Participation of specific PKC isozymes in the inhibitory effect of ET-1 on progesterone accumulation in cells isolated from early- and mid-phase corpora lutea

Expression of PKC alpha, beta I, beta II, epsilon and micro has been demonstrated in the whole bovine CL with PKC epsilon being differentially expressed as a function of development. In experiment 1 we have investigated the amount of mRNA encoding PKC epsilon at different stages of luteal development (days 1, 4, 10 and 17). In experiment 2, the cellular source of luteal PKC isozymes was determined. Enriched steroidogenic (SC) and endothelial (EC) cells from day-10 CL were used to examine this question by Western blot analysis and immuno-histochemistry. In experiment 3, Western blot analysis was used to examine the ability of ET-1 to activate luteal PKC isozymes in day-10 CL. In experiment 4, the role of luteal PKC isozymes in the ET-1 mediated inhibition of P(4) accumulation in steroidogenic cell cultures from day-4 and day-10 CL was examined. Abundance of PKC epsilon mRNA gradually increased from day-1 to -10 with no further increase on day-17. In experiment 2, PKC epsilon was exclusively detected in SC (LLC and SLC). In contrast, PKC alpha, beta I and beta II were detected in both SC and EC, with EC expressing higher amounts of PKC isozymes. In day-10 CL, ET-1 induced cellular redistribution of PKC alpha, beta I, epsilon but not beta II. Inhibitors specific for conventional PKC isozymes as well as PKC epsilon were able to negate the inhibitory effects of ET-1 on P4 accumulation in the day 10 CL. In the day-4 CL, the inhibitory effect of ET-1 might be mediated via conventional PKC. Thus, an exclusive presence of PKC epsilon in luteal steroidogenic cells, its higher expression along with the ability of ET-1 to stimulate its activation in day-10 CL strongly suggests that this PKC isoform may play an important regulatory role in decreasing P(4) during luteal regression. Inhibition of P(4) by ET-1 in the early CL may be mediated via conventional PKC isozymes.     

Detection of genes encoding virulence factors and bacteriocins in fecal enterococci of poultry in Portugal

Seventy-six Enterococcus isolates (43 E. faecalis, 30 E. faecium, two E. durans, and one E. hirae) recovered from fecal samples of poultry in a slaughterhouse (one isolate per fecal sample and one fecal sample per lot of animals) were studied for bacteriocin production and for the presence of genes encoding bacteriocins and virulence factors. The presence of genes encoding virulence factors (cpd, geE, fsr, ace, agg, and esp) and bacteriocins (entA, entB, entP, entQ, entAS-48, entL50A/B, cyl, and bac31) were studied by polymerase chain reaction in all enterococci. At least two virulence genes were detected in all 43 E. faecalis isolates, cpd and gelE being the most frequently detected genes (97.7%) followed by ace (62.8%), agg (39.5%), fsr (27.9%), and esp (2.3%). No virulence genes were detected in the other enterococcal species with the exception of one E. faecium and one E. durans isolates that harbored the gelE gene. Antimicrobial activity against eight indicator bacteria (including Listeria monocytogenes) was assayed in the enterococci, and 23 (30.3%) showed inhibitory activity against L. monocytogenes, the other 22 enterococci showing activity against indicator bacteria other than L. monocytogenes. Only the entA, entB, and cyl genes were detected in our study (entA + entB in nine E. faecium isolates and the cyl gene in seven E. faecalis isolates). A wide variety of virulence genes have been detected in fecal E. faecalis isolates from poultry, but not in the other enterococcal species. However, the presence of known bacteriocin structural genes is associated more with the E. faecium species.     

Dispersal of conidia of Fusicladium eriobotryae and spatial patterns of scab in loquat orchards in Spain

Dispersal of conidia of Fusicladium eriobotryae, the causal agent of loquat scab, was investigated in two loquat orchards in Spain from 2010 to 2012. A volumetric spore sampler, horizontally and vertically orientated microscope slides, and rain collectors were placed in loquat fields to trap conidia of F. eriobotryae. No conidia were collected in the volumetric sampler, and highly variable numbers of conidia were collected in the rain gatherers. Large numbers of conidia were collected by microscope slides, particularly by those held in a horizontal orientation compared with those held in a vertical orientation. Approximately 90 % of the F. eriobotryae conidia were collected during rainy periods. Based on ROC and Bayesian analysis, using ≥ 0.2 mm rainfall as a cut-off value resulted in a high probability of correctly predicting actual conidial dispersal, and had a low probability of failing to predict actual conidial dispersal. Based on the index of dispersion and the binary power law, the incidence of loquat scab on fruit was highly aggregated in space between and within trees, and aggregation was influenced by disease incidence. Our results demonstrate, for the first time, that F. eriobotryae is dispersed mainly in rain splash. The results will be integrated into a mechanistic, weather-driven, disease prediction model that should help growers to minimize fungicide application for the management of loquat scab.     

Neutrophil function and plasma opsonic capacity in colostrum-fed and colostrum-deprived neonatal kittens

OBJECTIVE: To determine whether passive transfer of IgG in neonatal kittens affects plasma opsonic capacity and neutrophil phagocytic and oxidative burst responses to bacteria in vitro. ANIMALS: 22 kittens from 6 specific pathogen-free queens. PROCEDURE: Kittens were randomized at birth into the following treatment groups: colostrum-fed, colostrum-deprived, or colostrum-deprived supplemented with feline or equine IgG. Blood samples were collected at intervals from birth to 56 days of age. Plasma IgG concentrations were determined by radial immunodiffusion assay. Neutrophil function was assessed by a flow cytometry assay providing simultaneous measurement of bacteria-induced phagocytosis and oxidative burst. The opsonic capacity of kitten plasma was determined in an opsonophagocytosis assay with bacteria incubated in untreated or heat-inactivated plasma. RESULTS: Among treatment groups, there were no significant differences in neutrophil phagocytic and oxidative burst responses to bacteria or opsonic capacity of plasma. In all samples of plasma, inactivation of complement and other heat-labile opsonins significantly reduced the opsonic capacity. Plasma IgG concentrations in kittens did not correlate with neutrophil function or plasma opsonic capacity before or after inactivation of complement. CONCLUSIONS AND CLINICAL RELEVANCE: The plasma opsonic capacity and neutrophil phagocytic and oxidative burst responses in vitro of kittens receiving passive transfer of IgG via colostrum intake or IgG supplementation and those deprived of colostrum were similar. The alternate complement pathway or other heat-labile opsonins may be more important than IgG in bacterial opsonization and phagocytosis.     

Molecular Mapping of the Stb4 Gene for Resistance to Septoria tritici Blotch in Wheat

ABSTRACT Breeding wheat for resistance is the most effective means to control Septoria tritici blotch (STB), caused by the ascomycete Mycosphaerella graminicola (anamorph Septoria tritici). At least eight genes that confer resistance to STB in wheat have been identified. Among them, the Stb4 locus from the wheat cv. Tadinia showed resistance to M. graminicola at both seedling and adult-plant stages. However, no attempt has been made to map the Stb4 locus in the wheat genome. A mapping population of 77 F10 recombinant-inbred lines (RILs) derived from a three-way cross between the resistant cv. Tadinia and the susceptible parent (Yecora Rojo x UC554) was evaluated for disease resistance and molecular mapping. The RILs were tested with Argentina isolate I 89 of M. graminicola for one greenhouse season in Brazil during 1999, with an isolate from Brazil (IPBr1) for one field season in Piracicaba (Brazil) during 2000, and with Indiana tester isolate IN95-Lafayette-1196-WW-1-4 in the greenhouse during 2000 and 2001. The ratio of resistant:susceptible RILs was 1:1 in all three tests, confirming the single-gene model for control of resistance to STB in Tadinia. However, the patterns of resistance and susceptibility were different between the Indiana isolate and those from South America. For example, the ratio of RILs resistant to both the Indiana and Argentina isolates, resistant to one but susceptible to the other, and susceptible to both isolates was approximately 1:1:1:1, indicating that Tadinia may contain at least two genes for resistance to STB. A similar pattern was observed between the Indiana and Brazil isolates. The gene identified with the Indiana tester isolate was assumed to be the same as Stb4, whereas that revealed by the South American isolates may be new. Bulked-segregant analysis was used to identify amplified fragment length polymorphism (AFLP) and microsatellite markers linked to the presumed Stb4 gene. The AFLP marker EcoRI-ACTG/MseI-CAAA5 and microsatellite Xgwm111 were closely linked to the Stb4 locus in coupling at distances of 2.1 and 0.7 centimorgans (cM), respectively. A flanking marker, AFLP EAGG/ M-CAT10, was 4 cM from Stb4. The Stb4 gene was in a potential supercluster of resistance genes near the centromere on the short arm of wheat chromosome 7D that also contained Stb5 plus five previously identified genes for resistance to Russian wheat aphid. The microsatellite marker Xgwm111 identified in this study may be useful for facilitating the transfer of Stb4 into improved cultivars of wheat.     

Seroepidemiology of neosporosis in dairy cattle in Uruguay

A seroepidemiological survey of Neospora caninum was done in a dairy farm in Uruguay employing indirect fluorescent antibody test (IFAT). Sera from 217 cattle (155 cows, 31 heifers and 31 calves) were tested for N. caninum antibodies in July 2000 and it was found that abortions were significantly associated with the seropositivity of the cows by chi(2)-test (P = 0.025). The cows with an IFAT titre of 1:3200 were at a significantly higher risk of abortion than seronegative cows (P = 0.0013). Neospora organisms were detected in the brains of two aborted foetuses by immunohistochemical examination. The infection of N. caninum was considered postnatal because of the even distribution of seropositive cows (60%), low seroprevalence (20%) in calves and no significant correlation in the serostatus between calves and their dams (P = 0.43). A fitted binomial generalised linear model revealed that there was an increasing risk of abortion with increasing IFAT titre and increasing age between 2 and 6 years.     

Detritivorous earthworms modify microbial community structure and accelerate plant residue decomposition

The impact of detritivorous earthworms (Eisenia andrei) on microbial community structure and function in grape marc, a lignocellulosic enriched plant residue, was investigated in a mesocosm experiment. Analysis of carbon and nitrogen pools was also carried out in order to evaluate how changes in microbial communities affect plant residue decomposition. The grape marc was completely processed after fifteen days as a result of the high density of earthworms present and the rapid gut transit time. Eisenia andrei had a large impact on the structure of the microbial community, as revealed by phospholipid fatty acid analysis. Earthworm activity reduced the abundance of both bacterial (except for Gram-negative bacteria) and fungal PLFA biomarkers relative to the control values. Decreases in microbial activity and in protease and cellulase activities were also attributable to the presence of earthworms. Moreover, earthworms strongly modified the substrate utilization patterns of microbial communities, as revealed by BIOLOG analysis. The presence of earthworms led to an increase in the utilization of some amino acids and polymers, which reached a higher substrate diversity value than that in the control mesocosm. The differences in microbial communities were accompanied by a reduction in the total C content and the labile C pool, relative to the control, although there were no significant differences in either cellulose or hemicellulose contents. However, total N content increased in both mesocosms – with and without earthworms – and the concentration of NH₄⁺ was also enhanced by earthworm activity. The results indicate that detritivorous earthworms play a key role in decomposing fresh plant residues in the short term via their intensive interactions with microbial communities.     

Chemical composition of the Lippia origanoides essential oils and their antigenotoxicity against bleomycin-induced DNA damage

The present work evaluated the chemical composition of the essential oils (EO) obtained from Lippia origanoides and their DNA protective effect against bleomycin-induced genotoxicity. L. origanoides EO chemical composition was determined by gas chromatography–mass spectrometry (GC–MS). The major compounds of the L. origanoides EOs were thymol (34–58%) and carvacrol (26%). The antigenotoxic effects of the EOs, major compounds and standard compound (epigallocatechin gallate) were assayed in co-incubation procedures using the SOS chromotest in Escherichia coli. Both EOs and their major compounds protected bacterial cells against bleomycin-induced genotoxicity indicating that these two compounds were principally responsible for the antigenotoxicity detected in the oils. Thymol and carvacrol antigenotoxicity was lower than those observed with epigallocatechin gallate. The results were discussed in relation to the chemopreventive potential of L. origanoides EOs and their major components, carvacrol and thymol.     

Evaluation of Natural Chemical Compounds Against Root-lesion and Root-knot Nematodes and Side-effects on the Infectivity of Arbuscular Mycorrhizal Fungi

The survival of two species of plant parasitic nematodes: the root-lesion nematode Pratylenchus brachyurus, and the root-knot nematode Meloidogyne javanica, was evaluated in saturated atmospheres of 12 natural chemical compounds. The infectivity of two isolates of arbuscular mycorrhizal fungi: Glomus mosseae and Glomus intraradices, under identical experimental conditions, was also determined. All the compounds tested exerted a highly significant control against M. javanica and among them, benzaldehyde, salicilaldehyde, borneol, p-anisaldehyde and cinnamaldehyde caused a mortality rate above 50% over P. brachyurus. The infectivity of G. intraradices was inhibited by cinnamaldehyde, salicilaldehyde, thymol, carvacrol, p-anisaldehyde, and benzaldehyde, while only cinnamaldehyde and thymol significantly inhibited mycorrhizal colonization by G. mosseae.     

Humoral response (IgG) of goats experimentally infected with Fasciola hepatica against cysteine proteinases of adult fluke

The use of cysteine proteinases from Fasciola hepatica adult flukes for the serodiagnosis of caprine fasciolosis by means of an indirect ELISA test was studied. Two proteolytic fractions from adult fluke homogenates, with apparent molecular weights of 28 and 34 kDa (P28 and P34 respectively), were characterised as cysteine proteinases using azocasein assays and gelatin gel analysis. Both P28 and P34 fractions were electroluted and used as antigens in two different indirect ELISA tests. Serum IgG levels against P28 and P34 in goats given an experimental primary infection with 200 metacercariae or in goats given two experimental infections with 200 metacercariae were determined and compared with those observed in an uninfected control group. ELISA tests using both cysteine proteases showed a rapid and consistent detection of specific IgG in all experimentally infected goats. The IgG response to P28 was the first to be detected as early as 2-3 weeks post-infection and remained elevated throughout the experiment. The response to P34 was detected later (4-6 wpi) and disappeared in some animals at 18 wpi, while flukes were still present in the bile ducts. No significant differences were observed between the anti-P28 and anti-P34 IgG responses between animals receiving a primary or a challenge infection. The results of our study, although preliminary, are promising since the P28 ELISA described here may be a reliable method for the immunodiagnosis of F. hepatica infection in goats.