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BackgroundJohne’s disease is caused by Mycobacterium avium subsp. paratuberculosis (Map) and it is one of the most important diseases in cattle worldwide. Several laboratory tests for Map detection are available; however, these are limited by inadequate sensitivity and specificity when used in subclinically infected populations. To identify Map shedders in subclinically infected cattle, we used a new, high-yield method for DNA-extraction from Map in faeces combined with quantitative real-time PCR (qPCR) for amplification of the insertion sequence IS900 of Map (HYDEqPCR). Evaluation of HYDEqPCR was carried out in comparison with faecal culture, milk qPCR, and milk enzyme-linked immunosorbent assay (ELISA), on 141 faecal and 91 milk samples, from 141 subclinically infected dairy cattle.ResultsThe qPCR proved to be highly sensitive, with a detection limit of 2 IS900 DNA copies/μl in 67 % of the reactions. It also showed 100 % specificity, as determined from 50 Map and non-Map strains, and by the sequencing of qPCR amplicons. The detection limit of HYDEqPCR was 90 Map/g Map-spiked faeces, which corresponds to 2.4 colony forming units/g Map-spiked faeces, with an estimated efficiency of 85 % (±21 %). When tested on the field samples, HYDEqPCR showed 89 % of the samples as positive for Map, whereas faecal culture, milk qPCR, and milk ELISA detected 19 %, 36 % and 1 %, respectively. Fisher’s exact tests only show statistical significance (p ≤0.05) for the correlation between HYDEqPCR and faecal culture. The agreement between HYDEqPCR and milk qPCR and milk ELISA was poor, slight, and non-significant.ConclusionsThis study highlights the advantages of HYDEqPCR for detection of Map in subclinically infected populations, in comparison with faecal culture, milk qPCR and milk ELISA. HYDEqPCR can detect low-level Map shedders that go undetected using these other methods, which will thus underestimate the proportions of Map-shedders in herds. Identification of these shedding animals is extremely important for prevention of the spread of Map infection in an animal population. Due to the relatively high sensitivity and specificity of HYDEqPCR, it can be applied to test for Map at the herd or individual level, regardless of animal age or production stage. HYDEqPCR will allow early detection and control of Map in any population at risk.

Clostridium difficile (CD) can cause a significant and transmissible disease in animals and humans, with poorly understood epidemiology. Animals have been suggested as a possible source of infection and environment contamination. It is necessary that a precise and rapid diagnostic tool is available for the detection of CD from clinical and/or environmental samples. A quantitative real-time PCR (qPCR) protocol for CD detection defined by Penders et al. (FEMS Microbiol Lett, 243, 2005, 141-147) was modified. The modified protocol, supported by a novel extraction method, was tested on CD-spiked cattle feces and clinical fecal samples from calves. Quantification was performed targeting CD 16S rRNA gene. Three different commonly used TaqMan universal PCR master mixes were also compared. Results indicate that the modified protocol is very sensitive with an LOD of 7.72 CD cells per g CD-spiked feces. The protocol is capable of precise quantification with an LOQ of 77.2 CD cells per g CD-spiked feces, R(2) between 0.9957 and 0.9968, isolation efficiency from 87.89% to 90.96%, and an interassay CV ranging from 3.71% to 9.57%. The qPCR protocol for the detection and quantification of CD from animal feces investigated and described in this article using MIQE guidelines has the lowest detection and quantification limits published to date. Therefore, it can be implemented for precise epidemiological investigations of CD infections in animals and humans.

The objective of the present work was to establish effective and rapid diagnostic methods for the detection of Bacillus anthracis, a highly virulent zoonotic pathogen, in the air, soil and animal (or human) tissue samples. Liquid culture of B. anthracis was aerosolized and four air sampling procedures were employed. Detection of B. anthracis in the air samples was successful with RCS High Flow sampler (culturebased detection) and when sampling through the air filter (molecular detection using SmartHelix Complex Samples DNA Extraction Kit). Liquid B. anthracis culture was also employed for spiking the homogenised bovine lymphatic gland tissue and soil samples. DNA extraction was performed using three different commercial kits for each sample type. High Pure PCR Template Preparation Kit was the most effective for DNA extraction from animal tissue samples. Detection in the soil was successful when PowerSoil DNA Isolation Kit was used. Our results indicate that B. anthracis can be monitored in different matrices by rapid molecular methods when appropriate sampling and DNA extraction procedures are employed prior to PCR assay. The selected rapid protocols can be implemented in specialized veterinary or human diagnostic laboratories with moderate costs