Prevalence and distribution of parasites and pathogens of Triatominae from Argentina, with emphasis on Triatoma infestans and Triatoma virus TrV

Chagas’ disease is the most important endemic arthropod-zoonosis in Argentina with an estimated 1.6 million people infected with the causative agent Trypanosoma cruzi. Triatoma infestans is the main vector of Chagas’ disease in Argentina. A survey for parasites and pathogens of Triatominae was conducted from August 2002 to February 2005. Collections of insects were made in domiciles, peridomiciles, and in the natural habitats of the Triatominae. Insects from these collections were dissected and their organs and tissues examined for flagellates. Frass from these insects was collected and examined for detection of the entomopathogenic virus Triatoma virus (TrV) using AC-ELISA and PCR. Triatominae belonging to four species, T. infestans (n = 1646), Triatoma guasayana (n = 4), Triatoma platensis (n = 1) and Triatoma sordida (n = 5) were collected from 62 sites located in 13 provinces of Argentina. Triatoma virus and two protozoan species, Blastocrithidia triatomae and T. cruzi, the etiological agent of Chagas disease, were found infecting Triatominae. The total prevalence of TrV in 1646 T. infestans analyzed by ELISA was 9.66% (159/1646) from 7 to 13 provinces where collections were made. Triatoma virus positive triatomines were found in 17 of 62 populations when examined by AC-ELISA but in 38 of 62 populations when PCR was used for detection. The prevalence of B. triatomae in T. infestans was 0.43% (7/1646), while the prevalence of T. cruzi was 1.3% (21/1646). This is the first study on the diversity, distribution and prevalence of flagellated protozoa and TrV of Triatominae in endemic Chagas’ disease regions of Argentina.     

Cold activity of Beauveria and Metarhizium, and thermotolerance of Beauveria

Heat and cold are environmental abiotic factors that restrict the use of entomopathogenic fungi as agents for biological control of insects. The thermotolerance and cold activity of 60 entomopathogenic fungal isolates, including five species of Beauveria and one isolate of Engyodontium albus (=Beauveria alba) were examined as to tolerance of temperatures that might be encountered during field use. In addition, cold activity of eight Metarhizium spp. isolates was evaluated. The isolates were from various geographic regions, arthropod hosts or substrates. High variability in conidial thermotolerance was found among the Beauveria spp. isolates after exposure to 45 °C for 2 h, as evidenced by low (0-20%), medium (20-60%), or high germination (60-80%). The thermal death point (0% germination) for three rather thermotolerant B. bassiana isolates (CG 138, GHA and ARSEF 252) was 46 °C for 6 h. At low temperatures (5 °C), with few exceptions (e.g. CG 66, UFPE 479, CG 227, CG 02), most of the B. bassiana isolates germinated well (ca. 100%). On the other hand, only one isolate of Metarhizium sp. was cold-active (i.e. ARSEF 4343 from Macquarie Island, 54.4°S, Australia). This probably is a M. frigidum isolate. The E. albus isolate (UFPE 3138) was the most susceptible isolate to both heat and cold stress. Isolates ARSEF 252 and GHA of B. bassiana, on the other hand, presented exceptionally high thermotolerance and cold activity. Some isolates with high cold activity, however, were thermosensitive (e.g. ARSEF 1682) and others with high thermotolerance had low cold activity (e.g. CG 227). An attempt to correlate the latitude of origin with thermotolerance or cold activity indicated that B. bassiana isolates from higher latitudes were more cold-active than isolates from nearer the equator, but there was not a similar correlation for heat.     

Rapid Detection and Identification of Wuchereria bancrofti,Brugia malayi,B. pahangi,and Dirofilaria immitis in Mosquito Vectors and Blood Samples by High Resolution Melting Real-Time PCR

A simple, rapid, and high-throughput method for detection and identification of Wuchereria bancrofti, Brugia malayi, Brugia pahangi, and Dirofilaria immitis in mosquito vectors and blood samples was developed using a real-time PCR combined with high-resolution melting (HRM) analysis. Amplicons of the 4 filarial species were generated from 5S rRNA and spliced leader sequences by the real-time PCR and their melting temperatures were determined by the HRM method. Melting of amplicons from W. bancrofti, B. malayi, D. immitis, and B. pahangi peaked at 81.5±0.2℃, 79.0±0.3℃, 76.8±0.1℃, and 79.9±0.1℃, respectively. This assay is relatively cheap since it does not require synthesis of hybridization probes. Its sensitivity and specificity were 100%. It is a rapid and technically simple approach, and an important tool for population surveys as well as molecular xenomonitoring of parasites in vectors.     

The energy density of jellyfish: Estimates from bomb-calorimetry and proximate-composition

Two techniques are described to calculate energy densities for the bell, gonad and oral arm tissues of three scyphozoan jellyfish (Cyanea capillata, Rhizostoma octopus and Chrysaora hysoscella). First, bomb-calorimetry was used, a technique that is readily available and inexpensive. However, the reliability of this technique for gelatinous material is contentious. Second, further analysis involving the more labour intensive proximate-composition analysis (protein, fat and carbohydrate) was carried out on two species (C. capillata and R. octopus). These proximate data were subsequently converted to energy densities. The two techniques (bomb-calorimetry and proximate-composition) gave very similar estimates of energy density. Differences in energy density were found both amongst different species and between different tissues of the same species. Mean (± S.D.) energy density estimates for whole animals from bomb-calorimetry were 0.18 ± 0.05, 0.11 ± 0.04, and 0.10 ± 0.03 kJ g wet mass^− 1 for C. capillata, R. octopus, and C. hysoscella respectively. The implications of these low energy densities for species feeding on jellyfish are discussed.     

Structural characterization and protective effect against murine sepsis of fucogalactans from Agaricus bisporus and Lactarius rufus

Fucogalactans from edible Agaricus bisporus (RFP-Ab) and wild Lactarius rufus (RFP-Lr) mushrooms were obtained on aqueous extraction followed by purification. RFP-Ab had M_w 43.8 × 10⁴ g mol⁻¹ and RFP-Lr M_w 1.4 × 10⁴ g mol⁻¹. RFP-Lr had a (1 → 6)-linked α-d-Galp main-chain partially substituted at O-2 by nonreducing end-units of α-l-Fucp (29%). While RFP-Ab had a similar main chain, it was partially substituted at O-2 by nonreducing end-units of α-l-Fucp (2.8%) and β-d-Galp (14.5%), and partially methylated at HO-3. Both RFP-Lr and RFP-Ab were tested in mice against polymicrobial sepsis. Lethality rate, myeloperoxidase (MPO) activity and cytokine levels were determined. It was observed a reduction in late mortality rate by 62.5% and 50%, respectively, prevention of neutrophil accumulation in ileum and decreasing in TNF-α and IL-1β serum levels.     

Flavonol tetraglycosides from fruits of Styphnolobium japonicum (Leguminosae) and the authentication of Fructus Sophorae and Flos Sophorae

The dried fruits and seeds of Styphnolobium japonicum (L.) Schott (syn. Sophora japonica L.) are used in traditional Chinese medicine and known as Fructus Sophorae or Huai Jiao. The major flavonoids in these fruits and seeds were studied by LC-MS and other spectroscopic techniques to aid the chemical authentication of Fructus Sophorae. Among the flavonoids were two previously unreported kaempferol glycosides: kaempferol 3-O-β-glucopyranosyl(1 → 2)-β-galactopyranoside-7-O-α-rhamnopyranoside and kaempferol 3-O-β-xylopyranosyl(1 → 3)-α-rhamnopyranosyl(1 → 6)[β-glucopyranosyl(1 → 2)]-β-glucopyranoside, the structures of which were determined by NMR. Two further tetraglycosides were identified for the first time in S. japonicum as kaempferol 3-O-β-glucopyranosyl(1 → 2)[α-rhamnopyranosyl(1 → 6)]-β-glucopyranoside-7-O-α-rhamnopyranoside and kaempferol 3-O-β-glucopyranosyl(1 → 2)[α-rhamnopyranosyl(1 → 6)]-β-galactopyranoside-7-O-α-rhamnopyranoside; the latter was the main flavonoid in mature seeds. The chromatographic profiles of 27 recorded flavonoids were relatively consistent among fruits of similar ages collected from five trees of S. japonicum, and those of maturing unripe and ripe fruits were similar to a market sample of Fructus Sophorae, and thus provide useful markers for authentication of this herbal ingredient. The flower buds (Huai Mi) and flowers (Huai Hua) of S. japonicum (collectively Flos Sophorae) contained rutin as the main flavonoid and lacked the flavone glycosides that were present in flower buds and flowers of Sophora flavescens Ait., reported to be occasional substitutes for Flos Sophorae. The single major flavonoid in fruits of S. flavescens was determined as 3′-hydroxydaidzein.     

Characterization of Xenorhabdus isolates from La Rioja (Northern Spain) and virulence with and without their symbiotic entomopathogenic nematodes (Nematoda: Steinernematidae)

Eighteen Xenorhabdus isolates associated with Spanish entomopathogenic nematodes of the genus Steinernema were characterized using a polyphasic approach including phenotypic and molecular methods. Two isolates were classified as Xenorhabdus nematophila and were associated with Steinernema carpocapsae. Sixteen isolates were classified as Xenorhabdus bovienii, of which fifteen were associated with Steinernema feltiae and one with Steinernema kraussei. Two X. bovienii Phase II were also isolated, one instable phase isolated from S. feltiae strain Rioja and one stable phase from S. feltiae strain BZ. Four representative bacterial isolates were chosen to study their pathogenicity against Spodoptera littoralis with and without the presence of their nematode host. The four bacterial isolates were pathogenic for S. littoralis leading to septicemia 24 h post-injection and killing around 90% of the insect larvae 36 h post-injection, except for that isolated from S. kraussei. After 48 h of injection, this latter isolate showed a lower final population in the larval hemolymph (10⁷ instead of 10⁸ CFU per larvae) and a lower larval mortality (70% instead of 95-100%). The virulence of the nematode-bacteria complexes against S. littoralis showed similar traits with a significant insect larvae mortality (80-90%) 5 days post-infection except for S. kraussei, although this strain reached similar of larval mortality at 7 days after infection.     

Effects of native and invasive macroalgal canopies on composition and abundance of mobile benthic macrofauna and turf-forming algae

We examined the effects of native kelps, Laminaria longicruris de la Pylaie and L. digitata (Hudson) Lamouroux, and of the invasive alga, Codium fragile ssp. tomentosoides (Van Goor) Silva, on the composition and abundance of mobile benthic macrofauna and of turf algae by measuring the response of these assemblages to experimental removal of the respective macroalgal canopy. From June 2003 to November 2004, we censused macrofauna and measured canopy cover within 4 × 10 m strips of alternating Canopy Intact (control) and Canopy Removed treatments in both a Codium- and a Laminaria-dominated habitat in the rocky subtidal zone of a semi-protected embayment on the Atlantic coast of Nova Scotia, Canada. Macroalgal canopy cover fluctuated seasonally, peaking in September/October (69% cover in 2003; 55% cover in 2004) for Codium and in May 2004 (70% cover) for kelps, and with both canopy types reaching a winter minimum in January 2004 (22 and 28% cover, respectively). In both Codium and Laminaria habitats, significant effects of canopy removal on the overall macrofaunal assemblage were evident only during periods in which canopy cover in the Canopy Intact treatment was ≥ 50%. In the Codium habitat, 4 out of 11 characteristic taxa were more abundant in the Canopy Intact treatment, where taxonomic diversity also was higher. In contrast, 4 out of 11 characteristic taxa in the Laminaria habitat were more abundant in the Canopy Removed treatment and diversity was similar between treatments. Turf algae were sampled in November 2004 and, despite between-treatment differences in the light regime, there was no significant effect of canopy removal. Our results indicate that selection of algal habitats by mobile macrofauna is likely determined by the different shelter and foraging opportunities offered by these morphologically dissimilar ecosystem engineers.     

Efficacy of karanjin and phorbol ester fraction against termites (Odontotermes obesus)

The non-edible oil seeds of Jatropha curcas (physic nut) and Pongamia pinnata (karanja) contain some toxic components (phorbol esters in J. curcas and karanjin in P. pinnata), which may be used as biopesticides. In this study, the active components of J. curcas and P. pinnata oil were extracted and their efficacy against the termites Odontotermes obesus (Rambur), was tested. The phorbol ester fraction of J. curcas and karanjin of P. pinnata oil were found to be effective against termites. A mortality rate of 100% was achieved in 6 h with karanjin and in 12 h with phorbol ester fraction. The LC₅₀ levels of karanjin and phorbol esters fractions were 0.038 and 0.071 g ml⁻¹, respectively, after 24 h at a 95% (0.05) confidence limit.     

Genetic structure of Pyrenophora teres net and spot populations as revealed by microsatellite analysis

The population structure of the fungal pathogen Pyrenophora teres, collected mainly from different regions of the Czech and Slovak Republics, was examined using a microsatellite analyses (SSR). Among 305 P. teres f. teres (PTT) and 82 P. teres f. maculata (PTM) isolates that were collected, the overall gene diversity was similar (? = 0.12 and ? = 0.13, respectively). A high level of genetic differentiation (F_ST = 0.46; P < 0.001) indicated the existence of population structure. Nine clusters that were found using a Bayesian approach represent the genetic structure of the studied P. teres populations. Two clusters consisted of PTM populations; PTT populations formed another seven clusters. An exact test of population differentiation confirmed the results that were generated by Structure. There was no difference between naturally infected populations over time, and genetic distance did not correlate with geographical distance. The facts that all individuals had unique multilocus genotypes and that the hypothesis of random mating could not be rejected in several populations or subpopulations serve as evidence that a mixed mating system plays a role in the P. teres life cycle. Despite the fact that the genetic differentiation value between PTT and PTM (F_ST = 0.30; P < 0.001) is lower than it is between the populations within each form (F_ST = 0.40 (PTT); F_ST = 0.35 (PTM); P < 0.001) and that individuals with mixed PTT and PTM genomes were found, the two forms of P. teres form genetically separate populations. Therefore, it can be assumed that these populations have most likely undergone speciation.     

Identification and differentiation of human schistosomes by polymerase chain reaction

Recent increasing number of travelers, immigrants and foreign workers from schistosomiasis endemic area has thus resulted in the importation of schistosomiasis to non-endemic countries. To avoid ova-induced pathogenicity, sensitive and specific diagnostic means at an early stage of infection are therefore crucial. In this study, we developed polymerase chain reaction (PCR) primers specific for human schistosome species. The PCR products were obtained in a species-specific manner (479 bp, Schistosoma mansoni; 365 bp, S. haematobium; 614 bp, S. japonicum; 303 bp, S. mekongi) and were detectable from 0.01 pg of total worm DNA (S. haematobium, S. japonicum, S. mekongi). The primer sets were also available for multiplex use. Although some difficulties were experienced in amplifying the parasite DNA from the infected animals, schistosome DNA could be detected from one day post infection. The PCR method described herein will therefore be beneficial to detect human schistosomiasis, after some improvements in this method.     

Blood parasites of livestock in certain Regions in Saudi Arabia

Blood samples from camels, sheep, goats and cattle from six Regions in Saudi Arabia were examined for blood parasites. Asir Region camels were disinfected while those of the Eastern, Jazan, Northern Frontiers, Riyadh and Tabouk Regions were infected with Trypanosoma evansi (5–40%), those of Riyadh and the Eastern Regions were infected with Dipetalonema evansi (1–6%) and those of the Eastern, Jazan and Riyadh Regions were infected with Eperythrozoon species (8–20%). Sheep and goats of all tested regions were infected with Theilaria hirci (4–20% and 6–14%, respectively), Theilaria ovis (5–19% and 6–24%, respectively) and Eperthrozoon ovis (2–9% and 2–8%, respectively). Sheep of the Eastern and Northern Frontiers Regions were also infected with Anaplasma ovis (2%) and also those of the Eastern Region were infected with Babesia motasi (4%) as well. Cattle of Asir and Eastern Regions were infected with Anaplasma marginale (1–3.4%) and those of the Eastern, Jazan and Riyadh Regions were infected with Theileria annulata (11.3–25%) and Eperthrozoon wenyoni (1–4%). Moreover, Jazan cattle were infected with Babesia bigemina (6%) and a benign Theileria species (27%). Some of these parasites are recorded in new localities indicating that they are spreading in the country. Also, this is the first report in Saudi Arabia of D. evansi in camels, A. ovis and B. motasi in sheep and A. marginale and B. bigemina in cattle. These parasites may be introduced into the country with infected livestock infested with the vectors of these parasites. The suspected vectors of the detected parasites in Saudi Arabia is discussed. Follow up surveys of blood parasites are recommended to assess their distribution and infection rates in the livestock of all Regions of Saudi Arabia, to make plans for control measures against their vectors.     

Black yeast habitat choices and species spectrum on high altitude creosote-treated railway ties

Polyextremotolerant black yeast-like fungi thrive in moderately hostile environments where they are concomitantly subjected to several types of stress, such as toxicity, scarce nutrient availability, and high or low temperature extremes. Their ability to assimilate alkylbenzenes (toxic environmental pollutants) enhances their growth in harsh conditions, including on railway ties. Samples were collected using cotton swabs, premoistened with physiological saline, from 658 oak and concrete railway ties at six train stations in Turkey at altitudes ranging between 1026 and 1427 m. The samples were inoculated on malt extract agar supplemented with chloramphenicol, and incubated at 26 °C for 4 weeks. Twenty-four samples (3.6 %), 17 from oak and 7 from concrete (5.6 % vs. 2 %; P = 0.02), tested positive for fungi. Exophiala crusticola was found to be the most common species (n = 13), followed by Exophiala phaeomuriformis (n = 7) and Exophiala heteromorpha (n = 4). These results suggest that hydrocarbons, particularly creosote-treated oak woods, support the growth of black yeasts, some of which are opportunists in humans.     

Decomposition and nutrient release in halophytes of a Mediterranean salt marsh

This study dealt with the decomposition and nutrient release from the halophytes Atriplex portulacoides, Arthrocnemum macrostachyum, Limoniastrum monopetalum, and Spartina densiflora, the dominant species in the Castro Marim salt marsh, Portugal. Environmental effects on decomposition were also assessed. The study was carried out for one year using the in situ litterbag technique. S. densiflora showed a lower decomposition rate (k = 0.003 day⁻¹) than the other study species (k = 0.005-0.009). Study species showed similar decomposition patterns, that is, the weight loss mostly occurred during the autumn-winter period (study beginning in November). This indicates that temperature in this period did not hamper the decomposition process. The decomposition rate was positively affected by the initial N concentration (r² = 0.87, P < 0.05) and negatively by the C:N ratio (r² = 0.86, P < 0.05) in decomposing materials. At the end of the study, S. densiflora and L. monopetalum, the species with lower initial N concentrations, retained much higher proportion of initial N (89-109%) than the others (5-14%). Also, S. densiflora with the lowest P concentration retained higher proportion of initial P (48%) than the others (5-20%). Release of K and Mg were also slower from S. densiflora and was associated with their initial low concentration in this species. The lowest Mn release was observed from A. macrostachyum and also in relation to the lowest initial concentration. Our study supports the hypothesis that decomposition patterns of marsh species are mostly associated with differences regarding their morphology and chemical composition. Given the higher resistance of S. densiflora to decomposition, its progressive spreading may result in accumulation of organic detritus overtime in invaded salt marshes.     

Differential in vivo response of soft-shell clam hemocytes against two strains of Vibrio splendidus: Changes in cell structure, numbers and adherence

Host-pathogen interaction models in aquatic species are useful tools for understanding the pathogenicity of diseases in cultured and wild populations. In this study we report the differential in vivo response of soft-shell clam (Mya arenaria) hemocytes against two strains of Vibrio splendidus. Responses were measured 24 h after injecting into the posterior adductor muscle either an endemic wild-type strain (7SHRW) or a strain associated with oyster mortalities (LGP32-GFP). Changes in hemocyte structure (percentage of rounded cells) were assessed microscopically. Changes in adherence and hemocyte numbers were analyzed by flow-cytometric cell counting. Increased percentages of rounded cells were found in response to both strains. However, values from the group infected with LGP32-GFP were significantly higher (p < 0.01) than with 7SHRW. The cell adherence was markedly diminished (p < 0.001) by LGP32-GFP whereas 7SHRW did not change it significantly. Increased numbers of hemocytes (p < 0.001) were induced by LGP32-GFP, while no significant changes were found after infection with 7SHRW. These results show the regulatory capacity of soft-shell clams hemocytes to perform specific responses against different strains of V. splendidus.     

Chemotaxonomy of Hypericum genus from Portugal: Geographical distribution and essential oils composition of Hypericum perfoliatum, Hypericum humifusum, Hypericum linarifolium and Hypericum pulchrum

The geographical distribution and analysis of the essential oils of species from three sections of Hypericum L. (Guttiferae/Clusiaceae/Hypericaceae) from Portugal are presented. Hypericum perfoliatum (section Drosocarpium) grows wild in the centre and south of Portugal; Hypericum humifusum and Hypericum linarifolium are both from section Oligostema, the former occurring throughout the country, while the second is distributed mainly in the north and centre; Hypericum pulchrum (section Taeniocarpium) is confined to the littoral north of Portugal. The essential oils were obtained by distillation–extraction, hydrodistillation and distillation in a modified Marcusson apparatus from the dried aerial parts of the different populations and were analysed by GC and GC–MS. Monoterpene hydrocarbons constituted the main fraction in all oils (43–69%, 53–85%, 28–45% and 48–65% for H. perfoliatum, H. humifusum, H. linarifolium and H. pulchrum, respectively). Sesquiterpene hydrocarbons (2–13%, 6–18%, 21–27% and 16–18%, respectively) and a third fraction of non-terpenic compounds (20–29%, 3–16%, 2–14% and 5–11%, respectively) from the four species attained relatively high amounts in all oils. Within each species, no major differences were detected in the essential oil composition, despite the fact that different locations, phenological phases and extraction methodologies were used. Notwithstanding the dominance of α-pinene in all four species' oils, cluster and principal components analysis on the identified components showed that the range of α-pinene, β-pinene and n-nonane supported a separation of the four species. The essential oil composition of the four species showed some qualitative resemblances, which correlate well with the taxonomical classification based on morphological characters.     

Expression and characterization of the chitinases from Serratia marcescens GEI strain for the control of Varroa destructor, a honey bee parasite

Serratia marcescens GEI strain was isolated from the gut of the workers of Chinese honey bee Apis cerana and evaluated in the laboratory for the control of Varroa destructor, a parasite of western honey bee A. mellifera. The supernatant and the collected proteins by ammonium sulfate from the bacterial cultures showed a strong miticidal effect on the female mites, with 100% mite mortality in 5 days. Heat (100 °C for 10 min) and proteinase K treatment of the collected proteins destroyed the miticidal activity. The improved miticial activity of this bacterial strain on chitin medium indicated the involvement of chitinases. The expressed chitinases ChiA, ChiB and ChiC1 from S. marcescens GEI by recombinant Escherichia coli showed pathogenicity against the mites in the laboratory. These chitinases were active in a broad pH range (5-9) and the optimum temperatures were between 60 and 75 °C. Synergistic effects of ChiA and ChiB on the miticidal activity against V. destructor were observed. The workers of both honey bee species were not sensitive to the spraying and feeding chitinases. These results provided alternative control strategies for Varroa mites, by formulating chitinase agents and by constructing transgenetic honey bees.     

Loop-mediated isothermal amplification assays for identification of antiseptic- and methicillin-resistant Staphylococcus aureus

A method for rapid identification of antiseptic- and methicillin-resistant Staphylococcus aureus (MRSA) based on 3 loop-mediated isothermal amplification (LAMP) assays was developed. LAMP targeting the femB gene identified S. aureus with 100% specificity, and LAMP targeting the mecA gene associated with methicillin resistance identified methicillin-resistant staphylococci with 100% specificity. LAMP targeting the qacA/B gene encoding an efflux pump responsible for antiseptic resistance identified high-acriflavine-resistant (MIC ≥ 100 mg/L) MRSA (92.5% positive) and acriflavine-susceptible (MIC < 25 mg/L) MRSA (100% negative). They were performed under the same reaction conditions within 60 min at 63 °C. The combined LAMP assays will be useful for rapid identification of S. aureus isolates and determination of their antibiotic and antiseptic resistance patterns with regard to methicillin and organic cationic substrates.     

Isolation of Fungi from Heterodera glycines and in vitro Bioassays for Their Antagonism to Eggs

Twenty fungi were assayed in vitro for antagonism to eggs of Heterodera glycines. Eight of the fungi were isolated from cysts or eggs of H. glycines during the current study, one was isolated from Panagrellus redivivus, and eleven were obtained from other researchers or collections. The bioassays were conducted on eggs from nematodes that had been grown monoxenically on excised root tips. Phoma chrysanthemicola, one strain of Verticillium chlamydosporium, and one strain of V. lecanii caused a decrease (P < 0.01, P < 0.05, P < 0.05, respectively) in the number of viable eggs, although no hyphae were observed colonizing live eggs. Trichoderma polysporum infected live eggs but enhanced (P < 0.05) egg survival. Acremonium bacillisporum, Chaetomium sp., Drechmeria coniospora (two strains), Epicoccum sp., Exophiala jeanselmei, Fusarium sp., Neocosmospora vasinfecta, Scytalidium fulvum, Trichoderma harzianum (two strains), V. chlamydosporium (one strain), V. lecanii (three strains), and an unidentified fungus did not measurably affect egg viability, even though hyphae of five of these fungi were seen in live eggs. The bioassay provides a useful step in the selection of a biological control agent for this major nematode pest.     

A multicenter pilot external quality assessment programme to assess the quality of molecular detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae

An external quality assessment (EQA) panel consisting of a total of 13 samples in broncho alveolar lavage (BAL) or transport medium was prepared to assess the proficiency of laboratories in the correct detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae by nucleic acid amplification techniques (NAATs) (6 samples containing various concentrations (4.9-490 inclusion forming units (IFU)/ml) of C. pneumoniae, 5 samples containing various concentrations (20-5000 color-changing units (CCU)/ml) of M. pneumoniae and 2 samples negative for both).Seventy-nine laboratories from 18 countries participated in this EQA study. Sixty-four datasets were returned for C. pneumoniae (n = 5 conventional commercial, n = 10 conventional in-house, n = 4 real-time commercial, n = 43 real-time in-house, and n = 2 SDA). Sixty-seven datasets were obtained for M. pneumoniae (n = 5 conventional commercial, n = 10 conventional in-house, n = 4 real-time commercial, n = 46 real-time in-house, and n = 2 strand displacement amplification (SDA)). For the total panels, correct results per sample varied between 95.3% and 100% for C. pneumoniae and between 53.7% and 95.5% for M. pneumoniae. In general, commercial conventional NAATs showed possible sensitivity issues when compared to conventional in-house NAATs for both organisms. On the other hand, real-time commercial NAATs scored better than real-time in-house assays in terms of sensitivity for both organisms. For C. pneumoniae and M. pneumoniae, 0.8% and 2.2% true false-positive results and 1.9% and 2.0% false positives were reported in the samples spiked with the other organism.Analysis of the data for C. pneumoniae showed that the concentrations used were easily detectable by the vast majority of participants. The percentage of correct qualitative results for M. pneumoniae demonstrated that the concentrations included in this panel proved challenging for a number of participants.