Molecular characterization of Echinococcus isolates indicates goats as reservoir for Echinococcus canadensis G6 genotype in Neuquén, Patagonia Argentina

Human cystic echinococcosis is a highly endemic zoonotic disease in the province of Neuquén, Patagonia Argentina, although a hydatid control programme has been carried out since 1970. Human infection due to Echinococcus canadensis (G6 genotype) is frequent in Neuquén. However, the reservoir for this species remains undetermined in a region where camels are absent. We investigated the fertility, viability and molecular epidemiology of hydatid cysts obtained from local goats, pigs and sheep in order to identify the possible reservoirs of E. canadensis (G6). We also analyzed isolates from infected dogs. A total of 67 isolates were identified by the DNA sequencing of the mitochondrial cytochrome c oxidase subunit 1 gene. Cysts from sheep (n = 16), goats (n = 23) and pigs (n = 18) and adult worms from 10 infected dogs were analyzed. The fertility of the hydatid cysts was 78.6%; 90.4% and 94.4% for sheep, goats and pigs, respectively. We detected E. canadensis (G6) in 21 of 23 goat samples and in 1 dog isolate, E. canadensis (G7) in all the pig isolates, E. granulosus sensu stricto (G3) in 1 sheep and the G1 genotype in 15 sheep, 2 goats and 9 dog samples. The G1 haplotypes included the common sheep strain sequence and 2 microvariants of this sequence. E. granulosus sensu stricto (G3) is described for the first time in South America. We conclude that goats act as reservoir for E. canadensis (G6) in Neuquén, and that control strategies may have to be adapted to local molecular epidemiology to improve the control of parasite transmission.     

Entomopathogenic fungi (Hypocreales) for control of potato psyllid, Bactericera cockerelli (Šulc) (Hemiptera: Triozidae) in an area endemic for zebra chip disease of potato

Potato psyllid, Bactericera cockerelli, is a serious pest of potato and other solanaceous vegetables in the United States, Mexico, Central America, and New Zealand and is responsible for transmission of Candidatus Liberibacter solanacearum which causes a disease known as “zebra chip” (ZC). Entomopathogenic fungi could provide a viable component for an integrated pest management strategy for control of B. cockerelli and other potato pest insects. Three field trials of commercial formulations of Metarhizium anisopliae (F 52®, Novozymes Biologicals) and Isaria fumosorosea (Pfr 97®, Certis USA) and abamectin (Agri-Mek®, Syngenta, USA) were conducted in Weslaco, Texas. Rates are expressed in quantity of product delivered in 375–470 l of water/ha. F 52 applied at 0.51, 1.1, and 2.2 l/ha and Agri-Mek applied at 584 ml/ha produced reductions of B. cockerelli eggs and nymphs of 45%, 59%, 67%, and 63%, respectively. Only Agri-Mek significantly reduced plant damage. Pfr 97 at 1.1 kg/ha with and without 1% Trilogy® (neem oil, Certis, USA), and Agri-Mek at 584 ml/ha resulted in psyllid reductions of 78%, 76%, and 84%, respectively. Significantly decreased plant damage and ZC symptoms were observed for all treatments. Tuber yields for Pfr plus Trilogy and Agri-Mek were significantly higher than the control. F 52 applied at 1.1 and 2.2 l/ha and Pfr 97 at 1.1 and 2.2 kg/ha produced 62%, 62%, 66%, and 65% reduction, respectively. Tuber yield for both rates of Pfr and the high rate of F 52 were significantly higher than the control. All fungal treatments significantly reduced plant damage and ZC symptoms.     

Anthelminthic properties of mangostin and mangostin diacetate

Few anthelminthic drugs are available for human use despite the significant burden caused by helminth infections. We studied the activities of mangostin, a major bioactive xanthone isolated from the pericarp and fruit of Garcinia mangostana and of the synthetic derivative mangostin diacetate. Mangostin and mangostin diacetate lacked activity against the nematodes Heligmosomoides polygyrus (third-stage larvae (L3)), Ancylostoma ceylanicum L3, and Trichuris muris adults and showed only low activity against A. ceylanicum adults (IC₅₀s of 91 μg/ml) in vitro. Mangostin showed promising activities (IC₅₀ of 2.9–15.6 μg/ml) against the trematodes Schistosoma mansoni, Echinostoma caproni, and Fasciola hepatica in vitro. Single oral doses (400 mg/kg and 800 mg/kg) of the drugs achieved worm burden reductions ranging from 0 to 38% and 11–54% against S. mansoni and E. caproni in vivo, respectively. Pharmacokinetic studies would be helpful to understand the differences observed between in vitro and in vivo activities and lacking dose–response relationships.     

Hemodynamic effects of inducible nitric oxide synthase inhibition combined with sildenafil during acute pulmonary embolism

While endogenous nitric oxide (NO) may be relevant to the beneficial hemodynamic effects produced by sildenafil during acute pulmonary embolism (APE), huge amounts of inducible NO synthase (iNOS)-derived NO may contribute to lung injury. We hypothesized that iNOS inhibition with S-methylisothiourea could attenuate APE-induced increases in oxidative stress and pulmonary hypertension and, therefore, could improve the beneficial hemodynamic and antioxidant effects produced by sildenafil during APE. Hemodynamic evaluations were performed in non-embolized dogs treated with saline (n = 4), S-methylisothiourea (0.01 mg/kg followed by 0.5 mg/kg/h, n = 4), sildenafil (0.3 mg/kg, n = 4), or S-methylisothiourea followed by sildenafil (n = 4), and in dogs that received the same drugs and were embolized with silicon microspheres (n = 8 for each group). Plasma nitrite/nitrate (NOx) and thiobarbituric acid reactive substances (TBARS) concentrations were determined by Griess and a fluorometric assay, respectively. APE increased mean pulmonary arterial pressure (MPAP) and pulmonary vascular resistance index (PVRI) by 25 ± 1.7 mm Hg and by 941 ± 34 dyn s cm^?5 m^?2, respectively. S-methylisothiourea neither attenuated APE-induced pulmonary hypertension, nor enhanced the beneficial hemodynamic effects produced by sildenafil after APE (>50% reduction in pulmonary vascular resistance). While sildenafil produced no change in plasma NOx concentrations, S-methylisothiourea alone or combined with sildenafil blunted APE-induced increases in NOx concentrations. Both drugs, either alone or combined, produced antioxidant effects. In conclusion, although iNOS-derived NO may play a key role in APE-induced oxidative stress, our results suggest that the iNOS inhibitor S-methylisothiourea neither attenuates APE-induced pulmonary hypertension, nor enhances the beneficial hemodynamic effects produced by sildenafil.     

Characterization of Paecilomycescinnamomeus from the camellia whitefly, Aleurocanthus camelliae (Hemiptera: Aleyrodidae), infesting tea in Japan

The whitefly, Aleurocanthus camelliae Kanmiya and Kasai (Hemiptera: Aleyrodidae), is an invasive species in Japan that was first discovered in 2004 on tea in Kyoto. Soon after its arrival epizootics of an entomopathogenic fungus were observed in populations of the whitefly in many tea-growing regions. Here we identify this fungus as Paecilomyces cinnamomeus (Petch) Samson and W. Gams (Hypocreales: Clavicipitaceae) based on morphological characteristics and molecular analyses. This is the first record of P. cinnamomeus in Japan and also the first time it has been recorded from the genus Aleurocanthus. A isolate of P. cinnamomeus caused greater than 50% and 90% infection in whitefly nymphs at 1 × 10⁶ and 1 × 10⁷ conidia/ml respectively, while the commercial mycoinsecticides Preferd® (Isaria fumosorosea) and Mycotal® (Lecanicillium muscarium) caused <10% infection at their recommended field rates (5 × 10⁶ and 9 × 10⁶ conidia/ml, respectively), suggesting that P. cinnamomeus may be more useful as a control agent than the currently available mycoinsecticides. Optimum and upper limit temperatures for in vitro growth of P. cinnamomeus isolates were 22.5–25 °C and 32.5 °C, respectively. At field rates, the fungicide thiophanate-methyl caused some inhibition of in vitro growth of P. cinnamomeus isolates, and the bactericide copper oxychloride and the insecticides tolfenpyrad and methidathion were strongly inhibitory. The findings obtained in this study will be useful in the development of microbial control programs using P. cinnamomeus against A. camelliae.     

Apparent dominance of the G1–G3 genetic cluster of Echinococcus granulosus strains in the central inland region of Portugal

Infection by the larval stage of the cestode Echinococcus granulosus causes a disease known as cystic echinococcosis or hydatidosis, which is one of the most widespread zoonotic infections of veterinary and medical importance. Numerous studies have shown that E. granulosus exists as a complex of strains differing in a wide variety of criteria. Ten distinct genotypes (G1–G10) have been identified with a potential impact on the pathology, epidemiology and the effect of the measures implemented for the control of hydatidosis. Our main objective was to carry out a preliminary analysis of the genotypes of E. granulosus circulating in the central inland region of Portugal.Parasite samples (hydatid cysts, n = 27) were isolated from the liver and lung of sheep and cattle. The DNA extracted from protoscoleces isolated from the fertile cysts served as a template for the PCR amplification of the part of the mitochondrial cytochrome c oxidase subunit 1 (cox1), ATP synthase F0 subunit 6 (atp6) as well as the large (rrnL/16 S) and small (rrnS/12 S) ribosomal RNA genes. Similarity searches with homologous sequences in the databanks indicated a very high similarity with references assigned to the G1, G3 and/or G1–G3 complex of Echinococcus strains. Phylogenetic analysis (Bayesian approach) supported these observations, and confirmed the assignment of all the analyzed sequences to the G1–G3 genetic cluster.     

Isolation of endophytic endospore-forming bacteria from Theobroma cacao as potential biological control agents of cacao diseases

Sixty-nine endospore-forming bacterial endophytes consisting of 15 different species from five genera were isolated from leaves, pods, branches, and flower cushions of Theobroma cacao as potential biological control agents. Sixteen isolates had in vitro chitinase production. In antagonism studies against cacao pathogens, 42% inhibited Moniliophthora roreri, 33% inhibited Moniliophthora perniciosa, and 49% inhibited Phytophthora capsici. Twenty-five percent of isolates inhibited the growth of both Moniliophthora spp., while 22% of isolates inhibited the growth of all three pathogens. Isolates that were chitinolytic and tested negative on Bacillus cereus agar were tested with in planta studies. All 14 isolates colonized the phyllosphere and internal leaf tissue when introduced with Silwet L-77, regardless of the tissue of origin of the isolate. Eight isolates significantly inhibited P. capsici lesion formation (p = 0.05) in detached leaf assays when compared to untreated control leaves. ARISA with bacilli specific primers amplified 21 OTUs in field grown cacao leaves, while eubacteria specific primers amplified 58 OTUs. ARISA analysis of treated leaves demonstrated that inundative application of a single bacterial species did not cause a long-term shift of native bacterial communities. This research illustrates the presence of endospore-forming bacterial endophytes in cacao trees, their potential as antagonists of cacao pathogens, and that cacao harbors a range of bacterial endophytes.     

Microbial community structure of sandy intertidal sediments in the North Sea, Sylt-Rømø Basin, Wadden Sea

Molecular biological methods were used to investigate the microbial diversity and community structure in intertidal sandy sediments near the island of Sylt (Wadden Sea) at a site which was characterized for transport and mineralization rates in a parallel study (D. de Beer, F. Wenzhöfer, T. Ferdelman, S.E. Boehme, M. Huettel, J.E.E. van Beusekom, M.E. Böttcher, N. Musat, N. Dubilier, Transport and mineralization rates in North Sea sandy intertidal sediments, Sylt-Romo Basin, Wadden Sea, Limnol. Oceanogr. 50 (2005) 113–127). Comparative 16S rRNA sequence analysis revealed a high bacterial diversity. Most sequences retrieved by PCR with a general bacterial primer set were affiliated with Bacteroidetes, Gammaproteobacteria, Deltaproteobacteria and the Pirellula cluster of Planctomycetales. Fluorescence in situ hybridization (FISH) and slot-blot hybridization with group-specific rRNA-targeted oligonucleotide probes were used to characterize the microbial community structure over depth (0–12 cm) and seasons (March, July, October). We found high abundances of bacteria with total cell numbers up to 3×10⁹ cells ml⁻¹ and a clear seasonal variation, with higher values in July and October versus March. The microbial community was dominated by members of the Planctomycetes, the Cytophaga/Flavobacterium group, Gammaproteobacteria, and bacteria of the Desulfosarcina/Desulfococcus group. The high abundance (1.5×10⁷–1.8×10⁸ cells ml⁻¹ accounting for 3–19% of all cells) of presumably aerobic heterotrophic polymer-degrading planctomycetes is in line with the high permeability, deep oxygen penetration, and the high rates of aerobic mineralization of algal biomass measured in the sandy sediments by de Beer et al. (2005). The high and stable abundance of members of the Desulfosarcina/Desulfococcus group, both over depth and season, suggests that these bacteria may play a more important role than previously assumed based on low sulfate reduction rates in parallel cores (de Beer et al., 2005).     

In vitro activity of some natural honeys against Entamoeba histolytica and Giardia lamblia trophozoites

Considering the potentiality of honey in combating diseases, the present study was carried out aiming to assess the in vitro antiprotozoal activity of several honeys (Ziziphus spina-christi, Acacia nilotica, Acacia seyal, and Cucurbita maxima) against Entamoeba histolytica and Giardia lamblia by employing the sub-culture method. All the tested honeys inhibited the growth of trophozoites, and the level of inhibition varied according to the assayed concentrations and incubation times. Acacia seyal honey had completely stopped motility of E. histolytica trophozoites at a concentration ≤ 50 µg/ml after incubation for 72 h. Ziziphus spina-christi, Acacia seyal, and Acacia nilotica honeys had completely inhibited the growth of Giardia lamblia trophozoites at concentration ≤ 200 µg/ml after 72 h. These inhibitory activities were similar to that of Metronidazole? which showed IC₅₀ = 0.27. The mammalian cytotoxicity of these honeys against normal Vero cell line which determined by applying MTT method verified the nontoxicity of the examined honeys. Also the proximate composition of the samples indicated compliance with the natural honey standards. The findings of the study indicate the need for in vivo studies and further investigations to identify active principles with antiprotozoal activities from natural honeys.     

Flexible chain conformation of (1 → 3)-β-d-glucan from Poria cocos sclerotium in NaOH/urea aqueous solution

A water-insoluble polysaccharide (PCS3-II) extracted from sclerotium of Poria cocos was identified as a linear (1 → 3)-β-d-glucan by ¹³C NMR and gas chromatography. Aqueous 0.5 M NaOH/0.2 M urea was a good solvent for PCS3-II and the dependence of intrinsic viscosity ([η]) on weight-average molecular weight (M_w) was established in the M_w range from 7.68 × 10⁴ to 5.14 × 10⁵ to be [η] = 3.39 × 10^?2 $M_{\text{W}}^{0.62}{\text{cm}}^3{\text{g}}^{-1}$ at 25 °C by using laser light scattering and viscometry. The chain conformation parameters of PCS3-II in the 0.5 M NaOH/0.2 M urea solution was 2.3 (± 0.3) nm for persistence length (q), 580 g mol^?1 nm^?1 for molar mass per unit contour length (M_L), 0.8 (± 0.2) nm for the diameter of the chain (d) and 3.63 for limited characteristic ratio (C_∞). The results revealed, for the first time, that PCS3-II existed as a flexible chain in 0.5 M NaOH/0.2 M urea aqueous solution.     

The novel protein BboRhop68 is expressed by intraerythrocytic stages of Babesia bovis

The apical complex of intracellular hemoparasites contains organelles like micronemes and rhoptries, specialized structures required for adherence and invasion of host cells. Several molecules discharged from rhoptries have been identified from Plasmodium spp., but only a single rhoptry associated protein-1 (RAP-1) has been characterized from Babesia bovis. In silico search of the B. bovis genome allowed to identifying a sequence homologous to the gene that encodes a P. falciparum rhoptry protein PfRhop148. The intron-less 1830 bp novel gene, predicted a 68 kDa protein, and it was highly conserved among different B. bovis strains and isolates. The deducted protein from the B. bovis T2Bo strain, named BboRhop68, showed two putative transmembrane domains, at least seven B-cell epitopes, and a well conserved DUF501 super family domain. The bborhop68 gene was amplified, analyzed and compared among different B. bovis strains and isolates showing overall high sequence conservation. A fragment of bborhop68 was expressed as a recombinant fusion protein (rBboRhop68). The mice anti-rBboRhop68 serum identified the novel protein in intraerythrocytic trophozoites and merozoites by WB and ELISA, but not in free merozoites. Sera from naturally and experimentally infected bovines also recognized BboRhop68, suggesting that it is expressed and immunogenic during B. bovis infection. Fluorescence microscopy analysis using anti-rBboRhop68 antibodies showed a rod structure associated to trophozoites and merozoites infected erythrocytes, but this pattern of reactivity was not observed in free merozoites. The BboRhop68 was also not detected in ELISA based on solubilized merozoites. Thus, at least three independent lines of evidence support differential expression of BboRhop68 in intraerythrocytic stages of B. bovis and its possible functional role immediately after B. bovis erythrocyte invasion. The results of this work suggest that BboRhop68 could be considered as a novel additional target for developing improved methods to control bovine babesiosis.     

Prevotella bivia as a source of lipopolysaccharide in the vagina

ObjectivesTo compare vaginal lipopolysaccharides (LPS) concentrations between patients with and without bacterial vaginosis (BV), to evaluate the correlation between Prevotella bivia colonization density and LPS concentration, and to determine the impact of LPS on loss of dopamine neurons (DA).MethodsVaginal washes obtained from patients with (n = 43) and without (n = 59) BV were tested for quantity of P. bivia cells using quantitative PCR and for concentrations of LPS using the Limulus Amebocyte Lysate gel clot method. Prevotella bivia, Gardnerella vaginalis and Escherichia coli sonicated cell extracts were also tested for LPS production. DA neuron cells obtained from embryonic day (E) 14.5 pregnant rats were exposed to fluid from eight vaginal washes; tyrosine hydrolase immunoreactive staining was applied for visualization and cell counts.ResultsThe median LPS concentrations were dramatically higher among patients who had symptoms of BV compared to those who did not have symptoms (3235.0 vs 46.4 EU/ml, respectively, P < 0.001); patients who had BV also had much higher colonization densities of P. bivia (0.06 ± 0.36 vs 5.4 ± 2.2 log₁₀ CFU/ml, respectively, P < 0.001).Prevotella bivia cell lysates resulted in a higher LPS concentration (10,713.0 ± 306.6 EU/ml) than either E. coli (4679.0 ± 585.3 EU/ml) or G. vaginalis (0.07 ± 0.01 EU/ml of LPS).The loss of DA neuron was 20–27% in cultures treated with vaginal washes from BV-negative patients and 58–97% in cultures treated with vaginal washes from patients with BV.ConclusionP. bivia produces high LPS concentration, which may create a toxic vaginal environment that damages DA neurons.     

Combined application of botanical formulations and biocontrol agents for the management of Fusarium oxysporum f. sp. cubense (Foc) causing Fusarium wilt in banana

Plant products along with biocontrol agents were tested against Fusarium wilt of banana caused by Fusarium oxysporum f. sp. cubense (Foc). Of the 22 plant species tested, the leaf extract of Datura metel (10%) showed complete inhibition of the mycelial growth of Foc. Two botanical fungicides, Wanis 20 EC and Damet 50 EC along with selected PGPR strains with known biocontrol activity, Pseudomonas fluorescens 1, Pf1 and Bacillus subtilis, TRC 54 were tested individually and in combination for the management of Fusarium wilt under greenhouse and field conditions. Combined application of botanical formulation and biocontrol agents (Wanis 20 EC + Pf1 + TRC 54) reduced the wilt incidence significantly under greenhouse (64%) and field conditions (75%). Reduction in disease incidence was positively correlated with the induction of defense-related enzymes peroxidase (PO) and polyphenol oxidase (PPO). Three antifungal compounds (two glycosides and one ester) in D. metel were separated and identified using TLC, RP-HPLC (Reverse Phase-High Pressure Liquid Chromatography) and mass spectrometry. In this study it is clear that combined application of botanical formulations and biocontrol agents can be very effective in the management of Fusarium wilt of banana.     

Brain fixation for analysis of brain lipid-mediators of signal transduction and brain eicosanoids requires head-focused microwave irradiation: An historical perspective

Endothelin-1 (ET-1) has been reported to mediate prostaglandin (PG) F₂α (PGF₂α)-induced luteolysis. Prostaglandins E (PGE; PGE₁ + PGE₂) are associated with implantation, maternal recognition of pregnancy, and are antiluteolytic and luteotropic in vitro and in vivo. ET-1 increased PGE secretion by bovine luteal tissue in vitro from cows where estrus was not synchronized or when estrus was synchronized with lutalyse and did not affect luteal PGF₂α or progesterone secretion, which does not support the concept that ET-1 is luteolytic or mediates PGF₂α luteolysis. Therefore, the objective of this experiment was to determine whether ET-1 infused every 6 h from 2400 h on day 10–1800 h on day 18 of the ovine estrous cycle either into the interstitial tissue of the ovarian vascular pedicle (IP) or intrauterine (IU) adjacent to the luteal-containing ovary was luteolytic in ewes. Treatments were: Vehicle-IP; Vehicle-IU; ET-1-IP; or ET-1-IU. Weights of corpora lutea differed (P ≤ 0.05) among treatment groups. Weights of corpora lutea at 1800 h on day 18 were: VEH-IP—247 ± 38 mg; VEH-IU—195 ± 31 mg; ET-1-IP—626 ± 74 mg; and ET-1-IU—542 ± 69 mg. Luteal weights on day 18 in ET-1-IP or ET-1-IU-treated ewes did not differ (P ≥ 0.05), but were heavier (P ≤ 0.05) than in the Vehicle-IP or Vehicle-IU treatment groups which did not differ (P ≥ 0.05). Profiles of progesterone in jugular venous plasma of both control groups treated with Vehicle-IP or Vehicle-IU were lower (P ≤ 0.05) than in ewes treated with ET-1-IP or ET-1-IU, which did not differ (P ≥ 0.05) between ET-1-IP or ET-1-IU treatment groups. Treatment with ET-1-IP or ET-1-IU increased (P ≤ 0.05) the PGE:PGF₂α ratio when compared to the Vehicle-IP or Vehicle-IU treatment groups, which did not differ (P ≥ 0.05) between each other. In summary, ET-1 prevented the decrease in luteal weights and the decline in progesterone, but increased the PGE:PGF₂α ratio when compared to controls. Therefore, it is concluded that ET-1 is not luteolytic in ewes, but instead may be luteotropic or antiluteolytic by altering uterine secretion of the PGE:PGF₂α ratio, since PGE₁ or PGE₂ are luteotropic in vitro and in vivo, PGE₁ or PGE₂ prevent PGF₂α-induced luteolysis in vitro and in vivo, and PGE₁ and PGE₂ increase two-fold in ewe endometrium to prevent luteolysis during early pregnancy.     

UVB-induced DNA damage, generation of reactive oxygen species, and inflammation are effectively attenuated by the flavonoid luteolin in vitro and in vivo

Ultraviolet (UV) radiation induces DNA damage, oxidative stress, and inflammatory processes in human keratinocytes resulting in skin inflammation, photoaging, and photocarcinogenesis. The flavonoid luteolin is one of the most potent antioxidative plant polyphenols. We investigated the UV protective and antioxidant properties of luteolin in human keratinocytes in vitro, ex vivo, and in vivo. Spectrophotometric measurements revealed extinction maxima of luteolin in the UVB and UVA range. UV transmission below 370 nm was < 10%. In human skin, luteolin effectively reduced the formation of UVB-induced cyclobutane pyrimidine dimers. The free radical scavenging activity of luteolin was assessed in various cell-free and cell-based assays. In the cell-free DPPH assay the half-maximal effective concentration (EC₅₀) of luteolin (12 μg/ml) was comparable to those of Trolox (25 μg/ml) and N-acetylcysteine (32 μg/ml). In contrast, in the H₂DCFDA assay performed with UVB-irradiated keratinocytes, luteolin (EC₅₀ 3 μg/ml) was much more effective compared to Trolox (EC₅₀ 12 μg/ml) and N-acetylcysteine (EC₅₀ 847 μg/ml). Luteolin also inhibited both UVB-induced skin erythema and the upregulation of cyclooxygenase-2 and prostaglandin E₂ production in human skin via interference with the MAPK pathway. These data suggest that luteolin may protect human skin from UVB-induced damage by a combination of UV-absorbing, DNA-protective, antioxidant, and anti-inflammatory properties.     

Potential biocontrol activity of a strain of Pichia guilliermondii against grey mold of apples and its possible modes of action

The efficacy of Pichia guilliermondii strain M8 against Botrytis cinerea on apples was evaluated under storage conditions, and its possible modes of action were investigated both in vitro and in vivo experiments. After storage at 1 °C for 120 days, M8 reduced grey mold incidence from 45.3% (control) to 20.0%. In apple juice medium (AJM) and in wound-inoculated apples, M8 at 10⁹ and 10⁸ cells ml⁻¹ inhibited the spore germination of B. cinerea and the grey mold development. When co-culturing B. cinerea in vitro or in vivo in the presence of the yeast, neither inactivated cells nor culture filtrate of the yeast had any effect on spore germination or germ tube elongation. In AJM, the spore germination was significantly recovered by the addition of 1% glucose, sucrose and fructose, or 0.5% and 1% of (NH₄)₂SO₄, phenylalanine and asparagine. When the pathogen and the yeast were co-incubated in apple wounds with addition of the same nutrients, the inhibition of rots was significantly reduced by the supplemental nutrients. Light microscopy revealed that the yeast strongly adhered to the hyphae and spores of B. cinerea. M8 produced hydrolytic enzymes, including β-1,3-glucanase and chitinases in minimal salt media with different carbon sources. Pretreatment with M8 at 10⁸ cells ml⁻¹ followed by washing, significantly reduced grey mold lesions, suggesting an induction of defense responses. Direct attachment, competition for nitrogen and carbon sources, secretion of hydrolytic enzymes and induction of host resistance play a role in the biocontrol mechanism of P. guilliermondii M8 against B. cinerea.     

Physiological characteristics of several rumen protozoa grown in vitro with observations on within and among species variation

When fed equal amounts of substrate, two Epidinium caudatum clone cultures of markedly different size produced similar volumes of microbial protoplasm. Addition of up to 50% volume of 72 h culture medium had no inhibitory effects on growth of Epidinium. Two clone cultures of Epidinium caudatum from Australia had longer generation times and showed less substrate attachment when compared to Ohio clones of this same species. Substitution of alfalfa for orchardgrass in the normal substrate increased Epidinium concentrations, while feeding only ground orchardgrass or alfalfa resulted in a marked decrease or disappearance of the protozoa. Eudiplodinium impalae, isolated from rumen contents of a steer in Australia, was successfully cultured, with generation times for this species averaging 11.3 h. Reducing particle size of the substrates by ball-milling was detrimental for growth of Entodinium and Epidinium; however, Eudiplodinium increased in concentration. Significant concentration differences were observed among six clone cultures of Epidinium obtained from Europe. A generation time of 18.7 h was measured for Enoploplastron triloricatum when the culture was transferred every 12 h. Lowering the incubation temperature to 34 °C completely inhibited protozoal growth of Epidinium and Entodinium exiguum after 12 days, but not for Entodinium caudatum.     

Antileishmanial activity of a guaianolide from Tanacetum parthenium (L.) Schultz Bip

Leishmaniasis is one of the major infectious diseases affecting the poorest regions of the world. The present study evaluated the antileishmanial activity of a guaianolide purified from the hydroalcoholic extract of aerial parts of Tanacetum parthenium (L.) Schultz Bip. The isolated compound showed activity against the promastigote form of Leishmania amazonensis, with 50% inhibition (IC₅₀) of cell growth at a concentration of 2.6 μg/ml. For the intracellular amastigote form, this guaianolide reduced by 10% the survival index of parasites in macrophages when it was used at 20.0 μg/ml. The selective index (SI) ratio (CC₅₀ for J774G8 cells/IC₅₀ for protozoans) was 385, showing that it is more selective against the parasite than mammalian cells. Morphological alterations of protozoans treated with IC₅₀ included changes in size, shape, and structure (more than one nucleus and flagellum) under both light and scanning electron microscopies.     

Field evaluation of the MM3-SERO ELISA for detection of anti-Fasciola IgG antibodies in milk samples from individual cows and bulk milk tanks

We carried out a field evaluation of the MM3-SERO ELISA for the diagnosis of Fasciola hepatica infection, by analysing serum and milk samples from individual cows and samples from bulk milk tanks. The diagnostic performance of the assay was assessed with serum samples from all 257 cows in eight fluke-free herds, and 240 cows with natural fasciolosis (diagnosed in vivo and/or post-mortem). Assay performance for individual milk samples was determined by analysis of paired serum and milk samples from 947 lactating cows from 33 F. hepatica-infected farms. The diagnostic usefulness of the assay for bulk tank milk was evaluated by analysis of bulk milk from infected (33) and non-infected (35) farms. For serum samples, the sensitivity, specificity and diagnostic accuracy of the assay were respectively 99.2% (95% CI: 97.0%–99.9%), 100% (95% CI: 98.6%–100%) and 0.997 (95% CI: 0.987–1.000). The only two infected animals in which serum antibodies were not detected had very low parasitic burdens (with only 2 and 3 flukes observed). The performance of the MM3 SERO ELISA for individual milk samples was similar to that for serum samples, and the stepwise linear regression revealed a strong correlation between the results for the milk samples and the serum samples (R² = 0.84; p < 0.001). The agreement between results obtained with pairs of serum and milk samples was very high: there was matching classification in 96% (910/947) of paired samples (kappa = 0.92; p < 0.001). Individual milk samples may therefore be used, instead of serum samples, in the MM3-SERO ELISA, for reliable detection of seropositive cows. Testing bulk tank milk samples enabled detection of infected herds, even when the within-herd prevalence of infection was as low as 12%. We conclude that the MM3-SERO ELISA is a sensitive and highly specific test for serodiagnosis of bovine fasciolosis, and can be used with individual samples of either serum or milk. Use of the assay with bulk milk samples enables estimation of the within-herd prevalence of infection.