Quantification of Mycobacterium avium subspecies in pig tissues by real-time quantitative PCR

Background

Mycobacterioses in animals cause economical losses and certain Mycobacterium avium subspecies are regarded as potential zoonotic agents. The evaluation of the zoonotic risk caused by M. avium subspecies requires information about the quantities of Mycobacterium strains in infected animals. Because M. avium subspecies in pig tissues are difficult or even impossible to quantify by culturing, we tested the suitability of a culture-independent real-time quantitative PCR (qPCR) assay for this purpose.

Methods

Mycobacterial DNA was extracted from porcine tissues by a novel method and quantified by Mycobacterium genus specific qPCR assay targeting the 16S rRNA gene.

Results

The response of the qPCR assay to the amount of M. avium subspecies avium mixed with porcine liver was linear in the range of approximately log10⁵ to log10⁷Mycobacterium cells per 1 g of liver. The assay was validated with three other M. avium subspecies strains. When the assay was applied to porcine lymph nodes with or without visible lesions related to Mycobacterium avium subspecies infections, around 10⁴–10⁷ mycobacterial genomes per gram of lymph nodes were detected.

Conclusions

The qPCR assay was found to be suitable for the quantification of Mycobacterium avium subspecies in porcine lymph nodes and liver.     

Optimising the management of maize — Grevillea robusta fields in Kenya

The study optimised the management of a Grevillea robusta (A. Cunn.) stand growing in the central highlands of Kenya. The optimisations were conducted separately for even-aged and uneven-aged management system of trees. The management was also optimised with the requirement that maize production under the tree cover must be profitable every year. Technically, the optimisation problems were solved by linking a simulation program with the non-linear optimisation algorithm of Hooke and Jeeves. The simulation program calculated the tree growth, volumes of harvested trees, and maize yields with a given set of management parameters (decision variables). The maize yield predictions and simulated timber yields were converted into gross incomes of which the production costs were subtracted. In even-aged management the objective variable was the soil expectation value with 5% discounting rate. In uneven-aged management the mean annual net income was maximised. The optimal solutions indicated that with both management systems it is optimal to concentrate on wood production. The optimal stand densities were so high that profitable maize production was not possible under the tree cover. The mean annual wood production of the optimal management schedule was more than 50m³ ha^–1. Forcing profitable maize production in the solution decreased the wood production by 57% (even-aged forestry) or 27% (uneven-aged forestry) and net income by 45% (even-aged forestry) or 24% (uneven-aged forestry).This revised version was published online in November 2005 with corrections to the Cover Date.     

In vitro adhesion of an avian pathogenic Escherichia coli O78 strain to surfaces of the chicken intestinal tract and to ileal mucus

The role of fimbria in adherence of an avian pathogenic Escherichia coli (APEC) O78 strain 789 to chicken intestine was studied. Bacterial adhesion to tissue sections representing the regions within the chicken intestinal tract was determined by using immunohistochemical methods. E. coli 789 grown to express the type 1 fimbria adhered efficiently to the crop epithelium, to the lamina propria of intestinal villi, and to the apical surfaces of both the mature as well as the crypt-located enterocytes in intestinal villi, whereas no adhesion to mucus-producing goblet cells was detected. The adhesion was inhibited by mannoside and the role of type 1 fimbriae in the observed adhesion was confirmed with a recombinant strain expressing type 1 fimbriae genes cloned from E. coli and Salmonella enterica. E. coli 789 strain grown to favor AC/I fimbriae expression as well as the recombinant E. coli strain expressing the fac genes adhered to goblet cells but only poorly to the other epithelial sites. E. coli strain 789 as well as S. enterica serovar Typhimurium IR715 and S. enterica serovar Enteriditis TN2 strains were able to multiply in ileal mucus medium. The type 1 fimbria expressing bacteria adhered to the ileal mucus, whereas the AC/I fimbriated strains showed poor adherence to the mucus. The adhesion of E. coli 789 onto the crop epithelium and the follicle associated epithelium of the chicken ileum was efficiently inhibited by an adhesive strain ST1 of Lactobacillus crispatus isolated from chicken, whereas poor inhibition of E. coli adherence was observed with the weakly adhesive L. crispatus strain 134mi. The type 1 fimbriae may be important in colonization of the chicken intestine by APEC and Salmonella.     

Clinical findings,bronchoalveolar lavage fluid cytology and matrix metalloproteinase-2 and -9 in canine pulmonary eosinophilia

We characterized clinical and clinicopathological features, and the involvement of gelatinolytic matrix metalloproteinases (MMP-2 and -9) in canine pulmonary eosinophilia (PE). Study material consisted of 20 PE dogs and 16 healthy beagles. All dogs underwent a similar clinical examination and bronchoalveolar lavage (BAL). Analysis for cell count and differential cell count of BAL fluid (BALF), arterial blood gas analysis before and after BAL, and thoracic radiographs before BAL and after treatment were obtained. Twelve dogs were re-evaluated and six relavaged. MMP-2 and MMP-9 in BALF were analysed by zymography, Western immunoblotting and immunocytochemistry.In the PE dogs, BALF, cell count, number and percentage of eosinophils, and numbers of macrophages, lymphocytes, neutrophils, mast cells and epithelial cells were all significantly elevated. Blood eosinophilia was detected in half of the PE dogs. Three PE dogs had mild hypoxaemia. The BAL procedure had an equal effect on PE and healthy dogs' arterial blood gas values. Bronchointerstitial densities were seen in PE dogs' radiographs. Treatment of PE decreased BALF cell count, eosinophil count and percentage and diminished radiographic changes. Gelatinolytic activity was higher in PE dogs' BALF. BALF macrophages and epithelial cells were the principal sources of the MMP-9.     

Fifty years after deep-ploughing: Effects on yield,roots, nutrient stocks and soil structure

Deep-ploughing far beyond the common depth of 30 cm was used more than 50 years ago in Northern Germany with the aim to break root-restricting layers and thereby improve access to subsoil water and nutrient resources. We hypothesized that effects of this earlier intervention on soil properties and yields prevailed after 50 years. Hence, we sampled two sandy soils and one silty soil (Cambisols and a Luvisol) of which half of the field had been deep-ploughed 50 years ago (soils then re-classified as Treposols). The adjacent other half was not deep-ploughed and thus served as the control. At all the three sites, both deep-ploughed and control parts were then conventionally managed over the last 50 years. We assessed yields during the dry year 2019 and additionally in 2020, and rooting intensity at the year of sampling (2019), as well as changes in soil structure, carbon and nutrient stocks in that year. We found that deep-ploughing improved yields in the dry spell of 2019 at the sandy sites, which was supported by a more general pattern of higher NDVI indices in deep-ploughed parts for the period from 2016 to 2021 across varying weather conditions. Subsoil stocks of soil organic carbon and total plant-available phosphorus were enhanced by 21%–199% in the different sites. Root biomass in the subsoil was reduced due to deep-ploughing at the silty site and was increased or unaffected at the sandy sites. Overall, the effects of deep-ploughing were site-specific, with reduced bulk density in the buried topsoil stripes in the subsoil of the sandy sites, but with elevated subsoil density in the silty site. Hence, even 50 years after deep-ploughing, changes in soil properties are still detectable, although effect size differed among sites.