Sensitive and specific detection of viable Mycobacterium avium subsp. paratuberculosis in raw milk by the Peptide-mediated magnetic separation (PMS)-phage assay

This study describes further validation of a previously described Peptide-mediated magnetic separation (PMS)-Phage assay, and its application to test raw cows’ milk for presence of viable Mycobacterium avium subsp. paratuberculosis (MAP). The inclusivity and exclusivity of the PMS-phage assay were initially assessed, before the 50% limit of detection (LOD50) was determined and compared with those of PMS-qPCR (targeting both IS900 and f57) and PMS-culture. These methods were then applied in parallel to test 146 individual milk samples and 22 bulk tank milk samples from Johne’s affected herds. Viable MAP were detected by the PMS-phage assay in 31 (21.2%) of 146 individual milk samples (mean plaque count of 228.1 PFU/50 ml, range 6-948 PFU/50 ml), and 13 (59.1%) of 22 bulk tank milks (mean plaque count of 136.83 PFU/50 ml, range 18-695 PFU/50 ml). In contrast, only 7 (9.1%) of 77 individual milks and 10 (45.4%) of 22 bulk tank milks tested PMS-qPCR positive, and 17 (11.6%) of 146 individual milks and 11 (50%) of 22 bulk tank milks tested PMS-culture positive. The mean 50% limits of detection (LOD50) of the PMS-phage, PMS-IS900 qPCR and PMS-f57 qPCR assays, determined by testing MAP-spiked milk, were 0.93, 135.63 and 297.35 MAP CFU/50 ml milk, respectively. Collectively, these results demonstrate that, in our laboratory, the PMS-phage assay is a sensitive and specific method to quickly detect the presence of viable MAP cells in milk. However, due to its complicated, multi-step nature, the method would not be a suitable MAP screening method for the dairy industry.

Transcriptional dynamics of the developing sweet cherry (Prunus avium L.) fruit: sequencing, annotation and expression profiling of exocarp-associated genes

The exocarp, or skin, of fleshy fruit is a specialized tissue that protects the fruit, attracts seed dispersing fruit eaters, and has large economical relevance for fruit quality. Development of the exocarp involves regulated activities of many genes. This research analyzed global gene expression in the exocarp of developing sweet cherry (Prunus avium L., 'Regina'), a fruit crop species with little public genomic resources. A catalog of transcript models (contigs) representing expressed genes was constructed from de novo assembled short complementary DNA (cDNA) sequences generated from developing fruit between flowering and maturity at 14 time points. Expression levels in each sample were estimated for 34 695 contigs from numbers of reads mapping to each contig. Contigs were annotated functionally based on BLAST, gene ontology and InterProScan analyses. Coregulated genes were detected using partitional clustering of expression patterns. The results are discussed with emphasis on genes putatively involved in cuticle deposition, cell wall metabolism and sugar transport. The high temporal resolution of the expression patterns presented here reveals finely tuned developmental specialization of individual members of gene families. Moreover, the de novo assembled sweet cherry fruit transcriptome with 7760 full-length protein coding sequences and over 20 000 other, annotated cDNA sequences together with their developmental expression patterns is expected to accelerate molecular research on this important tree fruit crop.

Análisis del potencial epizoótico de cepas de Mycobacterium avium subsp. paratuberculosis aisladas de diversos hospedadores empleando modelos de infección in vitro

299 p.

Rapid identification and differentiation of Mycobacterium avium subspecies paratuberculosis types by use of real-time PCR and high-resolution melt analysis of the MAP1506 locus

High-resolution melt (HRM) analysis can identify sequence polymorphisms by comparing the melting curves of amplicons generated by real-time PCR amplification. We describe the application of this technique to identify Mycobacterium avium subspecies paratuberculosis types I, II, and III. The HRM approach was based on type-specific nucleotide sequences in MAP1506, a member of the PPE (proline-proline-glutamic acid) gene family.

Occurrence of Mycobacterium avium subspecies paratuberculosis across host species and European countries with evidence for transmission between wildlife and domestic ruminants

BACKGROUND Mycobacterium avium subspecies paratuberculosis (Map) causes an infectious chronic enteritis (paratuberculosis or Johne's disease) principally of ruminants. The epidemiology of Map is poorly understood, particularly with respect to the role of wildlife reservoirs and the controversial issue of zoonotic potential (Crohn's disease). Genotypic discrimination of Map isolates is pivotal to descriptive epidemiology and resolving these issues. This study was undertaken to determine the genetic diversity of Map, enhance our understanding of the host range and distribution and assess the potential for interspecies transmission. RESULTS 164 Map isolates from seven European countries representing 19 different host species were genotyped by standardized IS900--restriction fragment length polymorphism (IS900-RFLP), pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphisms (AFLP) and mycobacterial interspersed repeat unit-variable number tandem repeat (MIRU-VNTR) analyses. Six PstI and 17 BstEII IS900-RFLP, 31 multiplex [SnaBI-SpeI] PFGE profiles and 23 MIRU-VNTR profiles were detected. AFLP gave insufficient discrimination of isolates for meaningful genetic analysis. Point estimates for Simpson's index of diversity calculated for the individual typing techniques were in the range of 0.636 to 0.664 but a combination of all three methods increased the discriminating power to 0.879, sufficient for investigating transmission dynamics. Two predominant strain types were detected across Europe with all three typing techniques. Evidence for interspecies transmission between wildlife and domestic ruminants on the same property was demonstrated in four cases, between wildlife species on the same property in two cases and between different species of domestic livestock on one property. CONCLUSION The results of this study showed that it is necessary to use multiple genotyping techniques targeting different sources of genetic variation to obtain the level of discrimination necessary to investigate transmission dynamics and trace the source of Map infections. Furthermore, the combination of genotyping techniques may depend on the geographical location of the population to be tested. Identical genotypes were obtained from Map isolated from different host species co-habiting on the same property strongly suggesting that interspecies transmission occurs. Interspecies transmission of Map between wildlife species and domestic livestock on the same property provides further evidence to support a role for wildlife reservoirs of infection.

Single nucleotide polymorphisms in the IS900 sequence of Mycobacterium avium subsp. paratuberculosis are strain type specific

Insertion sequence IS900 is used as a target for the identification of Mycobacterium avium subsp. paratuberculosis. Previous reports have revealed single nucleotide polymorphisms within IS900. This study, which analyzed the IS900 sequences of a panel of isolates representing M. avium subsp. paratuberculosis strain types I, II, and III, revealed conserved type-specific polymorphisms that could be utilized as a tool for diagnostic and epidemiological purposes.

Discovery of stable and variable differences in the Mycobacterium avium subsp. paratuberculosis type I, II, and III genomes by pan-genome microarray analysis

Mycobacterium avium subsp. paratuberculosis is an important animal pathogen widely disseminated in the environment that has also been associated with Crohn's disease in humans. Three M. avium subsp. paratuberculosis genomotypes are recognized, but genomic differences have not been fully described. To further investigate these potential differences, a 60-mer oligonucleotide microarray (designated the MAPAC array), based on the combined genomes of M. avium subsp. paratuberculosis (strain K-10) and Mycobacterium avium subsp. hominissuis (strain 104), was designed and validated. By use of a test panel of defined M. avium subsp. paratuberculosis strains, the MAPAC array was able to identify a set of large sequence polymorphisms (LSPs) diagnostic for each of the three major M. avium subsp. paratuberculosis types. M. avium subsp. paratuberculosis type II strains contained a smaller genomic complement than M. avium subsp. paratuberculosis type I and M. avium subsp. paratuberculosis type III genomotypes, which included a set of genomic regions also found in M. avium subsp. hominissuis 104. Specific PCRs for genes within LSPs that differentiated M. avium subsp. paratuberculosis types were devised and shown to accurately screen a panel (n = 78) of M. avium subsp. paratuberculosis strains. Analysis of insertion/deletion region INDEL12 showed deletion events causing a reduction in the complement of mycobacterial cell entry genes in M. avium subsp. paratuberculosis type II strains and significantly altering the coding of a major immunologic protein (MPT64) associated with persistence and granuloma formation. Analysis of MAPAC data also identified signal variations in several genomic regions, termed variable genomic islands (vGIs), suggestive of transient duplication/deletion events. vGIs contained significantly low GC% and were immediately flanked by insertion sequences, integrases, or short inverted repeat sequences. Quantitative PCR demonstrated that variation in vGI signals could be associated with colony growth rate and morphology.

Genetic diversity of Mycobacterium avium isolates recovered from clinical samples and from the environment: molecular characterization for diagnostic purposes

Isolation of Mycobacterium avium complex (MAC) organisms from clinical samples may occur in patients without clinical disease, making the interpretation of results difficult. The clinical relevance of MAC isolates from different types of clinical samples (n = 47) from 39 patients in different sections of a hospital was assessed by comparison with environmental isolates (n = 17) from the hospital. Various methods for identification and typing (commercial probes, phenotypic characteristics, PCR for detection of IS1245 and IS901, sequencing of the hsp65 gene, and pulsed-field gel electrophoresis) were evaluated. The same strain was found in all the environmental isolates, 21 out of 23 (91.3%) of the isolates cultured from urine samples, and 5 out of 19 (26.3%) isolates from respiratory specimens. This strain did not cause disease in the patients. Testing best characterized the strain as M. avium subsp. hominissuis, with the unusual feature that 81.4% of these isolates lacked the IS1245 element. Contamination of certain clinical samples with an environmental strain was the most likely event; therefore, characterization of the environmental mycobacteria present in health care facilities should be performed to discard false-positive isolations in nonsterile samples, mainly urine samples. Molecular techniques applied in this study demonstrated their usefulness for this purpose.

Viable mycobacterium avium subsp. paratuberculosis in retail artisanal coalho cheese from northeastern Brazil.

2014

Investigating the causes of late fruit drop in ‘Regina’ sweet cherry (Prunus avium)

The presented study aimed to correctly describe the (late) fruit drop pattern of sweet cherry cv. Regina grafted on ‘Gisela 5’ and investigate its internal causes. In the first season, a method to describe the fruit drop pattern was defined and validated. The second season was devoted to a province-based screening of the phenomenon to identify potential influences of environmental, physiological and management factors. The multisite trial involved 6 commercial orchards located at different elevations, from 225 up to 1175m a.s.l. The third season was dedicated to find confirmation of the hypothesis formulated during the previous year. The multisite comparison was maintained but reduced to only two orchards to allow more frequent samplings. It emerged that late fruit drop is a complex phenomenon showing variable intensity: the percentage of late fruit drop ranged from 7 to 76% of the fruitlets set, depending on the orchard and on the year considered. Two main waves of fruitlets drop have been observed: the first one was composed by unfertilized parthenocarpic fruitlets, probably caused by late or missing fertilization, that immediately after bloom already showed smaller diameters and symptoms of senescence; the second one (the focus of this study) was composed by fully developed fruits that at a certain point decreased their growth rate and got senescent. All the late dropped cherries showed an aborted embryo. This sudden change has been observed to be concomitant both with prolonged periods of low temperatures (or sudden severe decreases in the daily Growing Degree Hours accumulation) and with extraordinary high temperatures close to or above 30°C. Other factors, such as the position of the limb within the canopy, its orientation (sunny vs. shady side) or nutrition played only a marginal role. Excessive vigor can increase late fruit drop intensity but is not its main cause.

Direct Detection of Mycobacterium bovis in Bovine Lymph Nodes by PCR

P>Thirty-five lymph node samples were taken from animals with macroscopic lesions consistent with Mycobacterium bovis infection. The animals were identified by postmortem examination in an abattoir in the northwestern region of state of Parana, Brazil. Twenty-two of the animals had previously been found to be tuberculin skin test positive. Tissue samples were decontaminated by Petroff`s method and processed for acid-fast bacilli staining, culture in Stonebrink and Lowenstein-Jensen media and DNA extraction. Lymph node DNA samples were amplified by PCR in the absence and presence (inhibitor controls) of DNA extracted from M. bovis culture. Mycobacterium bovis was identified in 14 (42.4%) lymph node samples by both PCR and by culture. The frequency of PCR-positive results (54.5%) was similar to that of culture-positive results (51.5%, P > 0.05). The percentage of PCR-positive lymph nodes increased from 39.4% (13/33) to 54.5% (18/33) when samples that were initially PCR-negative were reanalysed using 2.5 mu l DNA (two samples) and 1 : 2 diluted DNA (three samples). PCR sensitivity was affected by inhibitors and by the amount of DNA in the clinical samples. Our results indicate that direct detection of M. bovis in lymph nodes by PCR may be a fast and useful tool for bovine tuberculosis epidemic management in the region.

Is alcohol a cofactor of HIV and AIDS? Evidence from immunological and behavioral studies

The authors aim to critically examine empirical research on the effects of alcohol on HIV and AIDS from the immunological and behavioral fields. In vitro immunological studies demonstrate that social drinking increases the susceptibility of human cells to HIV infection. Animal studies show that acute and chronic alcohol ingestion increases rare of progression from retrovirus to clinical illness. In humans with HIV, no experimental evidence shows that alcohol is a cofactor of AIDS. Findings from behavioral studies show that a link between social drinking and risk of HIV is weak. No experimental evidence demonstrates that chronic drinking influences rate and course of disease progression to AIDS in humans who are HIV+. It is premature to promote the role of alcohol as a cofactor in HIV and AIDS.

PLUNC protein expression in major salivary glands of HIV-infected patients

Objective: To analyse and compare the expression of Palate, Lung, and Nasal Epithelium Clone (PLUNC) proteins in salivary glands from patients with and without AIDS (control group) using autopsy material. Methods: We analysed the expression of PLUNCs using immunohistochemistry in parotid (n = 45), submandibular (n = 47) and sublingual gland (n = 37) samples of AIDS patients [30 with normal histology, 21 with mycobacteriosis, 14 with cytomegalovirus (CMV) infection, 30 with chronic non-specific sialadenitis, and 30 HIV-negative controls. In situ hybridization (ISH) for SPLUNC 2 in the HIV-negative group was performed. Results: SPLUNC 1 expression was detected in the mucous acini of submandibular and sublingual glands, and SPLUNC 2 were seen in the serous cells. LPLUNC 1 expression was only positive in the salivary ducts. There was a higher expression of SPLUNC 2 in AIDS patients with CMV infection and mycobacteriosis when compared with all other groups. The intensity of staining for SPLUNC 2 was greater around the lesions than the peripheral ones. ISH for SPLUNC 2 showed perinuclear positivity in the serous cells in all HIV-negative cases. Conclusions: SPLUNC 1 and LPLUNC 1 proteins were similarly expressed in the salivary glands of AIDS patients and non-HIV patients. CMV infection and mycobacteriosis increase SPLUNC 2 expression in serous cells in the salivary gland of AIDS patients.

In vitro uptake and antimycobacterial activity of liposomal usnic acid formulation

The cellular uptake and antimycobacterial activity of usnic acid (UA) and usnic acid-loaded liposomes (UA-LIPOs) were assessed on J774 macrophages. The minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) of UA and UA-LIPO against Mycobacterium tuberculosis were determined. Concentrations required to inhibit 50% of cell proliferation (IC(50)) were 22.5 (+/- 0.60) and 12.5 (+/- 0.26) mu g/ml, for UA and UA-LIPO, respectively. The MICs of UA and UA-LIPO were 6.5 and 5.8 mu g/mL, respectively. The MBC of UA-LIPO was twice as low (16 mu g/mL) as that of UA (32 mu g/mL). An improvement in the intracellular uptake of UA-LIPO was found (21.6 x 10(4) +/- 28.3 x 10(2) c.p.s), in comparison with UA (9.5 x 10(4) +/- 11.4 x 10(2) c.p.s). In addition, UA-LIPO remains much longer inside macrophages (30 hours). All data obtained from the encapsulation of usnic acid into liposomes as a drug delivery system (DDS) indicate a strong interaction between UA-liposomes and J774 macrophages, thereby facilitating UA penetration into cells. Considering such a process as ruling the Mycobacterium-transfection by magrophages, we could state that associating UA with this DDS leads to an improvement in its antimycobacterial activity.

Structure activity relationship, acute toxicity and cytotoxicity of antimycobacterial neolignan analogues

Objectives The study`s aims were to evaluate the antimycobacterial activity of 13 synthetic neolignan analogues and to perform structure activity relationship analysis (SAR). The cytotoxicity of the compound 2-phenoxy-1-phenylethanone (LS-2, 1) in mammalian cells, such as the acute toxicity in mice, was also evaluated. Methods The extra and intracellular antimycobacterial activity was evaluated on Mycobacterium tuberculosis H37Rv. Cytotoxicity studies were performed using V79 cells, J774 macrophages and rat hepatocytes. Additionally, the in-vivo acute toxicity was tested in mice. The SAR analysis was performed by Principal Component Analysis (PCA). Key findings Among the 13 analogues tested, LS-2 (1) was the most effective, showing promising antimycobacterial activity and very low cytotoxicity in V79 cells and in J774 macrophages, while no toxicity was observed in rat hepatocytes. The selectivity index (SI) of LS-2 (1) was 91 and the calculated LD50 was 1870 mg/kg, highlighting the very low toxicity in mice. SAR analysis showed that the highest electrophilicity and the lowest molar volume are physical-chemical characteristics important for the antimycobacterial activity of the LS-2 (1). Conclusions LS-2 (1) showed promising antimycobacterial activity and very weak cytotoxicity in cell culture, as well as an absence of toxicity in primary culture of hepatocytes. In the acute toxicity study there was an indication of absence of toxicity on murine models, in vivo.