A proof of concept study to assess the potential of PCR testing to detect natural Mycobacterium bovis infection in South American camelids

Background

Cases of Mycobacterium bovis infection South American camelids have been increasing in Great Britain. Current antemortem immunological tests have some limitations. Cases at post mortem examination frequently show extensive pathology. The feasibility of detecting Mycobacterium bovis DNA in clinical samples was investigated.

Findings

A sensitive extraction methodology was developed and used on nasal swabs and faeces taken post-mortem to assess the potential for a PCR test to detect Mycobacterium bovis in clinical samples. The gross pathology of the studied South American camelids was scored and a significantly greater proportion of South American camelids with more severe pathology were positive in both the nasal swab and faecal PCR tests. A combination of the nasal swab and faecal PCR tests detected 63.9% of all the South American camelids with pathology that were tested.

Conclusions

The results suggest that antemortem diagnosis of Mycobacterium bovis in South American camelids may be possible using a PCR test on clinical samples, however more work is required to determine sensitivity and specificity, and the practicalities of applying the test in the field.     

Diversity of Bartonella species detected in arthropod vectors from animals in Australia

A variety of Bartonella species were detected in two species of ticks and three species of fleas collected from marsupial hosts; brush-tailed bettong or woylie (Bettongia penicillata) and western barred bandicoots (Perameles bougainville) and from a rodent host; Rattus fuscipes in Western Australia. Bartonella species were detected using nested-PCR of the gltA gene and the 16S–23S ribosomal internal transcribed spacer region (ITS), and species were characterized using DNA sequencing of the 16S rRNA, gltA, rpoB, ftsZ genes and the ITS region. Bartonella rattaustraliani and B. coopersplainsensis were detected in Ixodes spp. ticks and fleas (Stephanocircus pectinipes) respectively collected from rodents. Two novel Bartonella species were detected from marsupials; Candidatus Bartonella woyliei n. sp. was detected in both fleas (Pygiopsylla hilli) and ticks (Ixodes australiensis) collected from woylies and Candidatus Bartonella bandicootii n. sp. was detected in fleas (Pygiopsylla tunneyi) collected from western barred bandicoots. Concatenated phylogenetic analysis of all 5 loci clarified the marsupial cluster of Bartonella species in Australia and confirmed the species status of these two Bartonella species in ticks and fleas from woylies and western barred bandicoots, which are classified as threatened species and are vulnerable to extinction.     

Apigenin-induced suicidal erythrocyte death

Apigenin, a flavone in fruits and vegetables, stimulates apoptosis and thus counteracts cancerogenesis. Erythrocytes may similarly undergo suicidal cell death or eryptosis, characterized by cell shrinkage and phosphatidylserine exposure at the cell surface. Triggers of eryptosis include increase of cytosolic Ca(2+) activity ([Ca(2+)](i)), ceramide formation and ATP depletion. The present study explored the effect of apigenin on eryptosis. [Ca(2+)](i) was estimated from Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin V binding, hemolysis from hemoglobin release, ceramide utilizing antibodies, and cytosolic ATP with luciferin-luciferase. A 48 h exposure to apigenin significantly increased [Ca(2+)](i) (≥ 1 μM), increased ceramide formation (15 μM), decreased ATP concentration (15 μM), decreased forward scatter (≥ 1 μM), and increased annexin V binding (≥ 5 μM) but did not significantly modify hemolysis. The effect of 15 μM apigenin on annexin V binding was blunted by Ca(2+) removal. The present observations reveal novel effects of apigenin, i.e. stimulation of Ca(2+) entry, ceramide formation and ATP depletion in erythrocytes with subsequent triggering of suicidal erythrocyte death, paralleled by cell shrinkage and phosphatidylserine exposure.     

NMR and HPLC-UV profiling of potatoes with genetic modifications to metabolic pathways

Metabolite profiling has been carried out to assess the compositional changes occurring in potato tubers after genetic modifications have been made to different metabolic pathways. Most major features in the (1)H NMR and HPLC-UV profiles of tuber extracts have been assigned. About 40 GM lines and controls belonging to 4 groups of samples (derived from cv. Record or cv. Desirée and modified in primary carbon metabolism, starch synthesis, glycoprotein processing, or polyamine/ethylene metabolism) were analyzed. Differences were assessed at the level of whole profiles (by PCA) or individual compounds (by ANOVA). The most obvious differences seen in both NMR and HPLC-UV profiles were between the two varieties. There were also significant differences between two of the four Desirée GM lines with modified polyamine metabolism and their controls. Compounds notably affected were proline, trigonelline, and numerous phenolics. However, that modification gave rise to a very abnormal phenotype. Certain lines from the other groups had several compounds present in significantly higher or lower amounts compared to the control, but the differences in mean values amounted to no more than a 2-3-fold change: in the context of variability in the whole data set, such changes did not appear to be important.     

Decomposition and macroinvertebrates in experimental litter along a secondary chronosequence of tropical montane forest

This paper presents the results of a decomposition experiment performed in a secondary chronosequence of tropical montane cloud forest in Mexico. The experiment was designed to explore whether the age of the forest influences the decomposition process and macroinvertebrate community independently of the quality of the decomposition resources. Fresh Pinus chiapensis needles and Persea americana leaves were set to decompose in each of four successional stages (15, 45, 75 and 100 years old). Results do not support the hypothesis that decomposition rate declines with increasing nutrient deficiency as forest succession proceeds. However, the chemical composition in decomposing leaves differed between successional stages. Higher availability of Ca in the 15-year-old forest appears to promote a positive feedback in the release of this nutrient from Persea americana leaves. Additionally, in old forests, a soil community that is more capable of breaking down recalcitrant material (acid detergent lignin) appears to have developed compared to early successional stages. The diversity of macroinvertebrates and abundance of predatory (Aranea and Diplura), detrivorous (Diplopoda) and geophagous (Enchytaeidae) taxa were different between boxes placed in different successional stages. We conclude that the decomposition and associated biota differ between successional stages. Apart from differences in litter quality, other factors associated with the age of the forest, such as small differences in soil temperature and long-lasting effects of disturbance, may also play influential roles.     

Determination of organic and inorganic carbon, δ13C,and nitrogen in soils containing carbonates after acid fumigation with HCl

In carbonate‐containing soils a reliable determination of organic C requires a method that effectively separates organic and inorganic C without altering the organic matter. This study was conducted to determine whether HCl vapor completely removes carbonates even in dolomite‐rich soils and to what extent a widely used acid‐fumigation method has to be modified for humus‐rich soils. Furthermore, it was tested whether HCl fumigation alters organic‐C content. Since C and N parameters are often analyzed simultaneously we also tested the influence of acid‐vapor treatment on N content and on δ¹³C of soil organic matter. We applied fumigation with 37% HCl for 8 and 32 h using 9 carbonate‐containing soil samples. Inorganic C ranged from 7 to 124 and organic C from 9 to 267 g kg^–1. The maximum contents of dolomite and calcite were 940 and 640 g kg^–1, respectively. A time of 8 h was enough to completely remove all carbonates. Neither the content nor the δ¹³C of organic C were significantly affected by fumigation. In contrast, N contents were altered by acid treatment. Based on these results and on our experience in analyzing more than 1000 soil samples, a recommended procedure for acid fumigation of carbonate‐containing soils with a wide range of organic‐ and inorganic‐C contents was derived. Samples pretreated in this way can be analyzed reliably for their organic‐C content and δ¹³C. Furthermore, N and inorganic‐C contents can be determined with a quality sufficient for many purposes.