Exploring the Diverse Biological Properties of Cannabidiol: A Focus on Plant Growth Stimulation.

The aim of the current study was to compare some biological activities of edible oils enriched with 10 % of cannabidiol (CBD samples) from the Slovak market. In addition, hemp, coconut, argan, and pumpkin pure oils were also examined. The study evaluated the fatty acids content, as well as antibacterial, antifungal, antioxidant, cytotoxic, and phytotoxic activities. The CBD samples presented antimicrobial activity against the tested bacterial strains at higher concentrations (10000 and 5000 mg/L) and antifungal activity against Alternaria alternata, Penicillium italicum and Aspergillus flavus. DPPH⋅ and FRAP assays showed greater activity in CBD-supplemented samples compared to pure oils and vitamin E. In cell lines (IPEC-J2 and Caco-2), a reduced cell proliferation and viability were observed after 24 hours of incubation with CBD samples. The oils showed pro-germinative effects. The tested activities were linked to the presence of CBD in the oils.

Immunomodulatory effect of probiotic exopolysaccharides in a porcine in vitro co-culture model mimicking the intestinal environment on ETEC infection.

The aim of this study was to evaluate the immunomodulatory effect of EPS-L26 isolated from the probiotic strain Lactobacillus (Limosilactobacillus) reuteri L26 Biocenol™, in a model of infection with an enterotoxigenic E. coli (ETEC) by establishing monocultures consisting of the IPEC-J2 cell line or monocyte-derived dendritic cells (moDCs) and creating a 3D model of cell co-cultures established with IPEC-J2 cells and moDCs. The immunomodulatory and immunoprotective potential of used EPS-L26 was confirmed in monocultures in an experimental group of pretreated cells, where our study showed that pretreatment of cells with EPS-L26 and subsequent exposure to infection resulted in significantly down-regulated mRNA levels of genes encoding inflammatory cytokines compared to ETEC challenge in single cell cultures (in IPEC-J2, decreased mRNA levels for TNF-α, IL-6, IL-1ß, IL-12p35; in moDCs, decreased mRNA levels for IL-1ß). Similar to monocultures, we also demonstrated the immunostimulatory potential of the ETEC strain in the co-culture model on directly treated IPEC-J2 cells cultivated on insert chambers (apical compartment) and also on indirectly treated moDCs cultivated in the lower chamber (basolateral compartment), however in the co-culture model the expression of inflammatory cytokines was attenuated at the mRNA level compared to monocultures. Pretreatment of the cells on the insert chambers pointed to the immunoprotective properties of EPS-L26, manifested by decreased mRNA levels in both cell lines compared to ETEC challenge (in IPEC-J2 decreased mRNA levels for IL-12p35; in moDCs decreased mRNA levels for IL-1ß, IL-6). Our results suggest intercellular communication via humoral signals derived from IPEC-J2 cells by influencing the gene expression of indirectly treated moDC cells located in the basolateral compartment.

Antibacterial activity, quality and stability study of creams with new potential silver(I) complexes and in vivo case report.

The aim of this study was to evaluate the antibacterial activity, quality and stability of creams (at 1 % concentration) prepared with synthesized silver(I) complexes: [Ag(Nam)2]NO3·H2O ( AgNam), [Ag2(HGly)2]n(NO3)2n (AgGly) (Nam - nicotin-amide, Gly - glycine) and silver(I) sulfadiazine (AgSD), which is commercially available. Antibacterial activity was evaluated by agar well diffusion method and in in vivo case. The pure silver(I) complexes as well as all three tested creams loaded with AgGly, AgSD and AgNam showed antibacterial potential. Moreover, the creams loaded with AgGly and AgNam showed higher antibacterial effects against S. aureus and B. subtilis than the cream loaded with AgSD. In terms of appearance, all cream samples were opaque and odourless, and no phase separation was observed. Creams were soluble in water (o/w emulsions) and they had a pseudoplastic behaviour. The pH of the creams was in the range of 4.87-5.75. No visible changes were observed in the case of commercially used AgSD cream during one month testing period at conditions -16 ± 1 °C; 6 ± 1 °C and 56 % relative humidity; 20 ± 1 °C and 58 % relative humidity and 40 ± 1 °C and 75 % relative humidity. However, creams containing AgGly and AgNam changed their colour depending on the tested conditions.

In Situ Gel with Silver Nanoparticles Prepared Using Agrimonia eupatoria L. Shows Antibacterial Activity.

Silver nanoparticles (Ag NPs) with antibacterial activity can be prepared in different ways. In our case, we used ecological green synthesis with Agrimonia eupatoria L. The plant extract was used with Ag NPs for the first time to prepare termosensitive in situ gels (ISGs). Such gels are used to heal human or animal skin and mucous membranes, as they can change from a liquid to solid state after application. Ag NPs were characterized with various techniques (FTIR, TEM, size distribution, zeta potential) and their antibacterial activity was tested against Staphylococcus aureus and Escherichia coli. In accordance with the TEM data, we prepared monodispersed spherical Ag NPs with an average size of about 20 nm. Organic active compounds from Agrimonia eupatoria L. were found on their surfaces using FTIR spectroscopy. Surprisingly, only the in situ gel with Ag NPs showed antibacterial activity against Escherichia coli, while Ag NPs alone did not. Ag NPs prepared via green synthesis using plants with medicinal properties and incorporated into ISGs have great potential for wound healing due to the antibacterial activity of Ag NPs and the dermatological activity of organic substances from plants.

Screening Evaluation of Antiproliferative, Antimicrobial and Antioxidant Activity of Lichen Extracts and Secondary Metabolites In Vitro.

Lichen metabolites represent a wide range of substances with a variety of biological effects. The present study was designed to analyze the potential antiproliferative, antimicrobial and antioxidative effects of several extracts from lichens (Pseudevernia furfuracea, Lobaria pulmonaria, Cetraria islandica, Evernia prunastri, Stereocaulon tomentosum, Xanthoria elegans and Umbilicaria hirsuta) and their secondary metabolites (atranorin, physodic acid, evernic acid and gyrophoric acid). The crude extract, as well as the isolated metabolites, showed potent antiproliferative, cytotoxic activity on a broad range of cancer cell lines in 2D (monolayer) and 3D (spheroid) models. Furthermore, antioxidant (2,2-diphenyl-1-picryl-hydrazylhydrate (DPPH) and in vitro antimicrobial activities were assessed. Data showed that the lichen extracts, as well as the compounds present, possessed biological potential in the studied assays. It was also observed that the extracts were more efficient and their major compounds showed strong effects as antiproliferative, antioxidant and antibacterial agents. Moreover, we demonstrated the 2D and 3D models' importance to drug discovery for further in vivo studies. Despite the fact that lichen compounds have been neglected by the scientific community for long periods, nowadays they are objects of investigation based on their promising effects.

Mechanochemical synthesis of non-stoichiometric copper sulfide Cu1.8S applicable as a photocatalyst and antibacterial agent and synthesis scalability verification.

An effort to prepare different non-stoichiometric CuxSy compounds starting from elemental precursors using mechanochemistry was made in this study. However, out of the 7 stoichiometries tested, it was only possible to obtain three phases: covellite CuS, chalcocite Cu2S and digenite Cu1.8S and their mixtures. To obtain the digenite phase with the highest purity, the Cu : S stoichiometric ratio needed to be fixed at 1.6 : 1. The reaction between copper and sulfur was completed within a second range, however, milling was performed for up to 15 minutes until the equilibrium in phase composition between digenite and covellite was reached. The possibility of preparing the product in a 300 g batch by eccentric vibratory milling in 30 minutes was successfully verified at the end. The estimated crystallite sizes for the digenite Cu1.8S obtained via lab-scale and scalable experiments were around 12 and 17 nm, respectively. The obtained products were found to be efficient photocatalysts under visible light irradiation in the presence of hydrogen peroxide, being capable of the complete degradation of the Methyl Orange dye in a concentration of 10 mg L-1 in 2 hours. Finally, the antibacterial potential of both lab-scale and large-scale industrial products was proven and, regardless of the manufacturing scale, the nanoparticles retained their properties against bacterial cells.

Immunomodulatory Effect of Lactobacillus reuteri (Limosilactobacillus reuteri) and Its Exopolysaccharides Investigated on Epithelial Cell Line IPEC-J2 Challenged with Salmonella Typhimurium.

The gastrointestinal tract is the largest and most complex component of the immune system. Each component influences the production and regulation of cytokines secreted by intestinal epithelial cells. The aim of this study was to see how the probiotic strain Limosilactobacillus reuteri L26 and its exopolysaccharide (EPS) affect porcine intestinal-epithelial cells IPEC-J2 infected with Salmonella Typhimurium. The results revealed that Salmonella infection up-regulated all studied pro-inflammatory cytokines such as TNF-α, IL-8, IL-6 and TLR4, TLR5 signaling pathways, while decreasing the expression of TGF-ß. An immunosuppressive activity was found in EPS-treated wells, since the transcriptional levels of the studied pro-inflammatory cytokines were not increased, and the pretreatment with EPS was even able to attenuate up-regulated pro-inflammatory genes induced by Salmonella infection. However, there was a significant increase in the expression of mRNA levels of IL-8 and TNF-α in L26-treated cells, although this up-regulation was suppressed in the case of pretreatment. The immunoregulatory function of L. reuteri was also confirmed by the increased level of mRNA expression for TGF-ß, a known immunosuppressive mediator. The most relevant finding of this ex vivo study was a case of immunity modulation, where the probiotic strain L. reuteri stimulated the innate immune-cell response which displayed both anti- and pro-inflammatory activities, and modulated the expression of TLRs in the IPEC-J2 cell line. Our findings also revealed that the pretreatment of cells with either EPS or live lactobacilli prior to infection has a suppressive effect on the inflammatory response induced by Salmonella Typhimurium.

Ternary and Quaternary Nanocrystalline Cu-Based Sulfides as Perspective Antibacterial Materials Mechanochemically Synthesized in a Scalable Fashion.

Twelve Cu-based ternary (Cu-Me1-S, Me1 = Fe, Sn, or Sb) and quaternary (Cu-Me2-Sn-S, Me2 = Fe, Zn, or V) nanocrystalline sulfides are shown as perspective antibacterial materials here. They were prepared from elemental precursors by a one-step solvent-free mechanochemical synthesis in a 100 g batch using scalable eccentric vibratory ball milling. Most of the products have shown strong antibacterial activity against Escherichia coli and Staphylococcus aureus bacteria. For instance, stannite Cu2FeSnS4 and mohite Cu2SnS3 were the most active against E. coli, whereas kesterite Cu2ZnSnS4 and rhodostannite Cu2FeSn3S8 exhibited the highest antibacterial activity against S. aureus. In general, stannite has shown the best antibacterial properties out of all the studied samples. Five out of twelve products have been prepared using mechanochemical synthesis for the first time in a scalable fashion here. The presented synthetic approach is a promising alternative to traditional syntheses of nanomaterials suitable for biological applications and shows ternary and quaternary sulfides as potential candidates for the next-generation antibacterial agents.

Green Synthesis of Silver Nanoparticles with Antibacterial Activity Using Various Medicinal Plant Extracts: Morphology and Antibacterial Efficacy.

A green synthetic route for the production of silver nanoparticles (AgNPs) using five different aqueous plant extracts, namely, Berberis vulgaris, Brassica nigra, Capsella bursa-pastoris, Lavandula angustifolia and Origanum vulgare, was investigated in this study. The present work demonstrates the influence of plant extract composition (antioxidant and total phenolic content) on the size and morphology of the produced AgNPs. The biosynthetic procedure was rapid and simple and was easily monitored via colour changes and ultraviolet and visible (UV-Vis) spectroscopy. Subsequently, measurement of zeta potential (ZP), photon cross-correlation spectroscopy (PCCS), X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM) and selected area electron diffraction (SAED) analysis were employed to characterise the as-synthesised nanoparticles. The XRD investigation confirmed the presence of Ag0 in the nanoparticles, and interactions between the bioactive compounds of the plants and the produced AgNPs were evident in the FTIR spectra. TEM indicated that the nanoparticles exhibited a bimodal size distribution, with the smaller particles being spherical and the larger having a truncated octahedron shape. In addition, the antimicrobial activity of the AgNPs was tested against five bacterial strains. All synthesised nanoparticles exhibited enhanced antimicrobial activity at a precursor concentration of 5 mM compared to the control substance, gentamicin sulphate, with the best results observed for AgNPs prepared with B. nigra and L. angustifolia extracts.

Mechanochemistry as an Alternative Method of Green Synthesis of Silver Nanoparticles with Antibacterial Activity: A Comparative Study.

This study shows mechanochemical synthesis as an alternative method to the traditional green synthesis of silver nanoparticles in a comparative manner by comparing the products obtained using both methodologies and different characterization methods. As a silver precursor, the most commonly used silver nitrate was applied and the easily accessible lavender (Lavandula angustofolia L.) plant was used as a reducing agent. Both syntheses were performed using 7 different lavender:AgNO3 mass ratios. The synthesis time was limited to 8 and 15 min in the case of green and mechanochemical synthesis, respectively, although a significant amount of unreacted silver nitrate was detected in both crude reaction mixtures at low lavender:AgNO3 ratios. This finding is of particular interest mainly for green synthesis, as the potential presence of silver nitrate in the produced nanosuspension is often overlooked. Unreacted AgNO3 has been removed from the mechanochemically synthesized samples by washing. The nanocrystalline character of the products has been confirmed by both X-ray diffraction (Rietveld refinement) and transmission electron microscopy. The latter has shown bimodal size distribution with larger particles in tens of nanometers and the smaller ones below 10 nm in size. In the case of green synthesis, the used lavender:AgNO3 ratio was found to have a decisive role on the crystallite size. Silver chloride has been detected as a side-product, mainly at high lavender:AgNO3 ratios. Both products have shown a strong antibacterial activity, being higher in the case of green synthesis, but this can be ascribed to the presence of unreacted AgNO3. Thus, one-step mechanochemical synthesis (without the need to prepare extract and performing the synthesis as separate steps) can be applied as a sustainable alternative to the traditional green synthesis of Ag nanoparticles using plants.

Biological activity of selected lichens and lichen-based Ag nanoparticles prepared by a green solid-state mechanochemical approach.

Lichens dispose a wide spectrum of bioactive compounds known as secondary metabolites. Their biological effects like antioxidant and antibacterial activities are widely studied. Green synthesis of silver nanoparticles (AgNPs) is a method where the compounds/substances present in plants are used for reduction of AgNO3instead of toxic chemicals. However, this methodology is usually a two-step process (extract preparation step and the synthesis step) performed under the elevated temperatures nad in the case of lichens, the redicing compounds are insoluble in water. These disadvantages can be overcome by a solid-state mechanochemical synthesis applied in the present study. As microorganisms are becoming more resistant to commercial antibiotics, AgNPs prepared in an environmentally friendly way represent an interesting alternative. In the present study, we compared the processing of lichen material of Pseudevernia furfuracea and Lobaria pulmonaria for extraction as well as for synthesis of AgNPs, and tested the antibacterial and antioxidant activity of the extracts. Both selected lichen species could be successfully used as reducing agents to produce AgNPs. Six different bacterial strains were tested for antibacterial activity of AgNPs-containing products and it was highly effective on all strains. However, the antioxidant activity of lichen extracts showed the lowest effect even if AgNPs are present which positively correlated with the content of total phenols and flavonoids. Both phenols and flavonoids are natural antioxidants and react with silver nitrate. Due to this fact, we observed a decrease of total phenols, total flavonoids as well as antioxidant activity when processing of lichen extracts with silver nitrate was used. We demonstrated that the formation of AgNPs increased the antibacterial activity but on the other hand reduced the antioxidant activity. Thus, antibacterial and antioxidant effects have to be treated differentially.

Sustainable One-Step Solid-State Synthesis of Antibacterially Active Silver Nanoparticles Using Mechanochemistry.

A combination of solid-state mechanochemical and green approaches for the synthesis of silver nanoparticles (AgNPs) is explored in this study. Thymus serpyllum L. (SER), Sambucus nigra L. (SAM) and Thymus vulgaris L. (TYM) plants were successfully applied to reduce AgNO3 to AgNPs, as confirmed by X-ray diffraction analysis, with SER being the best reducing agent, and TYM being the worst. The experiments were performed via a one-step planetary milling process, where various AgNO3:plant mass ratios (1:1, 1:10, 1:50 and 1:100) were investigated. Atomic absorption spectrometry indicated that the stability of the mechanochemically produced AgNPs increased markedly when a sufficiently large quantity of the reducing plant was used. Furthermore, when larger quantities of plant material were employed, the crystallite size of the AgNPs decreased. TEM analysis revealed that all AgNPs produced from both AgNO3:plant ratios 1:1 and 1:10 exhibit the bimodal size distribution with the larger fraction with size in tens of nm and the smaller one below 10 nm in size. The antibacterial activity of the produced AgNPs was observed only for AgNO3:plant ratio 1:1, with the AgNPs prepared using SER showing the greatest antibacterial properties.

Comprehensive mapping of the cell response to E. coli infection in porcine intestinal epithelial cells pretreated with exopolysaccharide derived from Lactobacillus reuteri.

Bacterial exopolysaccharides (EPSs) are known to modulate immunity. To date, a plethora of studies have reported the effect of EPSs on intestinal cells; however few works have revealed a complete picture of the signalling events in intestinal epithelial cells induced by bacterial EPSs. Here, using transcriptomics, we comprehensively mapped the biological processes in porcine intestinal epithelial cells challenged with EPS derived from Lactobacillus reuteri alone, enterotoxigenic Escherichia coli (ETEC) or ETEC after pretreatment with EPS. The Gene Ontology analysis of differentially expressed genes (DEGs) showed that ETEC is able to evoke biological processes specifically involved in cell junction reorganization, extracellular matrix degradation, and activation of the innate immune response through the activation of pattern recognition receptors, such as TLRs and CTRs. A total of 495 DEGs were induced in ETEC-challenged cells. On the other hand, EPS pretreatment was able to attenuate overexpression of the genes induced by ETEC infection. The most relevant finding of this study is that EPS has a suppressive effect on the inflammatory response evoked by ETEC infection. On the basis of high-throughput RNA-seq, this report is the first to describe the effects of EPSs derived from L. reuteri used as a pretreatment of global gene expression in porcine epithelial cells.

Exopolysaccharides of Lactobacillus reuteri: Their influence on adherence of E. coli to epithelial cells and inflammatory response.

The aim of the study was to characterize exopolysaccharides (EPS) originated from Lactobacillus reuteri strain DSM 17938 (EPS-DSM17938) and L. reuteri strain L26 Biocenol™ (EPS-L26) and evaluate their influence on adherence of enterotoxigenic Escherichia coli (ETEC) to IPEC-1 cells and proinflammatory gene expression. Both EPS were d-glucan polysaccharides with higher molecular weight (Mw), but differing in spatial conformation and elicited variable cytokine profile. EPS-DSM17938, relatively linear polysaccharide with (1→4) and (1→6) glycosidic linkages, increased IL-1ß gene expression (0.1mg/mL; P<0.05), while EPS-L26, more branched polysaccharide with (1→3) and (1→6) glycosidic linkages, exerted slight but statistically significant up-regulation of NF-κB, TNF-α and IL-6 mRNA (P<0.05). The most significant finding is that preincubation of IPEC-1 cells with both EPS followed by ETEC infection inhibit ETEC adhesion on IPEC-1 cells (P<0.01) and ETEC-induced gene expression of proinflammatory cytokine IL-1ß and IL-6 (P<0.01).

Phytotoxic and Antibacterial Activity of Essential Oil of New Peppermint Cultivar.

A new menthol cultivar of Mentha x piperita L. bred in East Slovakia was evaluated for the biological activity of its essential oils (EOs). The content and composition of the EO components changed during plant development and the different effects of samples collected each month (April - September) within the growing season were noted. EOs are considered to be an important source of potential allelochemicals. Changes in EO composition influenced phytotoxic activity. Stimulation and inhibition of seed germination and root elongation occurred with different EO concentrations. The four tested bacterial strains: presented different resistance to the samples collected in different growing periods.

In vitro study of biological activities of anthocyanin-rich berry extracts on porcine intestinal epithelial cells.

BACKGROUND: Anthocyanins, compounds that represent the major group of flavonoids in berries, are one of the most powerful natural antioxidants. The aim of this study was to evaluate biological activities and comparison of anthocyanin-rich extracts prepared from chokeberry (Aronia melanocarpa), elderberry (Sambucus nigra), bilberry (Vaccinium myrtillus) and blueberry (V. corymbosum) on the porcine intestinal epithelial IPEC-1 cell line. RESULTS: The IC50 values calculated in the antioxidant cell-based dichlorofluorescein assay (DCF assay) were 1.129 mg L(-1) for chokeberry, 1.081 mg L(-1) for elderberry, 2.561 mg L(-1) for bilberry and 2.965 mg L(-1) for blueberry, respectively. We found a significant negative correlation (P < 0.001) between cyanidin glycosides content and IC50 values. Moreover, extracts rich in cyanidin glycosides stimulated proliferation of IPEC-1 cells and did not have cytotoxic effect on cells at an equivalent in vivo concentration. CONCLUSIONS: We found that the chokeberry and elderberry extracts rich in cyanidin glycosides possess better antioxidant and anticytotoxic activities in comparison to blueberry or bilberry extracts with complex anthocyanin profiles.

Flax-seed oil and Lactobacillus plantarum supplementation modulate TLR and NF-κB gene expression in enterotoxigenic Escherichia coli challenged gnotobiotic pigs.

The present study analyses the effect of flax-seed oil rich in n-3 polyunsaturated fatty acids (PUFAs), the probiotic strain Lactobacillus plantarum - Biocenol™ LP96 and their combination on the expression level of selected Toll-like receptor (TLR) genes (TLR2, TLR4, TLR5, TLR9) and their downstream molecules (myeloid differentiation factor 88, MyD88; nuclear factor-κB, NF-κB) in the jejunum of gnotobiotic pigs challenged with enterotoxigenic Escherichia coli (ETEC). The results show that both immunomodulators are able to modulate the RNA level of at least one of the target molecules and thus regulate pathogeninduced inflammation. We confirmed that not only probiotic lactobacilli or flaxseed oil alone but also their synergistic action has great potential in the prevention and treatment of porcine colibacillosis. The results give an insight into one of the possible mechanisms by which natural agents, such as probiotic lactobacilli and flax-seed oil, exert their immunoregulatory properties during pathogen-induced inflammation.

Variation in the coding region of the prion protein gene in Slovak cattle.

This study was conducted to investigate the presence of single nucleotide polymorphisms (SNPs) in the coding region of the bovine prion protein (PrP) gene among healthy and bovine spongiform encephalopathy (BSE-) affected cattle in Slovakia. Denaturing gradient gel electrophoresis (DGGE) and single-strand conformation polymorphism (SSCP) followed by DNA sequencing were used to identify SNPs and variations in octapeptide repeats. Altogether three single nucleotide polymorphisms (g234a, c339t and c576t) and variations in the number of octapeptide repeat units (5 or 6) were found in the analysed part of the prion protein gene. All single nucleotide polymorphisms were silent, causing no amino acid changes. Significant differences (P < 0.05) in the genotype distribution of g234a polymorphism were observed when the homozygous genotype with a mutated allele (caa/caa) was compared to the heterozygous genotype -/cag among healthy and BSE-affected cattle. The homozygous genotype caa/caa was characteristic of the group of BSE-affected cattle. Additionally, the homozygous genotype caa/caa was significant for the group of Simmental crossbreeds among healthy cattle. The allele and genotype distribution of the other polymorphisms was not significantly different among groups of healthy and BSE-affected cattle. The possible influence of a silent mutation on expression of the gene is not clearly determined and needs further investigations.

IS900-PCR-based detection and characterization of Mycobacterium avium subsp. paratuberculosis from buffy coat of cattle and sheep.

Johne's disease is one of the main causes of economic losses in ruminants and a major health hazard in the developing and developed world. Up till now, many microbiological, serological and molecular methods have been tried for the detection of Mycobacterium avium subsp. paratuberculosis (MAP). In this study, we attempt a PCR-based detection of IS900, distinct insertion sequences of MAP from the buffy coat of cattle (n=262) and sheep (n=78), and direct genotyping by single strand conformational polymorphism (SSCP). A total of 30 (11.45%) cattle and one sheep (1.28%) were positive for MAP-IS900. This IS900-based PCR detection proved highly specific, particularly when tested on other non-MAP strains. SSCP analysis grouped the MAP-IS900 into four distinct clusters based on different band patterns. Nucleotide sequence variability between MAPs detected from sheep (GenBank accession ) and cattle (GenBank accession -) was noticed in the study. Although, in recent years IS900-PCR-based detection of MAP from WBCs is being used in human, its use in animals is still limited. Our work not only supports its use in animals but also suggests further IS900-SSCP-based MAP-genotyping, coupled with DNA sequencing, as a promising tool for rapid and effective Johne's disease surveillance.