Polymorphism of 14α-demethylase Gene (CYP51) in the Cereal Eyespot Fungi Tapesia acuformis and Tapesia yallundae

Cereal eyespot fungi Tapesia acuformis and Tapesia yallundae are closely related species which show different behaviours upon treatment with sterol 14-demethylase inhibitors (DMIs). T. acuformis is naturally resistant to DMIs belonging to the triazole family and susceptible to the imidazole ones, whilst T. yallundae is sensitive to both inhibitors. Cloning of the target enzyme gene, CYP51, from the two species revealed an important polymorphism between them. Further sequencing of CYP51 from sixteen T. acuformis and eleven T. yallundae strains with different phenotypes with regards to resistance to DMIs confirmed that at least eleven variations are species related. Among them, a conserved phenylalanine residue at position 180, found both in T. yallundae and in all known CYP51 proteins from filamentous fungi and yeast, was replaced in T. acuformis by a leucine. Therefore, a leucine at 180 could be possibly involved in natural resistance of T. acuformis to triazoles. Other mutations were observed in some resistant strains, sometimes simultaneously, but in contrast to what was reported for other filamentous fungi, where a mutation at the 136 position of the CYP51 gene product seemed to correlate with resistance to DMIs, we did not find a clear relationship between a given mutation and a particular phenotype. This result suggests that resistance to DMIs could have a polygenic nature in Tapesia. We took advantage of species-related variations to develop a PCR-based assay allowing rapid and easy discrimination between field strains of the two species.     

New gene-based approaches for an AIDS vaccine

Vaccine approaches against AIDS have focused on inducing cellular immune responses, since many studies revealed the role of T cell responses in the control of human immunodeficiency virus or simian immunodeficiency virus (SIV) infections. The experimental infection of rhesus macaques with SIV or chimeric SHIV is routinely used as a model for AIDS. In such models, DNA immunization is a tool to elicit specific T cell responses and to study their protective efficacy. DNA immunogenicity in primates depends on parameters such as level of antigen expression, choice of the antigen among SIV proteins, use of fusion proteins, route of immunization, and addition of adjuvants. Recent results suggest that priming with DNA and boosting with attenuated recombinant viral vectors, each expressing corresponding SIV antigens, leads to improved specific immunity and, in some cases, affords protection against pathogenic challenge. After preclinical evaluations, DNA has entered clinical trials for a therapeutic or prophylactic gene-based AIDS vaccine.     

Efficacy of injectable and pour-on microdose ivermectin in the treatment of goat warble fly infestation by Przhevalskiana silenus (Diptera, Oestridae)

The prophylactic efficacy of microdoses of injectable and pour-on ivermectin formulations against larval stages of Przhevalskiana silenus was assessed in naturally infected goats in the region of Calabria (southern Italy).Sixty-eight goats from two goat farms were divided into five groups: one group remained untreated, while the other four groups were treated with microdoses of ivermectin (5 and 10 microg/kg injectable formulation and 10 and 20 microg/kg pour-on formulation).The microdoses of ivermectin were fully effective in the treatment of goat warble fly infestation (GWFI) as no larvae emerged from the warbles in the treated groups, while all the larvae emerged in the control groups. Irrespective of the type of formulation used, the difference between the treated groups and the control group was statistically significant (P< 0.001). By contrast, no statistical differences were found between the goats treated with the injectable formulation and those receiving the pour-on applications, and between the two doses of the injectable and pour-on formulations used. Given the plasma concentrations it attains at its lowest dose (0.052 - 0.042 ng/ml for the injectable formulation and 0.030 ng/ml for the pour-on) the injectable formulation seems to offer the most reliable route for the administration of ivermectin microdoses and it is acceptable for milk consumption. The introduction of ivermectin in the early eighties and the use of microdoses in some cases have made it possible to control cattle hypodermosis in large areas of Europe. As with cattle hypodermosis, the administration of ivermectin microdoses in goats is particularly interesting because of the low costs involved and the low levels of residues found in goat milk; it may thus constitute the basis for GWFI control campaigns in areas where the disease is prevalent.     

Effect of short-chain fatty acids on contraction of smooth muscle in the canine colon

OBJECTIVE: To determine effects of short-chain fatty acids (SCFA) on canine colonic smooth muscle. SAMPLE POPULATION: Colonic tissue obtained from 14 healthy dogs. PROCEDURE: Short-chain fatty acid (SCFA; acetate, propionate, and butyrate; 1 to 100 mmol/L)-induced contractions were compared with responses obtained with acetylmethylcholine (AMCh; 10(-4) mol/L). Roles of enteric neurons, cholinergic receptors, calcium stores in the sarcoplasmic reticulum, and extracellular calcium in the SCFA-induced responses were investigated by incubating muscle strips with tetrodotoxin (1 micromol/L), atropine (1 micromol/L), ryanodine (10 micromol/L), nifedipine (1 micromol/L), ethylene glycol-bis (beta-aminoethylether)-N,N,N',N'-tetra-acetate (EGTA; 0.1 mmol/L), or an extracellular calcium-depleted (zero extracellular calcium) solution prior to the addition of propionate or butyrate. RESULTS: Incubation with SCFA elicited isometric stress responses (0.25 to 2.15 x 10(4) N/m2) in colonic longitudinal smooth muscle. Maximal responses to butyrate and propionate (50 mmol/L) were 37 and 23%, respectively, of the maximal AMCh response. Acetate was least effective in stimulating contractile responses. Tetrodotoxin and atropine did not affect SCFA-induced contractions. Nifedipine and zero extracellular calcium solution abolished responses to butyrate and propionate, whereas EGTA attenuated (> 60%) but did not abolish those responses. Ryanodine did not affect SCFA-induced contractile responses. The SCFA did not affect colonic circular smooth muscle. CONCLUSIONS AND CLINICAL RESPONSE: The SCFA stimulate longitudinal but not circular colonic smooth muscle contractions via a direct effect on smooth muscle. The mechanism of the SCFA effect appears to involve the influx of extracellular calcium. These findings may account for some of the effects of fiber on canine colonic motility [corrected].     

The gamma-interferon test: its usefulness in a bovine tuberculosis survey in African buffaloes (Syncerus caffer) in the Kruger National Park

A survey to determine the bovine tuberculosis status of buffalo herds north of the Olifants River in the Kruger National Park was conducted, using a new diagnostic approach. Diagnosis of Mycobacterium bovis infection was accomplished using the gamma-interferon assay technique in 608 adult buffaloes out of a total of 29 discreet herds. The animals were immobilized in groups of 10-15, bled, individually marked and then revived and released on site. As soon as test results were available (after 26-36 h), the same buffalo herd was relocated by tracking the frequency of a radio-collar previously fitted to one adult cow per group during the initial operation. Bovine reactors were identified, darted and euthanased from the helicopter. Necropsy and culture findings of all culled buffaloes showed excellent correlation with the results of the ante-mortem gamma-interferon test. The survey revealed that over and above the two positive herds that had been identified during a previous survey carried out in 1996, there were three additional, but previously unidentified, infected herds in the region north of the Olifants River.     

Protective Effects Against Erwinia amylovora Induced by hrp Mutants in the Whole Plant are Only Partially Mimicked in Cultivated Cells

A virulent strain of Erwinia amylovora, the causal agent of fire blight of Maloideae, and two of its non-virulent hrp mutants (a secretory and a regulatory mutant) were inoculated into apple cell suspensions either alone or in mixed inoculations. In single inoculations, death of 4- to 5-day-old apple cells occurred only when the concentration of the virulent strain of E. amylovora reached a threshold inoculum concentration of 10⁴CFUml^–1, while high concentrations of the hrp mutants were unable to kill apple cells. When mixed inoculated with the virulent parental strain, both hrp mutants protected apple cells from death caused by the virulent strain. The protective effect was associated with a decrease in the population level of the virulent strain and it was dependent on the non-virulent to virulent inoculum concentration suggesting a competition between the non-virulent mutant and the virulent strain. However, no differential protective ability between the two types of mutants could be noticed, contrary to previous results obtained with apple seedlings or apple flowers in which the regulatory mutant was significantly more effective than the secretory mutant. In conclusion, inoculation of apple cell cultures with E. amylovora does not seem to be a model suitable for investigating mechanisms leading to protection.     

Spatial and temporal distribution of geminiviruses in leafhoppers of the genus cicadulina monitored by conventional and quantitative polymerase chain reaction

ABSTRACT Spatial and temporal distribution of Maize streak virus (MSV, family Geminiviridae, genus Mastrevirus) was monitored in the vector species Cicadulina mbila and the nonvector species C. china? using conventional and real-time quantitative polymerase chain reaction. Sustained feeding on MSV-infected plants showed that virus accumulation reaches a maximum in C. china?, but not in C. mbila. After a 3-day acquisition access feeding period (AAP), MSV was detected in the gut, the hemolymph, and the head of C. mbila, but only in the gut of C. china?. Similarly, Digitaria streak virus (genus Mastrevirus), which is not transmitted by either of the two species, was only detected in the gut. MSV was detected in the hemolymph of C. mbila 3 h after the beginning of the AAP. Although viral DNA progressively decreases in the vector and nonvector species after a 3-day AAP, MSV DNA remained stable in the salivary glands of C. mbila.