Development and use of real-time PCR to detect and quantify Mycoplasma haemocanis and “Candidatus Mycoplasma haematoparvum” in dogs

Two canine haemoplasma species have been recognised to date; Mycoplasma haemocanis (Mhc), which has been associated with anaemia in splenectomised or immunocompromised dogs, and “Candidatus Mycoplasma haematoparvum” (CMhp), recently described in an anaemic splenectomised dog undergoing chemotherapy. The study aim was to develop quantitative real-time PCR assays (qPCRs) incorporating an endogenous internal control to detect Mhc and CMhp and to apply these assays to DNA samples extracted from canine blood collected in Northern Tanzania (n = 100) and from dogs presented to a Trinidadian veterinary hospital (n = 185).QPCRs specific for Mhc and CMhp were designed using 16S rRNA gene sequence data, and each was duplexed with an assay specific for canine glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The assays detected ≤10 copies of a sequence-specific haemoplasma plasmid per reaction and neither assay showed cross-reactivity with 10⁶ copies of the sequence-specific plasmid from the non-target canine haemoplasma species.Nineteen of the 100 Tanzanian samples (19%) were positive for Mhc alone and one (1%) was dually infected. One Trinidadian sample was negative for canine GAPDH DNA and was excluded from the study. Of the 184 remaining Trinidadian samples, nine (4.9%) were positive for Mhc alone, five (2.7%) for CMhp alone, and two (1.1%) dually infected.This is the first report of canine haemoplasma qPCR assays that use an internal control to confirm the presence of amplifiable sample DNA, and their application to prevalence studies. Mhc was the most commonly detected canine haemoplasma species.     

Yersinia enterocolitica in sheep - a high frequency of biotype 1A

Background

Pigs are regarded as the main reservoir for human pathogenic Yersinia enterocolitica, which is dominated by bioserotype 4/O:3. Other animals, including sheep, have occasionally been reported as carriers of pathogenic strains of Y. enterocolitica. To our knowledge, this is the first study performed in the Nordic countries in which the presence of Y. enterocolitica in sheep is investigated.

Methods

Tonsils and faecal samples collected from sheep slaughtered on the island Gotland (Sweden) from September 2010 through January 2011 were analysed for presence of Y. enterocolitica. In an attempt to maximize recovery, several cultural strategies were applied. Various non-selective media were used and different temperatures and durations of the enrichment were applied before subculturing on Cefsulodin Irgasan Novobiocin (CIN) agar. Presumptive Y. enterocolitica colonies were subjected to urease, API 20E and agglutination test. Yersinia enterocolitica isolates were biotyped, serotyped, and tested for pathogenicity using a TaqMan PCR directed towards the ail-gene that is associated with human pathogenic strains of Y. enterocolitica.

Results

The samples collected from 99 sheep yielded 567 presumptive Y. enterocolitica colonies. Eighty urease positive isolates, from 35 sheep, were identified as Y. enterocolitica by API 20E. Thirty-four of 35 further subtyped Y. enterocolitica isolates, all from faecal samples, belonged to biotype 1A serotype O:5, O:6. O:13,7 and O:10. One strain was Yersinia mollaretii serotype O:62. No human pathogenic strains of Y. enterocolitica were found in the investigated sheep. Other species identified were Y. kristensenii (n = 4), Y. frederiksenii/intermedia (n = 3), Providencia rettgeri (n = 2), Serratia marcescens (n = 1) and Raoultella ornithinolytica (n = 1).

Conclusions

This study does not support the hypothesis that sheep play an important role in transmission of the known human pathogenic Y. enterocolitica in the studied geographical region. However, because there are studies indicating that some strains of Y. enterocolitica biotype 1A may cause disease in humans, the relative importance of sheep as carriers of human pathogenic strains of Y. enterocolitica remains unclear. Tonsils do not appear to be favourable sites for Y. enterocolitica biotype 1A in sheep.     

Development of qPCR assays to monitor the ability of <Emphasis Type="Italic">Gliocladium catenulatum</Emphasis> J1446 to reduce the cereal pathogen <Emphasis Type="Italic">Fusarium graminearum</Emphasis> inoculum in soils

A qPCR approach was developed to specifically monitor in soils Fusarium graminearum, the main agent responsible for Fusarium Head Blight, and the biocontrol agent Gliocladium catenulatum J1446 (Prestop®). For both fungi, the amplification efficacy of standard curves obtained by mixing pure fungal DNA and soil background DNA was high (qPCR efficacy>96% with R²?>?0.97) with a linear range from 10^?3 ng to 10 ng/μL. Our qPCR method allowed quantifying down to 1 μg of F. graminearum and G. catenulatum J1446 mycelium per g of soil. The strong correlation observed between fungal biomass and quantified DNA (R²?=?0.9927 and 0.9356 for F. graminearum and G. catenulatum J1446, respectively) supported the use of the primers to monitor both fungi in soils. Under our experimental conditions, the ability of Prestop® to reduce F. graminearum growth was significantly higher in autoclaved soil compared to living soils, suggesting that there is an antagonistic effect of the soil microbial communities. In contrast, G. catenulatum J1446 growth was mostly not affected by the presence of F. graminearum and was able to persist in both autoclaved and living soils after 15 days of incubation. These results indicate that our qPCR approach may be used to assess the success of soil colonization by a biocontrol agent and its control efficacy by monitoring the dynamics of the BCA and the targeted pathogen in soil.     

Nitrogen and carbon mineralization of surface-applied and incorporated winter wheat and peanut residues

Laboratory incubation experiments were conducted to study the C and N mineralization dynamics of crop residues (fine roots and straw) of the two main crops (winter wheat and peanut) in the Chinese Loess Plateau under different ways of incorporation. The C mineralization patterns of the soil amended with winter wheat residues differed greatly, and the highest C mineralization was observed in the treatment with winter wheat straw incorporated (39% of the total added C mineralized). The way of straw placement had only a minor effect on the pattern of C mineralization for peanut. Generally, winter wheat residues showed a stronger immobilization than peanut residues during the incubation period, without any net N release. Winter wheat straw incorporated showed the strongest N immobilization with 35 mg kg⁻¹ (equivalent to 27% of added N) immobilized at the eighth week. This study indicated that retaining crop residues at the soil surface in the dry land soils of the Chinese Loess Plateau is beneficial for C sequestration. It also showed that N immobilization occurs only during a limited period of time, sufficient to prevent part of the mineral N pool from leaching, and that net N mineralization can be expected during the subsequent cropping season, thus enhancing synchronization of N supply and demand.     

La laccase acide des champignons supérieurs. Relation entre la présence de cet enzyme et les conditions de développement chez Agaricus campestris

Résumé Il a été montré que les champignons supérieurs produisent une oxydase qui catalyse l'oxydation des polyphénols, de la p. phénylènediamine et de l'acide ascorbique en milieu acide. D'après la nature des subtrats oxydés cet enzyme s'apparente à la laccase. Les différentes valeurs du pH optimum des enzymes provenant d'espèces ces diverses et qui s'étagent entre 3,5 et 7,2 pourraient s'interpréter par la production, dans chaque espèce étudiée, d'un mélange en proportions variées de deux enzymes très voisins: une laccase normale et une laccase acide qui ne se sépareraient pas au cours du traitement de purification mis en oeuvre. Le pH optimum de la laccase produite par le mycélium d'A. campestris cultivé dans les conditions habituelles est 7,0, mais, lorsque le mycélium est cultivé sur certains milieux synthétiques, le pH optimum de la laccase synthétisée est 5,6.Ainsi, la production de laccase acide n'apparaît pas comme une propriété spécifique, elle est en rapport avec la composition du milieu de culture.

---

Summary It is shown, that in higher fungi an oxydase is produced which catalyses the oxydation of polyphenols, of p-phenylene-diamine and ascorbic acid in an acid medium.From the nature of substrates this enzyme is closely related to laccase. Different species of fungi show different values of the pH-optimum, between 3,5 and 7,2; this could be interpreted by assuming that every species contains a mixture of two related enzymes: a normal laccase and an acid laccase which could not be separated during the purification.The pH optimum of the laccase ofAgaricus campestris, cultivated in the usual medium, is 7,0; after cultivation in certain synthetic medium the pH optimum of the formed laccase is 5,6. Therefore the formation of an acid laccase is not a specific property but appears to be related with the composition of the culture medium.

---

Zusammenfassung Es wurde gezeigt, dass höhere Pilze Oxydase erzeugen, die die Oxydation der Polyphenole, des p-Phenylendiamins und der Ascorbinsäure in saurer Lösung katalysieren. Die Natur der Substrate zeigt an, dass das Enzym mit der Laccasse verwandt ist.Das pH-Optimum des Enzyms ist je nach der Pilzart verschieden, und liegt zwischen den Werten von pH 3,5 und 7,3; es wäre möglich die Verschiedenheit dieser Werte dahin zu deuten, dass es sich je nach der Pilzart um ein Gemisch von zwei sich sehr nahe stehenden Enzymen handelt: einer normalen und einer sauren Laccase, die durch Reinigung nicht trennbar sind.Die Laccase, die — durch das Myzel vonAgaricus campestris gezüchtet — auf den üblichen Nährlösungen gebildet wird, besitzt eine optimale Aktivität bei pH 7; diese optimale Aktivität liegt bei pH 5,6 wenn das Myzel auf synthetischen Nährlösungen wächst.Die Bildung der sauren Laccase ist also in diesem Fall keine spezifische Eigenschaft der Pilzart, sondern ist durch die Zusammensetzung der Nährlösung bestimmt.

     

Characterization of nitrogen dynamics in a pasture soil by electromagnetic induction

The aim of this study was to investigate how electromagnetic induction can be used to improve the characterization of N dynamics in a 1.2 ha pasture. The soil apparent electrical conductivity (ECa) was measured by electromagnetic induction using an EM38DD. At 116 locations, soil samples were taken according to a clustered sampling design, three times during one winter, and analyzed for the NO₃^––N content in the topsoil (0–60 cm). Management zones were delineated using a fuzzy k-means classification of the interpolated ECa measurements. Two ECa zones were found, reflecting mainly differences in soil texture. Since the mean NO₃^––N content was different for the two ECa zones (24 and 65 kg/ha in November 2002), the residuals were interpolated using stratified simple kriging. This allowed evaluating the NO₃^– dynamics during the winter in both zones; one ECa zone showed a higher risk for NO₃^– losses than the other calling for a site-specific N management. As a validation, NO₃^––N was interpolated using ordinary kriging without stratification. This resulted in similar zones confirming the usefulness of the ECa measurements to assess N-specific management zones, even within small fields.     

A sequence in M13 phage detects hypervariable minisatellites in human and animal DNA

The term "DNA fingerprint" has been used to describe the extensive restriction fragment length polymorphism associated with hypervariable minisatellites present in the human genome. Until now, it was necessary to hybridize Southern blots to specific probes cloned from human genomic DNA in order to obtain individual-specific restriction patterns. The present study describes the surprising finding that the insert-free, wild-type M13 bacteriophage detects hypervariable minisatellites in human and in animal DNA, provided no competitor DNA is used during hybridization. The effective sequence in M13 was traced to two clusters of 15-base pair repeats within the protein III gene of the bacteriophage. This unexpected use of M13 renders the DNA fingerprinting technology more readily available to molecular biology laboratories.     

Highlighting the Biotechnological Potential of Deep Oceanic Crust Fungi through the Prism of Their Antimicrobial Activity

Among the different tools to address the antibiotic resistance crisis, bioprospecting in complex uncharted habitats to detect novel microorganisms putatively producing original antimicrobial compounds can definitely increase the current therapeutic arsenal of antibiotics. Fungi from numerous habitats have been widely screened for their ability to express specific biosynthetic gene clusters (BGCs) involved in the synthesis of antimicrobial compounds. Here, a collection of unique 75 deep oceanic crust fungi was screened to evaluate their biotechnological potential through the prism of their antimicrobial activity using a polyphasic approach. After a first genetic screening to detect specific BGCs, a second step consisted of an antimicrobial screening that tested the most promising isolates against 11 microbial targets. Here, 12 fungal isolates showed at least one antibacterial and/or antifungal activity (static or lytic) against human pathogens. This analysis also revealed that Staphylococcus aureus ATCC 25923 and Enterococcus faecalis CIP A 186 were the most impacted, followed by Pseudomonas aeruginosa ATCC 27853. A specific focus on three fungal isolates allowed us to detect interesting activity of crude extracts against multidrug-resistant Staphylococcus aureus. Finally, complementary mass spectrometry (MS)-based molecular networking analyses were performed to putatively assign the fungal metabolites and raise hypotheses to link them to the observed antimicrobial activities.