High genetic relatedness among Mycobacterium avium strains isolated from pigs and humans revealed by comparative IS1245 RFLP analysis

Members of the Mycobacterium avium complex cause pig mycobacteriosis and opportunistic human infections. Infections due to environmental mycobacteria are increasing in both industrial and developing countries. Mycobacterium-infected pig carcasses can pass for human consumption due to the poor specificity of meat control by visual detection at the slaughter houses. The genetic relatedness of porcine and human MAC isolates in Finland has been unknown. M. avium isolates isolated from pig organs (n=16) and clinical samples (n=13) were compared by IS1245 RFLP analysis to evaluate the similarity of the isolates obtained from human and porcine samples. Nearly identical multicopy M. avium subsp. hominissuis IS1245 RFLP fingerprints were obtained for isolates of porcine and human origin. IS1245 RFLP patterns of 38% of the porcine and human M. a. hominissuis isolates were >90% similar. The RFLP patterns of two porcine and two human isolates showed >95% similarity. The high similarity of the IS1245 RFLP patterns of the human and porcine M. a. hominissuis isolates indicates close genetic relatedness, suggesting that M. a. hominissuis is transmitted between pigs and humans, or that pigs and humans share common environmental sources of infection. Porcine and human isolates with RFLP patterns differing by only one or two bands were found, which shows that the same M. a. hominissuis strains may infect both humans and pigs.     

Dendrochronological investigation of wood extractives

X-ray microdensitometry was applied to a set of Scots pinewood (i.e. low extractive content). Earlywood and latewood properties were determined as minimum and maximum densities of each tree ring and the potential influence of acetone-soluble extractives (i.e. non-structural and secondary constituents of wood) was estimated using tree-ring statistics. The occurrence of extractives in different portions of wood was determined using dendrochronological methods, by comparing the densities of unextracted and extracted wood. It was not only found that unextracted samples exhibited inflated earlywood and latewood density values, but the growth trends were also altered. Extractives flattened the inter-annual growth variability, both in earlywood and latewood, and influenced the estimation of intra-annual radial growth variations. Characterizing the varying amount of extractives is of inter-disciplinary importance. The results in this study describe their occurrence and show that the radial variations in extractives could be highly detailed by simply using densitometry-based dendrochronology.     

The susceptibility of herbal willow to <Emphasis Type="Italic">Melampsora</Emphasis> rust and herbivores

Eight Dark-leaved willow (Salix myrsinifolia) clones and two naturally hybridised clones (S. myrsinifolia × S. phylicifolia), that are considered to be suitable for herbal production, were cultivated for 2 years in Luikonlahti and Punkaharju. Both experimental sites are located in eastern Finland and the distance between the sites is 140 km. Different cultivation methods were used, including combinations of soil tillage, plastic mulch and fertilisation, with the aim of comparing the growth and susceptibility of plants to pathogens and willow-eating herbivores amongst the clones cultivated by different methods. In both study years 2001 and 2002, Melampsora rust-infected willows occurred in Luikonlahti and in Punkaharju. The extent of rust severity varied greatly between the years and experimental areas and amongst the clones. In 2002, fertilisation increased rust severity in Luikonlahti, but the effect was the converse in Punkaharju. Mulch effect on rust severity was clone-dependent. Vole feeding was observed in 56% of the plants in Luikonlahti during the winter 2001–2002 and the frequency of damaged plants was nearly twice as much amongst the willows grown in unmulched soil as those with plastic mulch. Cultivation method had no effect on feeding by leaf beetles or the abundance of aphids. Of the cultivation methods tested here, plastic mulch seems to have the most important influence on willow cultivation, particularly by improving willow growth and also by decreasing winter-feeding by voles.     

Proliferation of mycobacteria in a piggery environment revealed by mycobacterium-specific real-time quantitative PCR and 16S rRNA sandwich hybridization

Pig mycobacteriosis is the most common animal mycobacterial disease in Finland with a long-term average prevalence of 0.34% and temporary peaks as high as 0.85%. In the current study Mycobacterium-specific real-time qPCR and 16S rRNA sandwich hybridization were utilized for culture-independent detection and measurement of potentially infectious mycobacteria in selected piggeries. Participating herds (n=5) were selected according to prevalence of tuberculous lesions (>4%) in slaughtered carcasses. When DNA extracted from piggery bedding materials was analyzed by Mycobacterium-targeted qPCR using the SYBR green I dye for detection of amplification products, 10(5) to 10(7) cell equivalents of mycobacterial DNA were detected in unused bedding materials and 10(8) to 10(10)g(-1) dry weight in used bedding materials. When Mycobacterium-specific hybridization probes were used for detection of amplification products, 10(5) to 10(7) cell equivalents of mycobacterial DNA g(-1) dry weight were detected in unused bedding materials in four out of the five piggeries studied and up to 10(8) cell equivalents in used bedding material. The results were confirmed by the Mycobacterium-specific 16S rRNA sandwich hybridization assay. The present results show, that mycobacteria occur in organic materials commonly used on pig farms, and may proliferate in bedding materials during use. We also show that DNA- and RNA-based methods may be utilized for detection of environmental reservoirs of mycobacteria causing porcine and human infection.     

Comparison of Variable-Number Tandem-Repeat Markers typing and IS1245 Restriction Fragment Length Polymorphism fingerprinting of Mycobacterium avium subsp. hominissuis from human and porcine origins

Background

Animal mycobacterioses are regarded as a potential zoonotic risk and cause economical losses world wide. M. avium subsp. hominissuis is a slow-growing subspecies found in mycobacterial infected humans and pigs and therefore rapid and discriminatory typing methods are needed for epidemiological studies. The genetic similarity of M. avium subsp. hominissuis from human and porcine origins using two different typing methods have not been studied earlier. The objective of this study was to compare the IS1245 RFLP pattern and MIRU-VNTR typing to study the genetic relatedness of M. avium strains isolated from slaughter pigs and humans in Finland with regard to public health aspects.

Methods

A novel PCR-based genotyping method, variable number tandem repeat (VNTR) typing of eight mycobacterial interspersed repetitive units (MIRUs), was evaluated for its ability to characterize Finnish Mycobacterium avium subsp. hominissuis strains isolated from pigs (n = 16) and humans (n = 13) and the results were compared with those obtained by the conventional IS1245 RFLP method.

Results

The MIRU-VNTR results showed a discriminatory index (DI) of 0,92 and the IS1245 RFLP resulted in DI 0,98. The combined DI for both methods was 0,98. The MIRU-VNTR test has the advantages of being simple, reproducible, non-subjective, which makes it suitable for large-scale screening of M. avium strains.

Conclusions

Both typing methods demonstrated a high degree of similarity between the strains of human and porcine origin. The parallel application of the methods adds epidemiological value to the comparison of the strains and their origins. The present approach and results support the hypothesis that there is a common source of M. avium subsp. hominissuis infection for pigs and humans or alternatively one species may be the infective source to the other.     

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.     

Environmental Exposure to Cadmium and Lead in the Pediatric Age Group

Lead additives in automotive fuel, smog from a nearby industrial center warranted an investigation on cadmium and lead in Manisa, a city of tabacco processing. Hundred and one children were screened in view of lead and cadmium exposure. In 23 children between the ages of 0–2 years the mean (±SEM) serum lead level was 7.15 ± 0.10 μg/dl, in 28 between the ages of 3–6 years was 7.20 ± 0.10 μg/dl and in 50 between the ages of 7–15 years was 7.20 ± 0.10 μg/dl, respectively, with no significant differences. Serum cadmium levels in the same groups of children was 0.066 ± 0.008 ng/ml, 0.078 ± 0.008 ng/ml 0.088 ± 0.006 ng/ml, respectively. The difference in cadmium levels between the age groups of 0–2 years and 7–15 years was significant (p<0.038). This significant increase in blood cadmium level is also shown by simple linear regression analysis: Cadmium (ng/ml) = 0.049 + 0.005 (age), and p<0.0001, F Ratio = 50.578, coefficient of correlation = 0.581. Our study revealed that lead is not a serious environmental contaminant for children, yet; however, the increasing trend seen in exposure to cadmium warrants serious consideration and urgant preventive measures.