Antibacterial activities of naturally occurring compounds against Mycobacterium avium subsp. paratuberculosis.

The antibacterial activities of 18 naturally occurring compounds (including essential oils and some of their isolated constituents, apple and green tea polyphenols, and other plant extracts) against three strains of Mycobacterium avium subsp. paratuberculosis (a bovine isolate [NCTC 8578], a raw-milk isolate [806R], and a human isolate [ATCC 43015]) were evaluated using a macrobroth susceptibility testing method. M. avium subsp. paratuberculosis was grown in 4 ml Middlebrook 7H9 broth containing 10% oleic acid-albumin-dextrose-catalase, 0.05% Tween 80 (or 0.2% glycerol), and 2 µg/ml mycobactin J supplemented with five concentrations of each test compound. The changes in the optical densities of the cultures at 600 nm as a measure of CFU were recorded at intervals over an incubation period of 42 days at 37°C. Six of the compounds were found to inhibit the growth of M. avium subsp. paratuberculosis. The most effective compound was trans-cinnamaldehyde, with a MIC of 25.9 µg/ml, followed by cinnamon oil (26.2 µg/ml), oregano oil (68.2 µg/ml), carvacrol (72.2 µg/ml), 2,5-dihydroxybenzaldehyde (74 µg/ml), and 2-hydroxy-5-methoxybenzaldehyde (90.4 µg/ml). With the exception of carvacrol, a phenolic compound, three of the four most active compounds are aldehydes, suggesting that the structure of the phenolic group or the aldehyde group may be important to the antibacterial activity. No difference in compound activity was observed between the three M. avium subsp. paratuberculosis strains studied. Possible mechanisms of the antimicrobial effects are discussed.

Risk factors for the introduction and within-herd transmission of Mycobacterium avium subspecies paratuberculosis (MAP) infection in 59 Irish dairy herds

Since 1994, Irish cattle have been exposed to greater risks of acquiring Mycobacterium avium subspecies paratuberculosis (MAP) infection as a consequence of the importation of over 70,000 animals from continental Europe. In recent years, there has been an increase in the number of reported clinical cases of paratuberculosis in Ireland. This study examines the prevalence of factors that promote the introduction and within-herd transmission of Mycobacterium avium subspecies paratuberculosis (MAP) on selected Irish dairy farms in the Cork region, and the association between these factors and the results of MAP screening tests on milk sock filter residue (MFR). A total of 59 dairy farms, selected using non-random methods but apparently free of endemic paratuberculosis, were enrolled into the study. A questionnaire was used to collect data about risk factors for MAP introduction and transmission. The MFR was assessed on six occasions over 24 months for the presence of MAP, using culture and immunomagnetic separation prior to polymerase chain reaction (IMS-PCR). Furthermore, blood samples from all entire male and female animals over one year of age in 20 herds were tested by ELISA. Eighteen (31%) farms had operated as closed herds since 1994, 28 (47%) had purchased from multiple sources and 14 (24%) had either direct or indirect (progeny) contact with imported animals. Milk and colostrum were mixed on 51% of farms, while 88% of farms fed pooled milk. Thirty (51%) herds tested negative to MFR culture and IMS-PCR, 12 (20%) were MFR culture positive, 26 (44%) were IMS-PCR positive and seven (12%) were both culture and IMS-PCR positive. The probability of a positive MFR culture was significantly associated with reduced attendance at calving, and with increased use of individual calf pens and increased (but not significantly) if multiple suckling was practised. There was poor agreement between MFR culture and MFR IMS-PCR results, but moderate agreement between MFR culture and ELISA test results. This study highlights a lack of awareness among Irish dairy farmers about the effect of inadequate biosecurity on MAP introduction. Furthermore, within-herd transmission will be facilitated by traditional calf rearing and waste management practices. The findings of viable MAP in the presence of known transmission factors in non-clinically affected herds could be a prelude to long-term problems for the Irish cattle and agri-business generally.

Maximising capture efficiency and specificity of magnetic separation for Mycobacterium avium subsp. paratuberculosis cells

In order to introduce specificity for Mycobacterium avium subsp. paratuberculosis prior to a phage amplification assay, various magnetic-separation approaches, involving either antibodies or peptides, were evaluated in terms of the efficiency of capture (expressed as a percentage) of M. avium subsp. paratuberculosis cells and the percentage of nonspecific binding by other Mycobacterium spp. A 50:50 mixture of MyOne Tosylactivated Dynabeads coated with the chemically synthesized M. avium subsp. paratuberculosis-specific peptides biotinylated aMp3 and biotinylated aMptD (i.e., peptide-mediated magnetic separation [PMS]) proved to be the best magnetic-separation approach for achieving 85 to 100% capture of M. avium subsp. paratuberculosis and minimal (<1%) nonspecific recovery of other Mycobacterium spp. (particularly if beads were blocked with 1% skim milk before use) from broth samples containing 103 to 104 CFU/ml. When PMS was coupled with a recently optimized phage amplification assay and used to detect M. avium subsp. paratuberculosis in 50-ml volumes of spiked milk, the mean 50% limit of detection (LOD50) was 14.4 PFU/50 ml of milk (equivalent to 0.3 PFU/ml). This PMS-phage assay represents a novel, rapid method for the detection and enumeration of viable M. avium subsp. paratuberculosis organisms in milk, and potentially other sample matrices, with results available within 48 h.

Culture phenotypes of genomically and geographically diverse Mycobacterium avium subsp. paratuberculosis isolates from different hosts

Mycobacterium avium subsp. paratuberculosis causes paratuberculosis (Johne's disease) in ruminants in most countries. Historical data suggest substantial differences in culturability of M. avium subsp. paratuberculosis isolates from small ruminants and cattle; however, a systematic comparison of culture media and isolates from different countries and hosts has not been undertaken. Here, 35 field isolates from the United States, Spain, Northern Ireland, and Australia were propagated in Bactec 12B medium and Middlebrook 7H10 agar, genomically characterized, and subcultured to Lowenstein-Jensen (LJ), Herrold's egg yolk (HEY), modified Middlebrook 7H10, Middlebrook 7H11, and Watson-Reid (WR) agars, all with and without mycobactin J and some with sodium pyruvate. Fourteen genotypes of M. avium subsp. paratuberculosis were represented as determined by BstEII IS900 and IS1311 restriction fragment length polymorphism analysis. There was no correlation between genotype and overall culturability, although most S strains tended to grow poorly on HEY agar. Pyruvate was inhibitory to some isolates. All strains grew on modified Middlebrook 7H10 agar but more slowly and less prolifically on LJ agar. Mycobactin J was required for growth on all media except 7H11 agar, but growth was improved by the addition of mycobactin J to 7H11 agar. WR agar supported the growth of few isolates. The differences in growth of M. avium subsp. paratuberculosis that have historically been reported in diverse settings have been strongly influenced by the type of culture medium used. When an optimal culture medium, such as modified Middlebrook 7H10 agar, is used, very little difference between the growth phenotypes of diverse strains of M. avium subsp. paratuberculosis was observed. This optimal medium is recommended to remove bias in the isolation and cultivation of M. avium subsp. paratuberculosis.