversed phase liquid chromatography and detected and characterized using UV-vis and electrospray ionization mass spectrometry. Flavanol-flavanol and anthocyanin-flavanol adducts linked with glyceraldehyde yielded compounds with m/z ratios for their main ions, in positive ion mode, of 653.2 for the (+)-catechin dimer or the (-)-epicatechin dimer and 855.5 for Mv3gl/(+)-catechin or Mv3gl/(-)-epicatechin dimers. The possible occurrence of these compounds in wine is suggested, and the potential role of these and related reactions in wine aging is discussed.     

Mouse epidermal development: effects of retinoic acid exposure in utero

Epidermal morphogenesis was studied in vivo following prenatal exposure to retinoic acid (RA). In pregnant mice, a single oral dose of RA on day 11.5 of gestation failed to induce histological changes in fetal epidermal development except in epidermal thickness. Epidermal thickness increased from 16.5 days post-coitum (dpc) onwards, and temporal and spatial epidermal modifications in keratins K5 and K14 related to proliferative activity of keratinocytes were observed. An RA effect on cell proliferation was supported by a statistically significant increase in the number of epidermal S-phase cells, containing BrdU-incorporated DNA in RA-exposed mice compared with nonexposed animals. The prolonged in utero action of RA on epidermal proliferative activity in fetuses and newborns suggests a long-term RA effect that may play a role on the development and evolution of diseases in adult skin.     

Portal site metastasis of invasive mesothelioma after diagnostic thoracoscopy in a dog

CASE DESCRIPTION: A 10-year-old Golden Retriever evaluated because of recurrent pericardial and pleural effusion underwent thoracoscopy with biopsy of the pleura and mediastinum. CLINICAL FINDINGS: Before thoracoscopy, 5 L of serosanguinous fluid was removed from the pleural cavity via thoracocentesis. During thoracoscopic exploration, it was observed that the parietal pleura and mediastinum were covered by miliary white to tan nodules 1 to 3 mm in diameter. Biopsy specimens were obtained, and partial pericardiectomy was performed. Portal sites were closed routinely. Cytologic evaluation of the pleural fluid revealed high protein concentration and cellularity, with cellular changes consistent with an exfoliating carcinoma. Results of bacterial culture were negative. TREATMENT AND OUTCOME: Carboplatin was administered via intracavitary instillation, and prednisone was administered orally. Twenty-one days later, 1 firm, irregularly shaped 6.5 x 3-cm mass and 4 smaller masses were detected in the area of the left thoracic wall where the cannula had been inserted during thoracoscopy. Histologic analysis of tissue from the masses collected at necropsy confirmed that they were malignant tumors with similar appearance to the pleural mesothelioma and immunohistochemical staining properties identical to those of the primary tumor. CLINICAL RELEVANCE: Although thoracoscopy is associated with less postoperative pain, shorter hospitalization times, and faster patient recovery than sternotomy procedures, complications are also possible with minimally invasive endoscopic surgery. Portal site metastasis can develop from contamination of portal sites with cells on instruments or cannulas or via leakage of effusion fluid. Although rare, this potential complication should be discussed with owners prior to performing the procedure.     

Genetic analysis and molecular characterisation of laboratory and field mutants of Botryotinia fuckeliana (Botrytis cinerea) resistant to QoI fungicides

BACKGROUND: QoI fungicides, inhibitors of mitochondrial respiration, are considered to be at high risk of resistance development. In several phytopathogenic fungi, resistance is caused by mutations (most frequently G143A) in the mitochondrial cytochrome b (cytb) gene. The genetic and molecular basis of QoI resistance were investigated in laboratory and field mutants of Botryotinia fuckeliana (de Bary) Whetz. exhibiting in vitro reduced sensitivity to trifloxystrobin. RESULTS: B. fuckeliana mutants highly resistant to trifloxystrobin were obtained in the laboratory by spontaneous mutations in wild‐type strains, or from naturally infected plants on a medium amended with 1–3 mg L^?1 trifloxystrobin and 2 mM salicylhydroxamic acid, an inhibitor of alternative oxidase. No point mutations were detected, either in the complete nucleotide sequences of the cytb gene or in those of the aox and Rieske protein genes of laboratory mutants, whereas all field mutants carried the G143A mutation in the mitochondrial cytb gene. QoI resistance was always maternally inherited in ascospore progeny of sexual crosses of field mutants with sensitive reference strains. CONCLUSIONS: The G143A mutation in cytb gene is confirmed to be responsible for field resistance to QoIs in B. fuckeliana. Maternal inheritance of resistance to QoIs in progeny of sexual crosses confirmed that it is caused by extranuclear genetic determinants. In laboratory mutants the heteroplasmic state of mutated mitochondria could likely hamper the G143A detection, otherwise other gene(s) underlying different mechanisms of resistance could be involved. Copyright © 2012 Society of Chemical Industry     

Potentiation of triclabendazole action in vivo against a triclabendazole-resistant isolate of Fasciola hepatica following its co-administration with the metabolic inhibitor, ketoconazole

An in vivo study in the laboratory rat model has been carried out to monitor morphological changes in adult Fasciola hepatica over a 4-day period resulting from co-treatment with triclabendazole (TCBZ) and ketoconazole (KTZ), a cytochrome P450 inhibitor. Rats were infected with the triclabendazole-resistant Oberon isolate of F. hepatica, dosed orally with triclabendazole at a dosage of 10 mg/kg live weight and ketoconazole at a dosage of 10 mg/kg live weight. Flukes were recovered at 24, 48, 72 and 96 h post-treatment (p.t.) and changes to fluke ultrastructure were assessed using transmission electron microscopy (TEM). Results showed an increase in the severity of changes to the fluke ultrastructure with time p.t. Swelling of the basal infolds and the associated mucopolysaccharide masses became more severe with time. Golgi complexes, if present, were greatly reduced in size and number by 96 h p.t., and sub-tegumental flooding was seen from the 72 h time-period onwards. Some sloughing of the tegumental covering over the spines was observed at 96 h p.t. The results demonstrated that the Oberon isolate is more sensitive to TCBZ action in the presence of KTZ than to TCBZ alone, reinforcing the idea that altered drug metabolism is involved in the resistance mechanism. Moreover, they support the concept that TCBZ + inhibitor combinations (aimed at altering drug pharmacokinetics and potentiating the action of TCBZ) could be used in the treatment of TCBZ-R populations of F. hepatica.     

Evaluation of the antifungal susceptibility of Malassezia pachydermatis to clotrimazole, miconazole and thiabendazole using a modified CLSI M27-A3 microdilution method

Background – In this study, we evaluated the antifungal susceptibility of Malassezia pachydermatis to clotrimazole (CTZ), miconazole (MCZ), and thiabendazole (TBD), azole derivatives employed in aural formulations labeled for treatment of canine otitis. Methods – The procedure for in vitro testing was based on the indications of the Clinical and Laboratory Standards Institute (CLSI) M27‐A3 microdilution method. A lipid‐enriched medium was employed to enhance the yeast growth (Christensen’s urea broth, with 0.1% Tween 80 and 0.5% Tween 40 as the lipid sources), while the inoculums size corresponded to approximately 1–5 × 10⁵ yeast cells/mL. Microplates were incubated at 37°C and read 48 h after inoculation. Azole MICs inhibiting fungal growth were the lowest drug concentrations that showed an optical density of ≤50% of the (drug‐free) growth control, as assessed by spectrophotometer (630 nm filter). Results – All isolates were inhibited by the three azoles, with different minimum inhibitory concentration (MIC) values. Most isolates were inhibited by drug concentrations of 2–8 (CTZ), 1–4 (MCZ), or 16–32 (TBD) μg/mL. These results are partially in agreement with the findings of previous studies, in which substantially higher/lower MICs were occasionally reported. This is likely because of the different methodologies employed. Such discrepancies may not apply to clinical situations, where the compounds are applied topically. Conclusion and clinical importance – The concept that clinical failure is linked to increased MICs is debatable, because significantly higher concentrations (in most cases at least 1,000 × the MIC) of the antifungals that were included in our study are routinely used in formulated products.     

Relationship between increased albendazole systemic exposure and changes in single nucleotide polymorphisms on the β-tubulin isotype 1 encoding gene in Haemonchus contortus

Resistance to benzimidazole anthelmintics in the nematode Haemonchus contortus has been correlated with single nucleotide polymorphisms (SNPs) on the β-tubulin isotype 1 gene. Three mutations can be used as markers for the detection of resistance, namely SNPs at position 200 and 167 (both TTC to TAC) or at position 198 (GAA to GCA). Harbouring a resistance genotype at any one of these codons can lead to a resistant phenotype. Our objective in this study was to analyse the frequencies of the three mutations when the albendazole dose rate and selection pressure were increased. We used adult H. contortus (males and females) collected directly from the abomasum of untreated lambs, or lambs treated with the manufacturer's recommended dose rate (5mg/kg), three times the recommended dose rate (15 mg/kg), or nine times the recommended dose rate (45 mg/kg). Anthelmintic efficacy was determined by worm and egg count reductions. For the surviving worms of the four treatment groups, the frequencies of each resistance SNP at codons 167, 200 and 198 were measured using pyrosequencing. Our results showed a strong relationship between an increasing dose rate and an increase in the frequency of the (TAC)(200) SNP and a decrease in the (TAC)(167) SNP. All worms genotyped were GAA at codon 198.