font creator 12  - Free Activators

Copy & paste multiple lines of text to create new tasks from each line. 12. See every task in a project and its sub-projects by using “##” in a filter. Cutting Edge: IL-10-Independent STAT3 Activation by Toxoplasma gondii Mediates Suppression of IL-12 and TNF-α in Host Macrophages. Briefly, cells dissociated from colliculi of 14- to 15-d-old Swiss mouse embryos were plated in serum-free medium in 12-well culture dishes (0.8 × 106. font creator 12  - Free Activators

Font creator 12 - Free Activators -

Tell Your Story the Way You Imagined

Create Professional Level Edits Effortlessly

Intuitive editing tools that help you set objects in motion, completely transform a scene's location, and create cinematic effects.

  • *Click on title text to see preview

Let AI Do the Heavy Lifting

Simplify your creative workflow with AI—you’ll be amazed at how quickly you finish your projects.

  • Enhance or totally replace the sky for perfect scenic shots.

  • Intelligently apply masks to seamlessly remove objects from images.

  • Remove gusts of wind for crisp, clear, distraction-free audio.

  • Reduce unwanted background noise for refined, high-quality vocal tracks.

Limitless Possibilities, Right at Your Fingertips

Bring your imagination to life with our wide array of tools to help customize your projects. 

Vast Library of Creative Assets With New Monthly Releases

Move, Capture, Edit, Go!

Create videos that bring back the intensity and adrenaline captured with your action camera, with editing features designed specifically for sports enthusiasts.

  • Video Stabilizer

    Fix shaky footage instantly to correct unwanted camera movement.

    Lens Correction

    Offset imperfections such as vignetting and fish-eye distortion.

    Video Speed Controls

    Speed up or slow down the speed of your clips to create your own video effects.

    Zoom and Pan Effects

    Move closer to the action and create camera movement.

    Video Collage Designer

    Select from a range of templates and adjust each video's playback options.

    MultiCam Designer

    Synchronize up to four video clips and perform real-time mixing.

Finish Your Projects in No Time

Experience the industry's fastest video rendering thanks to CyberLink's optimization expertise for both software and hardware acceleration.

True 4K Rendering Speed

DCI Cinema 4K Footage

Sony Full HD Rendering Speed

Sony A55 Camera Full HD Footage

GoPro 2.7K Rendering Speed

GoPro 2.7K Full QHD Footage

MultiCam Rendering Speed

2x2 PiP Full HD AVCHD Footage

PowerDirector

Competitor  C

Competitor  A

Competitor  W

Competitor  V

Time(sec)

Software Encoding

Hardware Encoding

* Hardware encoding uses both a dedicated video graphics card and a CPU to encode the video file, whereas software encoding is only done by the CPU.

Fast

Up to

0.0

Faster video
rendering speeds

Learning Resources

Learn how to create professional masterpieces with our tutorials below:

  • How to Convert a GIF to Video [PC, Mac, App, & Online]

    Step 1: Import your GIF into PowerDirector. Step 2: Add the GIF to your timeline Step 3: Export your project as a video file.

    Read More
  • How to Edit Videos on Your Phone [iPhone & Android] Step by Step Guide

    How to trim a video - Step 1: Open the app and create a new project. Step 2: Add your video to the timeline and move the sliders on each side of the clip. Step 3: Tap the play button to preview your clip.

    Read More
  • How to Record an Audiobook

    Step 1: Record the narration. Step 2: Edit and Engineer the audio. Step 3: Enhance your audio for publishing standards

    Read More

More Tutorials

Источник: https://www.cyberlink.com/prog/product/html/32183/365/features.jsp

Transition-metal-free intramolecular Friedel–Crafts reaction by alkene activation: A method for the synthesis of some novel xanthene derivatives

Our starting alkenes 4al are original and were synthesized in four steps involving coupling, Grignard, oxidation, and Wittig reactions. We synthesized the novel unactivated alkenes 4al containing three aryl groups as the starting materials. The synthesis of 4a is demonstrated in Scheme 1. First, 2-phenoxybenzaldehyde (1a) was synthesized by coupling reaction of phenol with commercial 2-fluorobenzaldehyde. This reaction was carried out with very high yield by refluxing the reactants in the presence of K2CO3 in DMF.

[1860-5397-17-142-i1]

2-Phenoxybenzaldehyde (1a) was converted into secondary alcohol derivative 2a by adding a phenyl group using a Grignard reaction. This reaction was carried out with high yield by adding the freshly prepared Grignard compound of phenyl bromide to 2-phenoxybenzaldehyde. As a result, phenoxy secondary alcohol 2a containing three aromatic rings was obtained. In the third step, 2a was oxidized and the ketone derivative 3a was obtained in high yield. The oxidation reaction was carried out using PCC in DCM at room temperature. In the fourth and final step, phenoxydiphenylalkene derivative 4a was prepared by a Wittig reaction, which was carried out using methyltriphenylphosphonium bromide with ketone 3a, in basic medium, at room temperature, and dry THF.

After the structures of the starting compounds were elucidated, the method development trials for the synthesis of xanthene derivatives were carried out. For this purpose, catalyst researches were carried out using compound 4a. An intramolecular Friedel–Crafts reaction was tried by activating the alkene with various organic Brønsted acids and Lewis acids (Table 1). In the reaction, iron(III) chloride hexahydrate, trifluoroacetic acid (TFA), N-trifylphosphoramide (NTPA), benzoic acid, diphenyl phosphate (DPP), malonic acid, chloroacetic acid, copper(II) triflate, acetic acid, and p-toluenesulfonic acid (p-TSA) were used as catalysts. TFA gave the best yield of these catalysts with 78% (Table 1, entry 2). The second-best yield was 65% when FeCl3·6H2O was used (Table 1, entry 1).

[Graphic 1]
Entry Catalyst (10 mol %) Conv. (%)b
1 FeCl3·6H2O 65
2TFA78
3 NTPA 10
4 benzoic acid 0
5 DPP 2
6 malonic acid 0
7 chloroacetic acid 14
8 Cu(OTf)2 12
9 AcOH 0
10 p-TSA 3

aConditions: 4a (0.1 mmol) and catalyst (10 mol %) in CHCl3 (1 mL) were stirred at room temperature for 24 hours. bConversions were determined with GC–MS.

Then the solvent was investigated. Toluene, methyl alcohol, ethyl acetate, THF, DMF, dichloromethane, chloroform, acetone, and acetonitrile were tested as solvents. As a result, it was determined that the best conversion was with dichloromethane (Table 2). Later, quantity and time experiments were performed (Table 2, entries 10–15) and, at the end of these trials, it was determined that the reaction was completed with >99% conversion in 6 hours with 10 mol % catalyst at room temperature (Table 2, entry 14).

[Graphic 2]
Entry Cat. amount (mol %) Solvent Time Conv.b
1 10 CHCl3 24 h 78
2 10 acetone 24 h 75
3 10 toluene 24 h 70
4 10 CH2Cl2 24 h >99
5 10 THF 24 h 45
6 10 CH3CN 24 h 10
7 10 MeOH 24 h 0
8 10 EtAc 24 h 35
9 10 DMF 24 h 0
10 10 CH2Cl2 1 h 32
11 10 CH2Cl2 2 h 53
12 10 CH2Cl2 3 h 70
13 10 CH2Cl2 4 h 85
1410CH2Cl26 h>99
15 5 CH2Cl2 6 h 95

aConditions: 4a (0.1 mmol) and TFA in dry solvent (1 mL) were stirred at room temperature. bConversions were determined with GC–MS.

After determining the most suitable conditions for the intramolecular Friedel–Crafts reaction with alkene activation, the synthesis of the new xanthene derivatives was performed according to this method. The synthesized xanthene derivatives with their isolated yields are shown in Figure 1. Compounds 5bl were synthesized for the first time in this study. The first synthesis of 5a was prepared by reduction of the corresponding xanthydrol [53] and was also synthesized recently by a different method from our group [29].

[1860-5397-17-142-1]

Although in the reactions for FCA reagents and strong inorganic acids, such as AlCl3, H2SO4, or H3PO4, which have generally corrosive properties, were used, in this study, an intramolecular ring closure reaction was carried out under easy operating conditions with an organic Brønsted acid catalyst with high yields. So, the xanthene synthesis with alkene activation was performed for the first time using TFA. The reasonable mechanism of this reaction is delineated in Scheme 2.

[1860-5397-17-142-i2]

Despite this reaction occurring by the classical Friedel–Crafts mechanism, we believe that o-quinone methide is formed as an intermediate. Because of its very reactive structure, most of the xanthene synthesis is based on the o-quinone methide intermediate [54-58]. The carbocation formed by the activation of an alkene with acid turns into an intermediate o-quinone methide, resulting in a successful cyclization.

As seen in the mechanism, the acid catalyst adds to the vinyl group, allowing the formation of a tertiary carbocation. The carbocation is then transformed into the o-quinone methide intermediate, which undergoes cyclization to yield 9-methyl-9-arylxanthene by aromatization. When the yields of the synthesized compounds are examined, it is seen that the yields are high when there is no substituent at the ring to which the carbocation is attached or when there is an electron-donating group, such as a methoxy or methyl group (Figure 1). When there are electron-withdrawing groups, such as chlorine or cyano groups, in the ring, the yield is slightly reduced because they deactivate the ring in the transition state. The lowest yield was observed for compound 5j to which a cyclopentyl group is attached. Since the cyclopentyl group in this compound is directly attached to the carbon from which the carbocation is formed, byproducts are formed and the yield is reduced since conversions can occur.

Источник: https://www.beilstein-journals.org/bjoc/articles/17/142

Microsoft Office 2019 Activation Key + Crack ISO Free Download

Microsoft Office 2019 Activation Key + Crack ISO Free Download

Microsoft Office 2019 Activation Key is a cross-platform that is comprehensive for everyone with smart tools for specific users, teams, and businesses. It can open your apps, documents anywhere on numerous products. It offers brand new compliance and deployment protection, giving companies more significant control of sensitive data and greater flexibility in deployment and management. Microsoft has expanded offices for iPad and Android pills. Updated Office features on Mac, iPhone, plus the web.

Additionally, brand new applications have been added to your family, a swipe and office lens. All things are made to keep work everywhere. We have concentrated on customizing Office for different platforms in the last 12 months. Office on the Windows desktop is Central to our strategy when you’ve seen just how.

Microsoft Office 2019 Crack is the latest version. It is the most demanded software ever. Everyone uses MS Office for their projects. Simply put, now you can’t do anything without Microsoft Office 2019 Crack. And one of the many difficulties that primary users can’t activate this version quickly. For sure, the user needs the Microsoft Office 2019 activation key for the activation of the software. So then you will solve your problem on this important platform if you’re certainly one of them. Right here, you’ll find working and activation that is valid for lifetime activation.

Microsoft Office 2019 Crack Download Full ISO

Microsoft Office 2019 Crack is utterly appropriate for its wide range – from students to office staff and personnel, along with business professionals. Due to the different enhancements, the essential functions, and the development of new people, document creation, processing, and handling are now doable and faster than previously. In addition, mathematical calculations and expressions are improved and generally are far more easily incorporated into practical use within PowerPoint 2016, Excel 2016, and Word 2016.

Microsoft Office 2019 Activation Key is useful for both Windows as well as a Mac users. It can open, edit, make, and save files regarding the cloud from your desktop. And a search that is new for commands is available in MS PowerPoint, Excel, and Outlook. Updated choices include its user software that supports Retina Display and uses ribbons as well. In addition, users can now share documents via social networks and email messages directly through the MS Office 2016 Crack toolbar.

Key Features of Microsoft Office 2019 Crack:

  • Better along with Windows 10– allow you to complete work, the papers in full fidelity across your Windows 10 products.
  • Better Control over Resource Scheduling– ensures that scarce resources are now being utilized effectively throughout the company.
  • You are permitted by the application to enter everything you wish to do in your terms, then guide you on how best to do so – plus other resources.
  • Modern shapes benefit from newly redesigned forms in Visio professionals.
  • Real-time typing-view everyone’s edits and where these are typically into the document as you work simultaneously on the same document.
  • New chart types– present better visualization ability and enables you to identify the partnership that is statistical your computer data.
  • Improved data connectivity-connecting your diagram to Excel data is just one click away.
  • Themes – the latest themes give a fresh look and an amazing presentation.
  • Smart lookup checks your document and automatically teaches you the SERPs via your writing environment via the web.
  • One drive integration – enables you to store your details in one access and location other than anywhere, using any device.

What’s New in Microsoft Office 2019 Crack?

  • Powerpoint, one notes, Skype, PowerPoint, Access, Excel, Skype, and Outlook are the most popular and advanced applications.
  • Creates presentations and creates computer analysis records.
  • Tests the emails from Outlook.
  • It does not need VPN settings from anywhere to call.
  • In the layout parameter, it will also automatically customize the UI.
  • All papers, including PDF e-books, are very much sponsored.
  • Updates and edits the database and prints a selection of templates as well.
  • Use Excel to create tables and solve problems with mathematics.
  • It can also view and edit device-wide files.
  • Create amazing Microsoft PowerPoint 2019 presentations.
  • Besides, we can work on the same project with our team concurrently.
  • Get permission automatically and post the reports with one touch.
  • New charts for displaying nuanced details are also included.

Microsoft Office 2019 Activation Key

QZA3W-SE4XD-5CR6T-FV7BGY-8HUN9

J8BY7T-V6CR5-E4ZWS-EXD5CT-FVBG8H

UN9JIH-8UBYV7-CR65X-E4SZX-DCRTFV

BG8NH-UIJMK-OJIN9U-B8Y7TV-6C5RE

X4SDC-RTFVBG-8NHUI-JM0KO-M9NUB

8Y7TV6-DC5RE4-SXDCR-TFVBG-Y8NHU

Microsoft Office 2019 Key 2022

IJM9NH-8UBY7T-6DC5R-E4SEXD-C5RTF

VBY8NH-UIJ98N-HBGTFV6-E4SX-Z3WA

4SXDCE-TFV68N-HYU9IJ-MN8H-UTFV6

D5RE4S-ZW3E4-XTFV6B8-HYNU-J9IMN

HB8UTF-V6E4SX-ZW3AS-E4XTFV-6B8HY

NUJ8HB-YV7T5R-XE4SZA-W3ZSE-4XTFV

Microsoft Office 2019 Product key ISO

6B8YNH-U9B8Y-V7T6D5R-ESZWA-3SE4

XTFV68-NHUJ9-IH8BGF-DCXE4-SZ4XT

FV68NH-UBGFV6-DC5SX4-ZKWS-4XETF

V6BY8-NHU9IN-8YT76R-E54W-O3WAZ

Benefits of Using Microsoft Office 2019 Activation Key?

Also, several individuals will concurrently work on the paper or an Excel spreadsheet or PowerPoint. This functionality was updated by Microsoft and is gradually being marketed now. Not everybody will be compelled to go to the cloud, but there’s a lot of push with subscription providers. And one thing to do is, even now, the Office 365 Crack has such a feature as more, for example, or PowerPoint concept concepts, which are not routinely available for the Office 2016 Crack. It is a database adapted so that certain features bring users into the cloud. Office 2019 presents lots of new features and some of the coolest I’ll introduce. Many of them are not exactly new functions. But they were exclusive to office 365, so you can now get access to them if you do not want to pay the monthly fee.

Microsoft Office 2019 Keys

  • ZAQ3W-S4XED5-CRF6TV-B8HYN-UJ9IHU
  • 8BYV7T-6CD5RX-E4SZEX-TFV68N-HUIJM
  • K0OMIJ-98HUBVY-7TDC5-RESX4-SXTFV6
  • 8NHUIJ9-M0KJ9N-H8UTFV6-D5RXE-4STF
  • V6BG8H-UNIJM9-8NHBG-TFV6D-5RE4D5
  • XCRTFV-BGHUNIJ-9M09N-HBGY7-TFVDC
  • 5RXE4S-SX5DCR-TFV7BG-8HUN9IJ-MN8H
  • UTFV6D-5RESX4-X5DC6TF-V7B8H-UN9IJ
  • MNH8-UT6D5R-ESXDR-C6TFVGB-YHUNIJ

Microsoft Office 2019 Product Key

  • AZWSX-EDC5RF-V6TGY-BH87V-F6C5X
  • 4SZAW-SEXD5-CTFVG-YBHU8-FT6CD
  • 4ZA3W-Z4SEX-DCRTF-GBY8G-V7C6D
  • 5RZSE4-ZSE5D-RC6TF-V7BGY-H8VFC
  • 6D5XSZ-4AWE5-SXDCR-6GVY-BHUGV
  • FCD5R-SXEZ4A-WZ4E-SXDCT-FVGYB8
  • YFV6D-C5SXEA-4WZESX-DRCTF-VGYH

Installation Instructions for Office 2019 Crack:

  1. The download is given below to start.
  2. Extract zip file through WinZip.
  3. Click “setup” and proceed with the installation process.
  4. Complete configuration and close it.
  5. Open MS Toolkit located inside the crack directory.
  6. Click the button to the left of the Windows icon and go to the Service tab.
  7. Activate” Microsoft Office 2019.
  8. Done!
Источник: http://www.cci.ci/

Oral vaccination of piglets against Mycoplasma hyopneumoniae using silica SBA-15 as an adjuvant effectively reduced consolidation lung lesions at slaughter

Abstract

Mycoplasma (M.) hyopneumoniae is the main pathogen of porcine enzootic pneumonia (PEP). Its controlling is challenging, and requires alternative strategies. This study aimed to develop an oral vaccine against M. hyopneumoniae using a nanostructured mesoporous silica (SBA-15) as an adjuvant, and compare its effect with an intramuscular (IM) commercial vaccine (CV). Fifty 24 day-old M. hyopneumoniae-free piglets composed five equal groups for different immunization protocols, consisting of a CV and/or oral immunization (OI). Control piglets did not receive any form of immunization. All piglets were challenged with M. hyopneumoniae strain 232 on D49 by tracheal route. IgA antibody response in the respiratory tract, bacterial shedding and serum IgG were evaluated. The piglets were euthanized on 28 (D77) and 56 (D105) days post-infection. Lung lesions were macroscopically evaluated; lung fragments and bronchoalveolar fluid (BALF) were collected for estimation of bacterial loads by qPCR and/or histopathology examination. All immunization protocols induced reduction on Mycoplasma-like macroscopic lung lesions. IgA Ab responses anti-M. hyopneumoniae, the expression of IL-4 cytokine and a lower expression of IL-8 were induced by CV and OI vaccines, while IgG was induced only by CV. Oral immunization using silica as a carrier-adjuvant can be viable in controlling M. hyopneumoniae infection.

Introduction

Mycoplasma hyopneumoniae (M. hyopneumoniae) is the main causative pathogen of porcine enzootic pneumonia (PEP), a chronic respiratory disease in pigs, and one of the main pathogens involved in the porcine respiratory disease complex (PRDC)1. The infections caused by this bacterium are highly prevalent worldwide and result in financial losses for the pig industry, mainly due to the costs of treatment and vaccination, decreased performance, and increased mortality from secondary infections2.

The microorganism's adhesion to the respiratory epithelium, the stimulation of a prolonged inflammatory reaction, the suppression and modulation of innate and adaptive immune responses favoring the pathogen are recognized as important steps in the colonization and infection by this microorganism. As a result, infected animals become more susceptible to infections by other respiratory pathogens1. As in other animals, most porcine pathogens cross mucous surfaces when ingested or inhaled, due to contamination of food, environment and fecal matter. Systemic vaccination generally promotes little stimulation of mucosal associated lymphoid tissue (MALT) and, therefore, the host immune system can only fight against the pathogen after its entering into the body3,4.

In the mucosal lymphoid tissues, mature T cells and B cells are stimulated by antigen and induce IgA antibody response. These cells migrate from the submucosal lymphoid tissue by the bloodstream to the lamina propria, where B cells differentiate into plasma cells secreting dimeric IgA antibodies. Many of these cells return to the original mucosal surface, but others can be found at different mucosal surfaces, so that oral immunization can lead to a migration of IgA precursor B cells to the bronchi, which subsequently secreted IgA antibodies in the bronchial mucosa5.

Previous studies with other pathogens have demonstrated the feasibility of using oral immunization as a strategy for inducing protective immunity in the swine reproductive tract, reinforcing the interconnection between different mucosal sites6. The secretory IgA (SIgA) specific antibodies have been considered as a crucial factor in protecting pigs against infection by M. hyopneumoniae7, while local humoral immunity seems to play an important role in this infection. SIgA is the main effector of respiratory tract mucosa immunity, which can form a protective barrier to eliminate respiratory invading pathogens and prevent infection and active colonization8. Since mucosal immunity has the potential to control pathogens at their portal of entry, it would be advantageous to develop vaccines that trigger a mucosal and systemic immune response rather than simply stimulating the systemic immune system9.

Promising M. hyopneumoniae bacterin formulations have been identified based on their capacity to induce strong innate immune responses10. Limitations in the use of adjuvants for vaccine formulation can be found in the literature, such as toxicity, the ability to lead to an immune response against the agent and not against the adjuvant, induction of adverse reactions, among other factors11. Since the silica has the characteristic of incorporating and releasing molecules12, the mesoporous silica particles have gained attention due to its potential role as adjuvants. Its toxicity to respiratory cells has been described13,14 and depends on the physicochemical properties of particles, size and concentration15. On the other hand, its use may enhance antigen-specific cellular immune responses in dendritic and Langerhans cells15. In addition, it is able to stimulate the immune system in a similar or superior way than other adjuvants, such as the Incomplete Freund's Adjuvant16. Moreover, an improvement in the recruitment of defense cells was observed, which led to an increase in phagocytosis and processing by the gut antigen-presenting cells17,18,19,20.

Based on the importance of developing oral vaccine adjuvants that are efficient in presenting antigens to the cells of mucosal lymphoid tissues (MALT), the objectives of this study were (i) to develop an oral vaccine specific to M. hyopneumoniae by encapsulating a blend of proteins of this bacterium into the silica (SBA-15); (ii) to stimulate the immune responses of the respiratory mucosa; (iii) to evaluate the efficacy of the protection induced by this vaccine against experimental infection with a virulent strain of M. hyopneumoniae, compared to a commercial inactivated vaccine.

Results

Mycoplasma-like macroscopic and microscopic lung lesion score at slaughter

At slaughter, gross lesions were mainly located in the apical and cardiac lung lobes, cranio-ventral portions of diaphragmatic lobe, and in portions of the intermediate lobe. All immunized groups showed lower lung lesion scores when compared to the control.

The medians of macroscopic lung lesion scores at the first slaughter (28dpi), followed by the lowest and higher scores observed in each group, were: CV-4.8% (0–24.5%), OI-2.5% (2–9.8%), CV + OI-2.7% (0–13.5%), OI + OI-9.6% (0.5–10.9%) and CONT-32.0% (15.6–41.7%). At the second slaughter (56 dpi), the values obtained were: CV-0.0% (0–8.8%), OI-1.3% (0.8–2.9%), CV + OI-3.5% (0–14.3%), OI + OI-8.8% (2.6–12.9%) and CONT-21.3% (17.4–24.9%). All immunized groups showed significant differences (Dunn test, p-value = 0.0229) in the total Mycoplasma-like lung lesion area when compared with the percentage obtained in the control. Significant differences were observed between the immunized groups and the control, but not between the immunized groups at both post-infection time-points (Fig. 1A). A difference in consolidation lung lesion score of 85% in CV, 92% in OI, 91% in CV + OI, 70% in OI + OI was observed in the first slaughter, while in the second slaughter the difference was of 100% in CV, 94% in OI, 83% in CV + OI, and 59% in OI + OI. Representative photos of consolidation lung lesion from each group are shown below (Fig. 1B).

(A) Comparison of Mycoplasma-like macroscopic lung lesions extent observed at slaughter of piglets, 28- and 56-days post-infection with M. hyopneumoniae. Means followed by the same letter do not differ statistically from each other (Tukey test). (B) Representative photos of Mycoplasma-like macroscopic lung lesions extent observed at slaughter of piglets, 28- and 56-days post-infection with M. hyopneumoniae.

Full size image

Microscopically, all groups showed histological lesions score varying between 3 and 4, characterized as PEP-specific (Fig. 2A,B). No significant differences were found, neither between groups, nor between the two post-infection intervals. No significant correlations were observed between macro and microscopic analysis (Kruskall-Wallis, p-value = 0.341).

(A) Photomicrography of histological lung lesion characterized by (a) hyperplasia of lymphoid follicles (arrow) (100 ×); (b) inflammatory infiltrate predominantly compound by macrophages (400 ×); (c) amorphous and acidophilic material in addition to inflammatory infiltrate in the light of the alveoli (arrow) (100 ×); (d) inflammatory infiltrate in bronchioles (arrow) (400 ×); (e) light of the alveoli without noteworthy changes (arrow) (100 ×); (f) normal lymphoid follicle (arrow) (40 ×). (B) Percentage of animals showing different microscopic lung lesion score (0–4) according to each vaccination protocol. No significant differences were observed between groups.

Full size image

Detection of IgA and IgG anti-M. hyopneumoniae antibodies in nasal swabs, serum and BALF samples

All immunized groups showed IgA Ab response in nasal swabs at 14 days post immunization, while the control group only became positive for IgA Abs 28 days post infection (Fig. 3a). The mean values with the respective standard deviation of each group and day of sampling, as well as statistical differences, are shown in Supplementary Table S3. On D28, OI + OI was statistically different from the other groups (for p-values, see Supplementary Table S5 online). The IgA responses of CV and OI at D42 and D49 were significantly different from the others. At D56, CV was statistically different from the other groups, while OI and CV + OI were statistically similar to each other and different from CONT. At D70, only CV and CV + OI IgA Ab responses were significantly higher than other groups, and OI was different from CONT. From D91 onwards, no differences were found between experimental groups. All piglets from immunized groups were IgA positive before challenge. When comparing the moments of slaughter (28 dpi and 56 dpi), significant differences (Two Sample t-test) were found for CV group (p = 0.045), CV + OI group (p = 0.028) and for CONT (p = 0.0001). No significant correlation was observed for any experimental group between the IgA Ab levels and the macroscopic lung lesion scores either at 28 or 56 dpi intervals.

(a) Mucosal IgA antibody response related to different immunization protocols (D0) against M. hyopneumoniae along the experimental period. Dots represent the mean values of each group in each day of sampling. Positive S/P values > 0.4. (b) Serum IgG antibody response obtained in different immunization protocols (D0) against M. hyopneumoniae along experimental period. Piglets challenged with M. hyopneumoniae on D49 (red arrows). Dots represent the mean values of each group in each day of sampling. Positive S/P values > 0.3. Kruskall-Wallis test was used.

Full size image

The oral vaccine did not induce a serum IgG Ab response, whereas the commercial vaccine induced the highest levels of this antibody isotype (Fig. 3b). This result has to be further investigated by other experiments with larger concentration of antigens in SBA-15, providing no aggregation of proteins. It is important to point out that the concentration of antigen administered by the oral vaccine is much smaller than that the one used in the injected CV. The mean values with the respective standard deviation of each group and day of sampling, as well as statistical differences, are shown in Supplementary Table S4. All animals from the CV and CV + OI groups produced IgG anti-M. hyopneumoniae antibodies, and the Ab response started between D14 and D28, remaining with high levels until slaughter. From the moment that seroconversion was detected, the levels of IgG Ab were statistically similar for these two groups, but they differed from the others until the end of the experimental period (for p-values, see Supplementary Table S6 online). The animals from groups OI, OI + OI, and CONT seroconverted for IgG antibodies only four weeks after challenge with the pathogen (D70), and produced levels of IgG Ab statistically similar until the end of the experimental period. Comparing the differences between the moments of slaughter, significant differences (Two sample t-test) were found between the levels of IgG antibodies for CV (p = 0.008) and OI (p = 0.0003). No significant correlation was found for any experimental group between the IgG Ab levels and the macroscopic lung lesion scores, neither at 28 or 56 dpi intervals.

Regarding BALF, all groups showed positive responses to both IgA and IgG anti-M. hyopneumoniae Ab on 28 dpi and 56 dpi (Fig. 4). No statistical differences were found between groups for any of the evaluated immunoglobulins. All correlation analysis tested between variables are shown in Supplementary Table S7.

ELISA S/P (mean ± sd) results for BALF on 28- and 56 dpi regarding (a) IgA antibody response against M. hyopneumoniae; (b) IgG antibody response against M. hyopneumoniae. Positive S/P values > 0.4.

Full size image

Detection and quantification of p102 gene of M. hyopneumoniae by qPCR

The samples of nasal swabs tested by qPCR indicated that at 7th dpi there were piglets already shedding the pathogen in all experimental groups in different proportions, and by the end of the evaluated period, most of the piglets from all groups were shedding intermittently M. hyopneumoniae (Table 1). No significant differences were found between groups along the experiment.

Full size table

Mycoplasma hyopneumoniae was detected and quantified in lungs and BALF of all piglets from all groups, and the respective estimate loads and standard deviation are shown in Table 2. No statistical differences (Two Sample t-test) were found for all groups in both sampled time-points (28 and 56 dpi). Considering the correlation between macroscopic lung lesion and M. hyopneumoniae estimate quantification in lungs, a lower quantification was found to be strongly correlated with a reduced macroscopic lung lesion in CV + OI at 28 dpi (Spearman’s correlation coefficient R = 1; p = 0.01667), with a trend on group CV (p = 0.0538, R = 0.87). A lower quantification was also correlated with a reduced M. hyopneumoniae quantification in the BALF (28dpi) of CV (R = 0.99, p = 0.0051), OI (R = 0.97, p = 0.0299), CV + OI (R = 0.96, p = 0.03), and on 56 dpi in OI + OI (R = 0.99, p = 0.0004).

Full size table

Cytokine coding gene’s expression and correlation with antibody levels and bacterial loads

Interleukin 8 was downregulated in immunized groups at 28 dpi compared to the control group, with a slight non-significant difference in the CV + OI group (Kruskall-Wallis test). Similarly, IFN-γ was less expressed in the immunized groups when compared to the control group, especially in CV, OI and CV + OI, with no significant differences between them. A significant and negative correlation was found between IFN-γ and IgA Ab response at 28 dpi (Pearson’s correlation coefficient R = − 0.43; p-value = 0.028). On the other hand, IL-4 expression was up regulated in all immunized groups, while it was less expressed in the control group. TGF-β expression was reported in all groups, with non-statistical differences between them (Fig. 5). TGF-β expression was positively correlated with IgA Ab response in the upper respiratory tract (nasal swabs) of piglets (Pearson’s correlation coefficient R = 0.95; p-value = 0.0401). All correlation analyses between variables are shown in Supplementary Table S7.

Bar graphs representing the fold change of cytokine gene expression (Fold Change mean ± sd) in lung lesion samples of five pigs per group, 28 days after experimental infection with M. hyopneumoniae strain 232, previously submitted to different immunization protocols. Target gene expression was normalized based on rpl-4 gene expression. No statistical differences were found between groups (Kruskall-Wallis test).

Full size image

Discussion

Oral vaccines are desirable in pig industry due to the relative ease of administration for large populations. In the present study, an oral vaccine for weaned piglets was developed by using the SBA-15 nanostructured mesoporous silica as a vehicle for a blend of M. hyopneumoniae proteins. Four immunization protocols were tested, followed by an experimental inoculation of M. hyopneumoniae strain 232, to evaluate the action of the oral and the intramuscular vaccines, alone or combined, and the results were compared with a non-immunized group. According to the manufacturer (MSD Animal Health), the commercial vaccine contains the proprietary dual adjuvant EMUNADE, capable of producing a rapid and prolonged immune response due to the aluminum hydroxide and an oil emulsion, respectively. As we have no information regarding the bacterial load present in M + PAC® and different routes of administration were performed, comparing its efficacy to the experimental oral vaccine is challenging. However, this IM vaccine was found to be an interesting option as a basis for comparison since it has been used worldwide and provided satisfactory results. In addition, most works with oral or aerosol vaccination against M. hyopneumoniae in piglets did not perform experimental challenge with this pathogen, which can limit the comparison of our results, such as bacterial shedding and quantification in the lugs, with other vaccines.

All immunization protocols showed reduced Mycoplasma-like lung lesion, and all groups presented a significant difference in lung lesion score when compared with the control group, being the highest differences observed in the medians of OI and CV + OI in the first slaughter (reduction of 92% and 91% of consolidated area, respectively), and in CV and OI (reduction of 100% and 94%, respectively) at the second slaughter. The oral and the commercial vaccines offered effective protection individually, but no combination effect with the two immunization ways were observed. These are promising findings, especially when compared to previous works in which no significant differences in lung lesions were found between vaccinated and non-vaccinated groups on the field with natural infection21, and with homologous and heterologous vaccines followed by experimental infection with M. hyopneumoniae22. On the other hand, microscopic lung lesions were statistically similar in all groups, vaccinated or not, with histological findings characteristics of M. hyopneumoniae infection, as also observed by Almeida23, after experimental infection using the same M. hyopneumoniae 232 virulent strain. An oral vaccine with recombinant E. rhusiopathiae strain expressing the M. hyopneumoniae P97 protein reduced the severity of pneumonic lung lesions caused by M. hyopneumoniae infection24. Similarly, a number of M. hyopneumoniae commercial inactivated vaccines administered by IM route provided such type of protection25,26,27.

Mycoplasma hyopneumoniae induces innate and adaptive immune responses, which is able to prevent significant systemic spread of the organisms. However, the immune system is unable to rapidly clear pulmonary airways infection, resulting in a prolonged localized inflammatory and cellular immune response, responsible for the majority of gross and microscopic lesions1. The vaccines and vaccination programs tested in the current study were able to induce IgG (commercial vaccine only) and IgA (commercial and experimental oral vaccines) antibodies against M. hyopneumoniae. Only the groups that received IM vaccination were able to produce serum IgG Ab detectable by ELISA test. It is well known that systemic anti-M. hyopneumoniae antibodies are considered to play a minor role in protection against PEP28,29. On the contrary, it is believed that specific locally secreted IgA may play a protective role by preventing the adhesion of the pathogen to the ciliate epithelium30. Independently on the presence of serum IgG Ab, all vaccinated animals showed a significant reduction of consolidation lung lesions, which allows to infer that mucosal IgA Ab probably participates in the protection and prevention against M. hyopneumoniae invasion and adherence, corroborating Martelli30. In this study, both oral and IM vaccines were capable of inducing IgA Ab production, which was found in the respiratory tract, as all immunized piglets were positive for this Ab at 14 days post-vaccination. Feng31 also observed the presence of IgA Ab in nasal cavities 14 days after administration of an aerosol vaccine against M. hyopneumoniae, although no challenge was performed by these authors.

Our results differ from previous ones that only detected significant antibody immune response after challenge infection24, and from studies using commercial vaccine which were not able to induce IgA Ab responses31,32. We have found that vaccinated pigs presented significantly higher antibody levels than the non-vaccinated ones, which may indicate a memory humoral immune response, either for IgA or IgG Abs. It is noteworthy that antibodies induced by IM vaccination may occur between 3 to 4 weeks after vaccination33, while seroconversion in natural M. hyopneumoniae infected pigs usually occurs around 8–24 weeks of age34,35,36. In the current study, seroconversion in the challenged piglets occurred 3 weeks post-infection. This early immune response, when compared to naturally infected ones, may be due to the differences in experimental and natural bacterial loads. The infecting dose under field conditions is expected to be lower, especially in farms with good management and biosecurity practices37. Determining the ideal time point to experimentally infect the piglets after vaccination is challenging, once vaccination and infections occur dynamically in field conditions. Furthermore, the primary or boosted immune response must be considered in the dynamics of M. hyopneumoniae infection. In the present study, the infectious challenge occurred two weeks after the booster vaccination, and the piglets from CV + OI and OI + OI could be still experiencing the effects of the immunization at that time. For this reason, further studies evaluating different moments of immunization in the field are necessary to a better understanding of this situation.

When considering the Ab immune response in BALF at slaughter (28 dpi and 56 dpi), all groups showed high S/P values for both IgA and IgG Ab, despite the statistically non-significance. As these samples were taken only at slaughter, it was not possible to determine the contribution of each vaccination protocol in inducing the antibody immune response in BALF, once all piglets were challenged with the pathogen and an immune response was expected at 28 dpi. However, it is possible to observe that a substantial Ab detection occurred in piglets previously submitted to both IM and oral vaccination when compared to the control. It may be due not only to the capacity of vaccines to induce memory cells, but also to a previous local presence of these antibodies, as observed in the respiratory tract by nasal swabs. It was expected that vaccinated animals showed higher mucosal IgA Ab responses compared to the non-vaccinated ones after challenge28,38.

The number of organisms colonizing a pig possibly depends on cumulated infectious doses, capacity of the M. hyopneumoniae strain to multiply in the lungs, and time1. In the present study, a high dose of inoculum containing M. hyopneumoniae organisms was provided. M. hyopneumoniae replication in the lungs was lower in vaccinated pigs in both slaughter points compared to control, and quantification in lungs and BALF was lower at 56 dpi in vaccinated groups than in control, despite non-significant differences in bacterial load estimate quantification. The lower lung lesion score at 56 dpi was expected for the immunized groups, as previously observed39. However, it also indicates that vaccination alone does not significantly reduce the bacterial load in the lower respiratory tract of pigs, and would not eliminate infection with this pathogen from pig herds24,27, which may justify nasal shedding not differing between groups along the time37. In addition, several factors such as the challenge dose, the time post-infection, the strain virulence and individual immune responses of pigs may influence the number of M. hyopneumoniae organisms and its nasal shedding23,33.

The evaluation of cytokines production 30 days after challenge with M. hyopneumoniae could better illustrate the resistance status in the vaccinated pigs after infection40. Thus, in the current study, the expression of some cytokine genes at 28 dpi of vaccinated animals point out to a possible role of T cell response after pathogen exposure, so that the oral administration of M. hyopneumoniae antigens incorporated to the silica adjuvant may induce an activation of Treg lymphocytes, which can reduce part of the inflammatory response caused by this pathogen. In this context, it is known that mesenteric Treg lymphocytes of animals submitted to tolerance by the oral route secrete TGF-β with variable amounts of IL-441. Additionally, TGF-β expression was positively correlated with IgA Ab response in the upper respiratory tract, raising the hypothesis that the presence of TGF-β further enhances both secreted IgA and the number of IgA producing cells, as reported42.

T helper (Th) cells are essential to initiate the B-cell activation and generation of antibody responses, which will result in antibody production for T-dependent antigens5. The higher gene expression level of IL-4 in lungs of immunized piglets could also be associated with a positive regulation of a Th2-mediated immune response30, positively influencing the IgA Ab local secretion. Th lymphocytes activation is an important pathway for generating protective immune response against M. hyopneumoniae43.

Interferon-γ secretion is important for the control of several infectious diseases44, and it is usually evident between 4 to 8 weeks post vaccination against M. hyopneumoniae30,38. Although the piglets were tested only at 28 days post-infection, its lower expression in immunized groups may be involved with an immunosuppressed environment caused by the inhibition of Th1 differentiation, since IL-4 suppresses the production of IFN-γ by Th1 cells45. In addition, IFN-γ was negatively correlated with IgA Ab response in the upper respiratory tract. These findings may be justified either by the suppressive effect of IL-4 or the effect of TGF-β on IFN-γ secretion and the direct correlation of IL-4 and IgA Ab response. IL-8 was found to be less expressed in all immunized groups of this study, and its suppression at this time point may be similar to IFN-γ. This chemokine is mainly produced by tissue macrophages in response to infection, and it is an important neutrophil recruiter46. M. hyopneumoniae organisms stimulate the production of proinflammatory and immunoregulatory cytokines by the alveolar macrophages and lymphocytes, inducing lung inflammation and lymphoid hyperplasia. As colonization by M. hyopneumoniae progresses, there is an increase in cytokine secretion due to the increase in the number of inflammatory cells recruited1. In non-vaccinated challenged piglets23, IL-8 gene expression was positively correlated with M. hyopneumoniae bacterial loads, suggesting an intense inflammatory response being required at lung lesion sites due to the presence of the pathogen. In our study, IL-8 and IFN-γ were found downregulated in vaccinated piglets, which may be due to modulation effect caused by the vaccines, preventing an intense inflammatory and immune responses in lungs, consequently leading to a lower consolidation lung lesion.

Under the conditions of this study, the immune response induced by IM and oral vaccination involved both humoral and likely T-cell immune responses. Thus, oral vaccination with a blend of M. hyopneumoniae antigens incorporated into the silica induced local humoral immunity in the gut, measured in this study as IgA anti-M. hyopneumoniae antibodies in respiratory secretions, which were comparable to those obtained by the intramuscular administration of the commercial inactivated vaccine with oil adjuvant. All vaccination protocols reduced the severity of macroscopic lung lesions in the challenged pigs. It suggests that mucosal antibodies and the inflammatory responses were involved in the mechanism of immune-protection, or by raising IL-4 and reducing the IL-8 expression, or even likely by down-regulating the expression of other pro-inflammatory cytokines in the vaccinated animals, which were not evaluated in the current study.

As the protection induced by M. hyopneumoniae vaccines evaluated here and conferred by others conventional vaccines do not prevent colonization and shedding of this microorganism, the immune-protection induced by these vaccines is often incomplete47. Considering that, better results may be achieved whether vaccination is combined with good management and biosecurity procedures in pig farms. Moreover, under field conditions, improved results should be expected for the vaccines tested in this study, once a lower infection challenge dose probably results in lower number of microorganisms in the respiratory tract21. Thus, future studies applying the oral vaccination against M. hyopneumoniae in pigs reared in field conditions could elucidate this point and bring more consistent results for the use of inactivated oral vaccines in the control of this important respiratory pathogen for pigs, combined with the advantage of being a needle-free strategy of M. hyopneumoniae control.

The oral vaccine developed in this research with the SBA-15 nanostructured silica proved to effectively reduce macroscopic lung lesions in challenged pigs and induce mucosal humoral immune response. Moreover, its efficacy was similar to the one conferred by the commercial vaccine parentally administered. The immunogenicity characterization determined in the current study provided useful data for the further development of this oral vaccine, which requires studies under field conditions to elucidate its potential for the effective control and prevention of Mycoplasma hyopneumoniae injuries in the pig production.

Methods

Oral vaccine preparation

Cultivation and preparation of M. hyopneumoniae for protein obtainment

A pure pathogenic strain of M. hyopneumoniae (232) was purchased from Iowa State University48, certified free of any other pathogens, and a small fraction was removed for cultivation in Friis medium. Initially, this fraction was inoculated into two sterile graduated vials (Corning®, USA) containing 5 ml of Friis medium, kept in a shaking oven at 37 °C, which color was observed daily until indicating bacterial growth (CCU). After approximately five days, 2 ml of the culture were used to inoculate each of two flasks containing 200 ml of Friis medium, which were maintained in the same incubation conditions and presented color changing about a week later. The determination of the concentration of M. hyopneumoniae was carried out by successive dilutions of the samples in Friis medium, varying from 10–1 to 10–8, which reached a concentration of 107, and the negative control remained unchanged. Sterility tests were performed for all flasks on blood agar and McConkey media, left in an oven at 37 °C for three days, proving the absence of contamination with other pathogens by the absence of colonies growth.

The contents were centrifuged in appropriate tubes, previously autoclaved, in an ultracentrifuge (Sorvall) at 13,700×g for 45 min. The bacterial cells were deposited at the bottom of the tubes, forming pellets, which were resuspended in 15 mL of Phosphate Buffered Saline 1X (PBS, Sigma-Aldrich, USA), pH 7.4, for washing. The tubes were centrifuged three times at 21,000×g for 10 min until a clean pellet was obtained, which was resuspended in 10 mL of 1X PBS and stored in sterile falcon tubes. The resuspended content underwent a sonication process, and before sonication, an aliquot of whole cell preparation was taken for Dynamic Light Scattering (DLS) evaluation, which will be further discussed. For sonication, the flask was kept on ice and sonicated for three consecutive times in a sonicator (Soni-tech Ultrasonic Cleaning) in 20 Hz frequency for 1 min, with one-minute breaks between processes. To determine the concentration of proteins in the cell lysate, the Bradford method was used (Thermofisher Scientific, USA) followed by spectrophotometer reading (NanoDrop One, Thermofisher Scientific, USA), which provided a concentration of 1048 µg/mL.

Characterization and development of the oral vaccine

A partnership was established with the Department of Applied Physics, of the Physics Institute of the University of São Paulo (USP-SP), Department of Chemistry, Federal University of São Paulo (UNIFESP/Diadema) and the Butantan Institute (São Paulo), which have been conducting researches with an innovative immunogenic complex for human oral vaccines. They are based on the use of ordered mesoporous silica (OMS, SBA-15 type) as protective vehicle of antigens. The SBA-15 sample was synthesized according to Cavalcante49. It is composed by a bi-dimensional matrix of hexagonally ordered mesopores (diameter around 10 nm), high specific surface (around 1240 m2 g−1) and pore volume 1.8 cm3 g−1 (see Supplementary Fig. S1 online), with amorphous silica walls and rod-like morphology, formed by aggregates of rods connected as rope-like domains (Fig. 6), capable of encapsulating different molecules into their macropores and mesopores. The immunogenic complex has to be able to cross the digestive tract in order to be absorbed by the intestinal mucosa.

Scanning electron microscopy images of SBA-15 (Santa Barbara Amorphous silica) before antigen adsorption in macro pores. (a) 1000x magnification; (b) 10,000x magnification.

Full size image

To protect the oral vaccine from the harsh stomach medium, the commercial polymer Eudragit® (Evonik Industries) was used for coating. This polymer is insoluble in acidic pH and dissolves in contact with the intestinal basic medium, providing slow release of the desired proteins12.

The preparation and characterization of the vaccine particles were carried out at the Crystallography Laboratory of the Department of Applied Physics at USP. The process of including the antigen in the pores of the SBA-15 was carried out considering the concentration of the obtained proteins. The 1:35 antigen-to-silica ratio was established (w/w), based on previous work with Hepatitis B encapsulation in SBA-1518,20. In the present study, the silica SBA-15, from a closed recipient (to avoid contamination and humidity) was weighed on a precision scale (± 1 μg), and immediately macerated with a glass stick. After that, the total amount of the silica was mixed with the flask containing the liquid antigen (concentration determined previously). The wet mixture was spread in a glass recipient and partially covered by a glass plate, and placed in an oven at 35 (± 2) °C for total drying. This drying procedure took 48 h. Then, the product was covered with Eudragit L30 D-55 (Evonik®), by mixing the polymer with the silica + antigen, using a glass spatula to homogenize the final product. The amount of Eudragit was equal (in weight) to the amount of silica added to the liquid antigen until a paste was formed, which was taken again to an oven at 35 (± 2) °C for drying, during 48 h. The dry contents were stored in a refrigerator to protect the antigen until use. Since the polymer has the ability to dissolve at a pH higher than 5.5, the compound was supplied to piglets in acidified water (pH 4.5).

Dynamic light scattering (DLS) evaluation of particle diameter and nitrogen adsorption/desorption isotherms of SBA-15:Antigen sample

Dynamic Light Scattering (DLS) evaluation was performed at the Physics Institute, USP-SP, using a DLS (DynaPro NanoStar, Wyatt) equipment at 90° scattering angle with a 100 mW He–Ne laser and wavelength of 658 nm. An aliquot of the whole-cells (concentration of 106 CCU/mL) dispersed in 25 mL of PBS (pH 7.4) was used to fill the sample holder. The same process was done to investigate by DLS the sonicated sample (cell lysate), having a concentration of 1048 µg/ml. The size distribution was obtained by the equipment software, calculated by the CONTIN method50. The antigen encapsulation, depending on its size, can occur in the silica mesopores, with an average diameter of ~ 10 nm, or in the morphological macrospores, with dimensions greater than 50 nm. The average diameter of the molecules present in the whole-cell preparation sample was 460 nm, while the protein preparation was 218 nm, which allowed inferring that cell lysis occurred, and the antigen would be adsorbed on the silica macro porosity, regarding the sizes of the antigen in solution compared with the mean diameter of the mesopores.

By nitrogen adsorption/desorption isotherms of SBA-15:Antigen sample (Supplementary Figs. S2 and S3 online) were evidenced the preservation of mesostructured SBA-15, which exhibited a type IV isotherm and type H1 hysteresis loops, according to the IUPAC classification51 that are characteristic of hexagonal cylindrical channel mesoporous such as SBA-15, with a pore size about 10 nm, as reported in the literature52. The reduction of specific surface area (336 m2 g−1) and pore volume (0.91 cm3 g−1) of SBA-15:Antigen, comparing to pure SBA-15, has evidenced antigen into the macropores of SBA-15.

Oral vaccine administration

After a pilot test, a concentration of 200 µg of protein antigen of M. hyopneumoniae per animal was established. The doses of the compound were weighed on a precision scale, considering the proportion between antigen, silica and polymer. The vaccine was provided by gavage using an esophageal tube with the aid of a laryngoscope, administered in individual doses diluted in 5 ml of filtered water plus acidifier (Selko pH, Trouw Nutrition). Each dose was diluted at the moment of administration.

Experimental design and sample collection

Experimental design

Fifty piglets were randomly separated into five groups (n = 10) to receive different vaccine protocols, consisting of a commercial vaccine (CV) or oral immunization (OI). Random numbers were generated using the standard = RAND() function in Microsoft Excel. Group 1 (CV) piglets received a single dose commercial vaccine at 24 days of age (D0). Group 2 (OI) piglets received a single dose of oral vaccine at D0. Group 3 (CV + OI) received a dose of the commercial vaccine at D0 and a booster with the oral vaccine at D28. Group 4 (OI + OI) received a dose of the oral vaccine on D0 and a booster with the same vaccine on D28; piglets of Group 5 (CONT) were the control group, which did not receive any form of immunization. The commercial vaccine chosen for this work was M+PAC (Merck Animal Health, USA), which promotes an effective protection against M. hyopneumoniae as it contains aluminum hydroxide, responsible for a rapid immune response, and an oil emulsion, which promotes a prolonged immune response by its slow release in the organism. At about 70 days of age (D49), all piglets were challenged by the tracheal route with 5 mL of Friis medium containing 106 CCU/mL of M. hyopneumoniae virulent strain 232, homologous to the vaccine.

Weekly, nasal swabs were collected since D0 for IgA measurement (ELISA), and, after the challenge, were used to evaluate M. hyopneumoniae shedding as well. Fortnightly, all piglets underwent blood collections to obtain serum for IgG measurement (ELISA). Half of the animals in each group were euthanized 28 days post-infection (D77), and the other half was euthanized 56 days post-infection (D105). At slaughter, lungs of all animals were macroscopically evaluated following the European Pharmacopeia methodology, biological samples such as lung fragments were collected for qPCR and histopathology, and bronchoalveolar fluid (BALF) for qPCR. A schematic design is shown in the Supplementary Fig. S4 online.

Animal selection

Fifty 21-day-old piglets of commercial lineage (Landrace × Large White) and average weight between 6 and 7 kg were purchased from a certified M. hyopneumoniae-free commercial farm. The piglets were housed in the experimental barn of the Swine Medicine Laboratory of School of Agricultural and Veterinarian Sciences (FCAV), remaining in the nursery pens until 65 days of age (3 pigs/m2), and then transferred to fattening pens (1 pig/m2), where they remained until 130 days of age. Biosecurity standards were met to avoid cross-infections and minimize external interferences, such as shower-in/shower-out, specific and clean clothes for pig husbandry and no contact with any other pig during the experimental period. The animals received feed according to the production phase, free of antibiotics, and water ad libitum. Upon arrival, blood samples were taken from all piglets to obtain serum for measurement of antibodies, in addition to laryngeal swabs to confirm the absence of the pathogen. Piglets went through an adaptation period of three days before the beginning of the experiment.

All procedures described here were conducted in accordance with the Federal Council of Veterinary Medicine (Brazil), submitted for approval by the Ethics Committee on the Use of Animals (CEUA) of the School of Agricultural and Veterinarian Sciences, São Paulo State University—Campus Jaboticabal, being approved and registered under the protocol number 005174/18. The study was carried out in compliance with the ARRIVE guidelines53.

Blood serum and nasal swabs collection

The titers and duration of antibodies in serum and nasal swabs were assessed along the experimental period. The blood was collected every two weeks to obtain blood serum by puncture the jugular vein, using sterile disposable needles and syringes, deposited in tubes with clot activator and centrifuged at 1500×g for 10 min. The serum was aliquoted in duplicate and stored at − 20 °C until the time of analysis. Samples of nasal swabs were weekly collected to obtain quantitative data on the immune response induced by different protocols of immunization (ELISA), and on the dynamics of excretion of M. hyopneumoniae (qPCR). The animals were restrained, and the swab samples were collected from a light rubbing of the swab on the nasal mucosa and deposited in 2 mL graduated plastic microtubes (Kasvi, Brazil) containing 500 μL of 1 × PBS, and stored at − 80 °C until analysis.

Slaughtering of piglets, lung lesion scoring, BALF and lung fragments collection

Four and eight weeks after the challenge, half of the piglets of each group was euthanized with an intramuscular administration of a combination of ketamine and xylazine (6 mg/kg and 4 mg/kg, respectively), followed by intravenous administration of saturated potassium chloride solution (approved by Federal Council of Veterinary Medicine, Brazil). After evisceration, the respiratory set (trachea + lung) of each animal was separated to collect bronchoalveolar fluid (BALF), with the introduction of 20 mL of PBS 1 × in the cranial portion of the trachea. After pouring all the liquid, the lung was lightly massaged and the liquid was aspirated by the pipette, recovering an approximate volume of 10 mL. The aspirate was aliquoted in duplicate in 2 mL graduated microtubes, free of DNAses and RNAses (Kasvi, Brazil), while the remaining volume was deposited in sterile Falcon tubes (Kasvi, Brazil) and stored at – 20 °C until analysis by qPCR.

The lung was evaluated for the extent of lung lesions followed by photo documentation by a unique blinded investigator, and the extent of the lesions was quantified using the European Pharmacopeia method, in which the percentage of each lobe affected area is multiplied by the lobe relative weight and summed to provide the total weight percentage of affected lung54. The lung tissue fragments for the qPCR were collected with individual scalpel blades and sterile tweezers. The tweezers were kept in boiling water in the interval between collections, while each fragment was collected with a disposable scalpel blade. These fragments were collected in duplicate in the shortest possible time after the animal's death, and all samples were bathed in liquid nitrogen and later stored in a freezer at − 80 °C.

Histopathological evaluation

The histopathological analysis aimed at evaluating lung lesions caused by Mycoplasma hyopneumoniae in different groups, in order to classify qualitatively histological lesions as PEP-specific or non-specific. For that, during the necropsy, lung fragments with lesions caused by PEP resources were collected, mainly in the transition area between healthy and affected tissue. Samples of tissues apparently healthy were also collected for control. The fragments were collected with a thickness of approximately 5 mm and initially stored submerged in a 10% buffered formalin solution (pH 7.0) in an approximate ratio of 10: 1 formalin: tissue. After 24 h in formalin solution, the fragments were routinely processed for Hematoxylin/Eosin staining.

The slides were blind read under a light microscope and microscopic lesions on the tissues were classified into five different degrees55, in which: 0 = absence of lesion; 1 = lesions of interstitial pneumonia and/or purulent bronchopneumonia; 2 = light to moderate infiltrates of macrophages, lymphocytes and neutrophils into airways and alveoli; 3 = perivascular and peribronchiolar lymphoplasmacytic hyperplasia, type II pneumocyte hyperplasia, alveolar spaces with edema fluid, neutrophils, macrophages and plasma cells; 4 = lesions with characteristics of grade 3, together with peribronchial and perivascular lymphoid nodules. Grades 1 and 2 injuries were nonspecific, while injuries 3 and 4 were considered PEP-specific.

Detection and quantification of IgA and IgG antibodies by enzyme-linked immunosorbent assay

The detection and quantification of antibodies (IgA and IgG) in blood serum, nasal secretions and tracheobronchial lavage were carried out by enzyme-linked immunosorbent assay (ELISA). To detect serum IgG Ab, the commercial kit M.hyo Ab test (Idexx, USA) was used. For the detection of IgA Ab in nasal swab, and IgA and IgG Abs in BALF samples, standardization was performed using the sensitized plates and components of the kit mentioned above, with modifications. Initially, the plate was blocked with 1.5% ovalbumin in PBS, followed by incubation at 37 °C for 30 min. Then, a first procedure was performed to determine the ideal dilutions of the samples (positive and negative controls for anti-M. hyopneumoniae antibodies) and the anti-IgA or anti-IgG peroxidase conjugates. For standardization, samples of nasal swabs from animals known to be positive and negative for M. hyopneumoniae were used, and part of the negative and positive samples were homogenized in the form of a pool to compose the negative (CN) and positive (CP) controls, respectively.

To detect IgA Ab in nasal swabs, 100 μL of the sample's liquid fraction was used, as the swabs were deposited in 500 μL of PBS, which were quickly homogenized in a vortex and placed, without further dilution, in each microplate well. The same procedure was performed with the controls, which were tested in duplicate, followed by plate incubation for 60 min at room temperature. The conjugate from the kit was replaced by an immunoenzymatic conjugate of goat anti-Pig IgA Antibody HRP Conjugated (Bethyl Laboratories Inc., USA), at a dilution of 1:500 using the diluent provided by the kit, followed by incubation for 60 min at room temperature. The washing processes and all the following steps were performed according to the protocol of the kit M.hyo Ab test (Idexx, USA).

For BALF IgA Ab detection, the samples were diluted 1:10 using the diluent from the kit, and the same conjugate was used in the proportion of 1:800. For IgG detection in the BALF, the samples were diluted in the proportion of 1:2 and the conjugate (Pig IgG-Fc Fragment Antibody HRP, Bethyl Laboratories Inc., USA) in the proportion of 1:5000. In all microplates, the conjugate was tested in separate wells to determine its non-specific adsorption in the absence of samples. The plate was read in an absorbance microplate reader (iMark, Bio-Rad Laboratories Inc., USA), at a wavelength of 650 nm.

The mean optical densities (OD) for each of the test samples (ODs) were related with the OD found for the negative and positive controls (NC\(\overline{x }; PC\overline{x })\) in order to calculate the S/P values (sample/positive ratio) according to the formula: S/P = \(ODs- NC\overline{x }/PC\overline{x }- NC\overline{x }\). The threshold between positive and negative samples was calculated from the value of S/P \(NC\overline{x }\) + 2 × standard deviation. Serum samples were considered positive if S/P > 0.3; nasal swabs S/P > 0.4. BALF S/P > 0.4.

Detection and quantification of p102 gene fragment of M. hyopneumoniae by qPCR

DNA extraction from nasal swabs, lung and BALF samples

The DNA extraction was carried out by Tris–HCl protocol56. For nasal swabs and BALF samples, a centrifugation (Centrifuge 5804 R, Eppendorf, Germany) at 13,000×g at 4 °C for 20 min was performed previously to the DNA extraction protocol. For lung samples, 0.05 g of lung tissue were used. After DNA extraction, the samples were stored at − 20 °C until qPCR analysis. The measurement of the DNA concentration of the samples was made through spectrophotometry, with the aid of the Thermo Scientific NanoDrop 2000 Spectrophotometer (Thermo FisherScientific®, USA), having as exclusion factor the samples that did not reach the purity of 1.8 to 2.0 in the 260/280 ratio to perform the qPCR technique. To rule out the presence of inhibitors in the extracted DNA samples and the occurrence of false negatives in the qPCR for M. hyopneumoniae, all samples were submitted to a conventional PCR targeting the endogenous gene Glyceraldehyde-3-phosphate dehydrogenase (gapdh), and the conventional PCR technique57. The amplified products of gapdh gene with 437 bp were detected after horizontal electrophoresis on a 1% agarose gel stained with Ethidium Bromide (0.5 μL/mL) in TEB running buffer pH 8.0 at a current of 90 V/50 mA for 90 min.

qPCR assay

Absolute real-time quantitative PCR analysis (qPCR) was used to detect p102 gene fragment in nasal swab samples, and to detect and quantify it in lung fragments and bronchoalveolar fluids. For M. hyopneumoniae, the primers used in the reaction were based on the bacterium p102 adhesion protein gene sequence. All samples were tested in duplicate and the qPCR reaction was optimized from a previous published protocol58, adapted by Almeida23. The nucleotide sequences used were forward primer 5′-AAGGGTCAAAGTCAAAGTC-3′, reverse primer 5′-AAATTAAAAGCTGTTCAAATGC-3′ and hydrolysis probe 5′-FAM-AACCAGTTTCCACTTCATCGCC-§BHQ2-3′.

The results were only accepted for those with a standard deviation lesser than or equal to 0.5 cycle, and quantification data were used only if the efficiency obtained was between 90 and 105%57, otherwise, the samples were retested in triplicates. As a negative control in the qPCR reactions, sterile ultrapure water was used (Nuclease-Free Water, Promega®, Madison, Wisconsin, USA) q.s.p. Serial dilutions were made to determine the standard curve generated with different concentrations of synthetic DNA (GBlock®, IDT, USA) containing the target sequence (107 copies/μL to 101 copies/μL), that were also used as positive controls. The synthetic DNA was diluted according to the manufacturer's guidelines and maintained in a stock concentration of 107 molecules∕µL.

Quantification was performed using serial tenfold dilutions (starting at 107 until 101 copies∕ μL) of synthetic DNA (GBlock®, IDT, Iowa City, IA, USA) containing the 150 bp fragment amplified by the primer pair used in the qPCR. Quantification data based on the standard curve generated was only validated if the reaction efficiency was between 90 and 105%59. qPCR parameters are shown in Supplementary Table S1.

Cytokine coding gene expression in lung samples

RNA extraction and cDNA synthesis

Total RNA was extracted from 0.02 g of lung tissue samples collected on the first slaughter (28 dpi) using RNeasy Blood and Tissue Plus kit (Qiagen, USA), and 500 ng of extracted RNA were used per reaction to convert the extracted RNA into cDNA by Superscript IV First Strand Synthesis kit (Thermo Fisher, USA), both according to the manufacturer’s guidelines. RNA purity and integrity were assessed by Bioanalyzer® (Thermo Scientific, USA), and immediately stored at − 80 °C until use. Samples were only used for gene expression analysis if they had RNA Integrity Number (RIN) > 7.0. Oligo d(T)20 targeted the poly-A tail of mRNA for the synthesis of this molecule into cDNA, instead of other types of RNA. Reactions were all performed in a MyCycler thermocycler (Bio Rad, USA), and the cDNA was stored at − 20 °C until use for qPCR.

Detection and quantification of cytokine coding gene’s expression

The reference gene used to normalize the expression of target genes was standardized23, and the best result was obtained with Ribosomal protein L4 (rpl-4), which was also used in this study. Quantification of cytokine coding gene’s expression was performed using relative quantification of cDNA produced. Transcript levels of inflammatory (IL-8) and specific immunity regulation (IFN-γ IL-4 and TGF-β) cytokines evaluated to compare the immune response promoted by each type of immunization with the control group. Specific primers targeting the genes of IL-8 (CXCL-8) and IFN-γ were based on previous work23, while genes of IL-4 and TGF- β were designed based on the reference sequences deposited in GenBank and using software Primer360. To avoid genomic DNA interference, all primers were designed comprising the exon-exon span. The specific primers are shown in Table 3.

Full size table

The qPCR reactions were performed as previously described23, using the Quantitect® Sybr Green master mix (Qiagen, USA) and 1 μL of cDNA template, totalizing 10 μL of final volume per reaction, in a real time thermocycler CFX 96 (Bio Rad, USA). The dissociation curve was used to assess the specificity of the amplicons at the end of 40 cycles, with a maximum variation of ± 0.5 °C. Serial tenfold dilutions (107 copies∕μL until 101 copies∕μL) of positive controls (Gblock®, IDT, USA) of synthetic DNA containing the amplified fragment of each primer pair was used to assess the efficiency, which was only accepted between 90 and 105%59. In order to normalize the target gene expression, the 2−ΔΔCq calculation61 was performed, and, to attend this methodology, all qPCR reaction’s efficiency had to be close to 100% with a maximum difference of 5% between them. All parameters of cytokine gene expression qPCR are shown Supplementary Table S2.

Data analysis

The variables of each group for each moment were assessed for normality and homoscedasticity by the Shapiro–Wilk and Bartlet tests, respectively. The difference between the means was calculated using the Tukey test (p < 0.05). Variables that did not meet the assumptions were subjected to the Kruskal–Wallis non-parametric test (p < 0.05) and in cases which significance was observed, the Dunn test (Post hoc) was applied. Differences between the slaughter times for the quantitative variables were subjected to parametric analysis by the unpaired T-test, and the variables that did not meet the assumptions were subjected to the Wilcoxon–Mann–Whitney non-parametric test. The difference between the means (post hoc) was calculated by the last square mean adjusted by the Tukey method. For proportion analysis, the differences between groups by date were calculated using the Wilson score interval method. The correlation analysis of parametric data were subjected to Pearson's correlation test (p < 0.05), and non-parametric data, to Spearman's test. Correlation analysis to assess whether two variables can be considered dependent was performed using Kendall's nonparametric test. For this, the software R was used, with the following packages “agricolae”62; "LmerTest"63, "arm"64; "Emmeans"65; "Car"66; "Nortest"67; "MASS"68 with the software R69. Normalization of cytokine gene expression and the graphs were performed on software GraphPad Prism 6 (La Jolla, CA-USA).

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

References

  1. 1.

    Pieters, M. G. & Maes, D. Mycoplasmosis. In The Diseases of Swine (ed. Zimmermann, J. J.) (Wiley-Blackwell, 2019).

    Google Scholar

  2. 2.

    Holst, S., Yeske, P. & Pieters, M. Elimination of Mycoplasma hyopneumoniae from breed-to-wean farms: A review of current protocols with emphasis on herd closure and medication. J. Swine. Health. Prod.23, 6 (2015).

    Google Scholar

  3. 3.

    Murtaugh, M. P. Advances in swine immunology help move vaccine technology forward. Vet. Immunol. Immunopathol.159, 3–4. https://doi.org/10.1016/j.vetimm.2014.02.017 (2014).

    CASArticle Google Scholar

  4. 4.

    Pasternak, J. A., Ng, S. H. & Wilson, H. L. A single, low dose oral antigen exposure in newborn piglets primes mucosal immunity if administered with CpG oligodeoxynucleotides and polyphosphazene adjuvants. Vet. Immunol. Immunopathol.161, 3–4. https://doi.org/10.1016/j.vetimm.2014.08.006 (2014).

    CASArticle Google Scholar

  5. 5.

    Chase, C. & Lunney, J. K. Immune system. In The Diseases of Swine (ed. Zimmermann, J. J.) 264–291 (Wiley-Blackwell, 2019).

    Chapter Google Scholar

  6. 6.

    Hyland, K., Foss, D. L., Johnson, C. R. & Murtaugh, M. P. Oral immunization induces local and distant mucosal immunity in swine. Vet. Immunol. Pathol.102, 3. https://doi.org/10.1016/j.vetimm.2004.09.015 (2004).

    CASArticle Google Scholar

  7. 7.

    Lin, J. H., Weng, C. N., Liao, C. W., Yeh, K. S. & Pan, M. J. Protective effects of oral microencapsulated Mycoplasma hyopneumoniae vaccine prepared by co-spray drying method. J. Vet. Med. Sci.2, 7. https://doi.org/10.1292/jvms.65.69 (2003).

    Article Google Scholar

  8. 8.

    Hua, L. Z. et al. Comparative analysis of mucosal immunity to Mycoplasma hyopneumoniae in Jiangquhai porcine lean strain and DLY piglets. Genet. Mol. Res.13, 3. https://doi.org/10.4238/2014.July.7.13 (2014).

    CASArticle Google Scholar

  9. 9.

    Wilson, H. L. & Obradovic, M. R. Evidence for a common mucosal immune system in the pig. Mol. Immunol.66, 1. https://doi.org/10.1016/j.molimm.2014.09.004 (2015).

    CASArticle Google Scholar

  10. 10.

    Matthijs, A. M. et al. Systems immunology characterization of novel vaccine formulations for Mycoplasma hyopneumoniae bacterins. Front. Immunol.10, 1087. https://doi.org/10.3389/fimmu.2019.01087 (2019).

    CASArticlePubMedPubMed Central Google Scholar

  11. 11.

    Petrovsky, N. & Aguilar, J. C. Vaccine adjuvants: Current state and future trends. Immunol. Cell Biol.82, 5. https://doi.org/10.1111/j.0818-9641.2004.01272.x (2004).

    Article Google Scholar

  12. 12.

    Mariano-Neto, F. et al. Physical properties of ordered mesoporous SBA-15 silica as immunological adjuvant. J. Phys. D. Appl. Phys.47, 42 (2014).

    Article Google Scholar

  13. 13.

    Ko, J. W. et al. Silica dioxide nanoparticles aggravate airway inflammation in an asthmatic mouse model via NLRP3 inflammasome activation. Regul. Toxicol. Pharmacol.112, 104618. https://doi.org/10.1016/j.yrtph.2020.104618 (2020).

    CASArticlePubMed Google Scholar

  14. 14.

    Ravinayagam, V. & Jermy, B. R. Nanomaterials and their negative effects on human health. In Applications of Nanomaterials in Human Health (ed. Khan, F.) 249–273 (Springer, 2020).

    Chapter Google Scholar

  15. 15.

    Chen, L. et al. The toxicity of silica nanoparticles to the immune system. Nanomedicine13, 1939–1962. https://doi.org/10.2217/nnm-2018-0076 (2018).

    ArticlePubMed Google Scholar

  16. 16.

    Mercuri, L. P. et al. Ordered mesoporous silica SBA-15: A new effective adjuvant to induce antibody response. Small2, 2. https://doi.org/10.1002/smll.200500274 (2006).

    ADSCASArticle Google Scholar

  17. 17.

    Carvalho, L. V. et al. Immunological parameters related to the adjuvant effect of the ordered mesoporous silica SBA-15. Vaccine.28, 50. https://doi.org/10.1016/j.vaccine.2010.09.087 (2010).

    CASArticle Google Scholar

  18. 18.

    Scaramuzzi, K. et al. Nanostructured SBA-15 silica: An effective protective vehicle to oral hepatitis B vaccine immunization. Nanomed. Nanotechnol.12, 8. https://doi.org/10.1016/j.nano.2016.06.003 (2016).

    CASArticle Google Scholar

  19. 19.

    Lopes, J. L. S. et al. Antigenic and physicochemical characterization of Hepatitis B surface protein under extreme temperature and pH conditions. Vaccine.37, 43. https://doi.org/10.1016/j.vaccine.2019.09.005 (2019).

    CASArticle Google Scholar

  20. 20.

    Rasmussen, M. K. et al. 3D visualisation of hepatitis B vaccine in the oral delivery vehicle SBA-15. Sci. Rep.9, 1. https://doi.org/10.1038/s41598-019-42645-5 (2019).

    CASArticle Google Scholar

  21. 21.

    Villarreal, I. et al. The effect of vaccination on the transmission of Mycoplasma hyopneumoniae in pigs under field conditions. Vet. J.188, 1. https://doi.org/10.1016/j.tvjl.2010.04.024 (2011).

    Article Google Scholar

  22. 22.

    Villarreal, I. et al. Effect of challenge of pigs previously immunised with inactivated vaccines containing homologous and heterologous Mycoplasma hyopneumoniae strains. Vet. Res.8, 1. https://doi.org/10.1186/1746-6148-8-2 (2012).

    CASArticle Google Scholar

  23. 23.

    Almeida, H. M. et al. Cytokine expression and Mycoplasma hyopneumoniae burden in the development of lung lesions in experimentally inoculated pigs. Vet. Microbiol1, 108647. https://doi.org/10.1016/j.vetmic.2020.108647 (2020).

    CASArticle Google Scholar

  24. 24.

    Ogawa, Y. et al. Oral vaccination against mycoplasmal pneumonia of swine using a live Erysipelothrix rhusiopathiae vaccine strain as a vector. Vaccine.27, 33. https://doi.org/10.1016/j.vaccine.2009.04.081 (2009).

    CASArticle Google Scholar

  25. 25.

    Dawson, A., Thevasagayam, S. J., Sherington, J., Harvey, R. E. & Peters, A. R. Studies of the field efficacy and safety of a single-dose Mycoplasma hyopneumoniae vaccine for pigs. Vet. Rec.151, 18. https://doi.org/10.1136/vr.151.18.535 (2002).

    Article Google Scholar

  26. 26.

    Moreau, I. A., Miller, G. Y. & Bahnson, P. B. Effects of Mycoplasma hyopneumoniae vaccine on pigs naturally infected with M. hyopneumoniae and porcine reproductive and respiratory syndrome virus. Vaccine.22, 17–18. https://doi.org/10.1016/j.vaccine.2003.10.041 (2004).

    CASArticle Google Scholar

  27. 27.

    Meyns, T. et al. Comparison of transmission of Mycoplasma hyopneumoniae in vaccinated and non-vaccinated populations. Vaccine.24, 49–50. https://doi.org/10.1016/j.vaccine.2006.07.004 (2006).

    CASArticle Google Scholar

  28. 28.

    Djordjevic, S. et al. Serum and mucosal antibody responses and protection in pigs vaccinated against Mycoplasma hyopneumoniae with vaccines containing a denatured membrane antigen pool and adjuvant. Austr. Veter. J.https://doi.org/10.1111/j.1751-0813.1997.tb14383 (1997).

    Article Google Scholar

  29. 29.

    Thacker, E. L., Thacker, B. J., Boettcher, T. B. & Jayappa, H. Comparison of antibody production, lymphocyte stimulation, and protection induced by four commercial Mycoplasma hyopneumoniae bacterins. J. Swine Health Prod.6, 107–112 (1998).

    Google Scholar

  30. 30.

    Martelli, P. et al. Systemic and local immune response in pigs intradermally and intramuscularly injected with inactivated Mycoplasma hyopneumoniae vaccines. Vet. Microbiol.168, 2–4. https://doi.org/10.1016/j.vetmic.2013.11.025 (2014).

    CASArticle Google Scholar

  31. 31.

    Feng, Z. X. et al. Development and validation of an attenuated Mycoplasma hyopneumoniae aerosol vaccine. Vet. Microbiol.167, 3–4. https://doi.org/10.1016/j.vetmic.2013.08.012 (2013).

    CASArticle Google Scholar

  32. 32.

    Bai, Y. et al. Application of a sIgA-ELISA method for differentiation of Mycoplasma hyopneumoniae infected from vaccinated pigs. Vet. Microbiol.https://doi.org/10.1016/j.vetmic.2018.07.023 (2018).

    ArticlePubMed Google Scholar

  33. 33.

    Sibila, M. et al. Chronological study of Mycoplasma hyopneumoniae infection, seroconversion and associated lung lesions in vaccinated and non-vaccinated pigs. Vet. Microbiol.122, 1–2. https://doi.org/10.1016/j.vetmic.2007.01.010 (2007).

    CASArticle Google Scholar

  34. 34.

    Andreasen, M., Nielsen, J. P., Bækbo, P., Willeberg, P. & Bøtner, A. A longitudinal study of serological patterns of respiratory infections in nine infected Danish swine herds. Prev. Vet. Med.45, 3. https://doi.org/10.1016/S0167-5877(00)00122-7 (2000).

    Article Google Scholar

  35. 35.

    Djordjevic, S. P., Eamens, G. J., Romalis, L. F. & Saunders, M. M. An improved enzyme linked immunosorbent assay (ELISA) for the detection of porcine serum antibodies against Mycoplasma hyopneumoniae. Vet. Microbiol.39, 3–4. https://doi.org/10.1016/0378-1135(94)90163-5 (1994).

    Article Google Scholar

  36. 36.

    Leon, E. A., Madec, F., Taylor, N. M. & Kobisch, M. Seroepidemiology of Mycoplasma hyopneumoniae in pigs from farrow-to-finish farms. Vet. Microbiol.78, 4. https://doi.org/10.1016/S0378-1135(00)00303-5 (2001).

    Article Google Scholar

  37. 37.

    Villarreal, I. et al. Infection with a low virulent Mycoplasma hyopneumoniae isolate does not protect piglets against subsequent infection with a highly virulent M. hyopneumoniae isolate. Vaccinehttps://doi.org/10.1016/j.vaccine.2008.12.005 (2009).

    ArticlePubMed

Источник: https://www.nature.com/articles/s41598-021-01883-2

Free radicals, antioxidants and functional foods: Impact on human health

1. Aruoma OI. Methodological consideration for characterization for potential antioxidant actions of bioactive components in plants foods. Mutat Res. 2003;532:9–20. [PubMed] [Google Scholar]

2. Mohammed AA, Ibrahim AA. Pathological roles of reactive oxygen species and their defence mechanism. Saudi Pharm J. 2004;12:1–18.[Google Scholar]

3. Bagchi K, Puri S. Free radicals and antioxidants in health and disease. East Mediterranean Health Jr. 1998;4:350–60.[Google Scholar]

4. Aruoma OI. Nutrition and health aspects of free radicals and antioxidants. Food Chem Toxicol. 1994;32:671–83. [PubMed] [Google Scholar]

5. Cheeseman KH, Slater TF. An introduction to free radicals chemistry. Br Med Bull. 1993;49:481–93. [PubMed] [Google Scholar]

6. Young IS, Woodside JV. Antioxidants in health and disease. J Clin Pathol. 2001;54:176–86.[PMC free article] [PubMed] [Google Scholar]

7. Liu T, Stern A, Roberts LJ. The isoprostanes: Novel prostanglandin-like products of the free radical catalyzed peroxidation of arachidonic acid. J Biomed Sci. 1999;6:226–35. [PubMed] [Google Scholar]

8. Ebadi M. Antioxidants and free radicals in health and disease: An introduction to reactive oxygen species, oxidative injury, neuronal cell death and therapy in neurodegenerative diseases. Arizona: Prominent Press; 2001. [Google Scholar]

9. Lea AJ. Dietary factors associated with death rates from certain neoplasms in man. Lancet. 1966;2:332–3. [PubMed] [Google Scholar]

10. Harman D. Role of free radicals in aging and disease. Ann N Y Acad Sci. 1992;673:126–41. [PubMed] [Google Scholar]

11. Sies H. Oxidative stress: Introductory remarks. In: Sies H, editor. Oxidative Stress. San Diego: Academic Press; 1985. pp. 1–7. [Google Scholar]

12. Docampo R. Antioxidant mechanisms. In: Marr J, Müller M, editors. Biochemistry and Molecular Biology of Parasites. London: Academic Press; 1995. pp. 147–60. [Google Scholar]

13. Rice-Evans CA, Gopinathan V. Oxygen toxicity, free radicals and antioxidants in human disease: Biochemical implications in atherosclerosis and the problems of premature neonates. Essays Biochem. 1995;29:39–63. [PubMed] [Google Scholar]

14. Rock CL, Jacob RA, Bowen PE. Update o biological characteristics of the antioxidant micronutrients- Vitamin C, Vitamin E and the carotenoids. J Am Diet Assoc. 1996;96:693–702. [PubMed] [Google Scholar]

15. Mc Cord JM. The evolution of free radicals and oxidative stress. Am J Med. 2000;108:652–9. [PubMed] [Google Scholar]

16. Rao AL, Bharani M, Pallavi V. Role of antioxidants and free radicals in health and disease. Adv Pharmacol Toxicol. 2006;7:29–38.[Google Scholar]

17. Stefanis L, Burke RE, Greene LA. Apoptosis in neurodegenerative disorders. Curr Opin Neurol. 1997;10:299–305. [PubMed] [Google Scholar]

18. Esterbauer H, Pubi H, Dieber-Rothender M. Effect of antioxidants on oxidative modification of LDL. Ann Med. 1991;23:573–81. [PubMed] [Google Scholar]

19. Neuzil J, Thomas SR, Stocker R. Requirement for promotion, or inhibition of α- tocopherol of radical induced initiation of plasma lipoprotein lipid peroxidation. Free Radic Biol Med. 1997;22:57–71. [PubMed] [Google Scholar]

20. Poppel GV, Golddbohm RA. Epidemiologic evidence for β – carotene and cancer prevention. Am J Clin Nutr. 1995;62:1393–5. [PubMed] [Google Scholar]

21. Glatthaar BE, Horing DH, Moser U. The role of ascorbic acid in carcinogenesis. Adv Exp Med Biol. 1986;206:357–77. [PubMed] [Google Scholar]

22. Sokol RJ. Vitamin E deficiency and neurologic diseses. Annu Rev Nutr. 1988;8:351–73. [PubMed] [Google Scholar]

23. Ashok BT, Ali R. The aging paradox: Free radical theory of aging. Exp Gerontol. 1999;34:293–303. [PubMed] [Google Scholar]

24. Sastre J, Pellardo FV, Vina J. Glutathione, oxidative stress and aging. Age. 1996;19:129–39.[Google Scholar]

25. Cantuti-Castelvetri I, Shukitt-Hale B, Joseph JA. Neurobehavioral aspects of antioxidants in aging. Int J Dev Neurosci. 2000;18:367–81. [PubMed] [Google Scholar]

26. Freeman BA, Crapo JD. Biology of disease: Free radicals and tissue injury. Lab Invest. 1982;47:412–26. [PubMed] [Google Scholar]

27. Lovell MA, Ehmann WD, Buffer BM, Markesberry WR. Elevated thiobarbituric acid reactive substances and antioxidant enzyme activity in the brain in Alzemers disease. Neurology. 1995;45:1594–601. [PubMed] [Google Scholar]

28. Woo RA, Melure KG, Lee PW. DNA dependent protein kinase acts upstream of p53 in response to DNA damage. Nature. 1998;394:700–4. [PubMed] [Google Scholar]

29. Hattori Y, Nishigori C, Tanaka T, Ushida K, Nikaido O, Osawa T. 8 Hydroxy-2-deoxyguanosine is increased in epidermal cells of hairless mice after chronic ultraviolet B exposure. J Invest Dermatol. 1997;89:10405–9. [PubMed] [Google Scholar]

30. Halliwell B. How to characterize an antioxidant- An update. Biochem Soc Symp. 1995;61:73–101. [PubMed] [Google Scholar]

31. Shi HL, Noguchi N, Niki N. Comparative study on dynamics of antioxidative action of α- tocopheryl hydroquinone, ubiquinol and α- tocopherol, against lipid peroxidation. Free Radic Biol Med. 1999;27:334–46. [PubMed] [Google Scholar]

32. Levine M, Ramsey SC, Daruwara R. Criteria and recommendation for Vitamin C intake. JAMA. 1991;281:1415–23. [PubMed] [Google Scholar]

33. Matill HA. Antioxidants. Annu Rev Biochem. 1947;16:177–92. [PubMed] [Google Scholar]

34. German J. Food processing and lipid oxidation. Adv Exp Med Biol. 1999;459:23–50. [PubMed] [Google Scholar]

35. Jacob R. Three eras of vitamin C discovery. Subcell Biochem. 1996;25:1–16. [PubMed] [Google Scholar]

36. Knight J. Free radicals: Their history and current status in aging and disease. Ann Clin Lab Sci. 1998;28:331–46. [PubMed] [Google Scholar]

37. Moreau, Dufraisse Comptes Rendus des Séances et Mémoires de la Société de Biologie. 1922;86:321.[Google Scholar]

38. Wolf G. The discovery of the antioxidant function of vitamin E: The contribution of Henry A. Mattill. J Nutr. 2005;135:363–6. [PubMed] [Google Scholar]

39. Frie B, Stocker R, Ames BN. Antioxidant defences and lipid peroxidation in human blood plasma. Proc Natl Acad Sci. 1988;37:569–71.[Google Scholar]

40. Rice-Evans CA, Diplock AT. Current status of antioxidant therapy. Free Radic Biol Med. 1993;15:77–96. [PubMed] [Google Scholar]

41. Krinsky NI. Mechanism of action of biological antioxidants. Proc Soc Exp Biol Med. 1992;200:248–54. [PubMed] [Google Scholar]

42. Niki E. Antioxidant defenses in eukaryotic cells. In: Poli G, Albano E, Dianzani MU, editors. Free radicals: From basic science to medicine. Basel, Switzerland: Birkhauser Verlag; 1993. pp. 365–73. [Google Scholar]

43. Sies H. Oxidative stress: Oxidants and antioxidants. Exp Physiol. 1997;82:291–5. [PubMed] [Google Scholar]

44. Magnenat JL, Garganoam M, Cao J. The nature of antioxidant defense mechanisms: A lesson from transgenic studies. Environ Health Perspect. 1998;106:1219–28.[PMC free article] [PubMed] [Google Scholar]

45. Zelko I, Mariani T, Folz R. Superoxide dismutase multigene family: A comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) gene structures, evolution, and expression. Free Radic Biol Med. 2002;33:337–49. [PubMed] [Google Scholar]

46. Banniste J, Bannister W, Rotilio G. Aspects of the structure, function, and applications of superoxide dismutase. CRC Crit Rev Biochem. 1987;22:111–80. [PubMed] [Google Scholar]

47. Johnson F, Giulivi C. Superoxide dismutases and their impact upon human health. Mol Aspects Med. 2005;26:340–52. [PubMed] [Google Scholar]

48. Wuerges J, Lee JW, Yim YI, Yim HS, Kang SO, Djinovic Carugo K. Crystal structure of nickel-containing superoxide dismutase reveals another type of active site. Proc Natl Acad Sci. 2004;101:8569–74.[PMC free article] [PubMed] [Google Scholar]

49. Corpas FJ, Barroso JB, del Río LA. Peroxisomes as a source of reactive oxygen species and nitric oxide signal molecules in plant cells. Trends Plant Sci. 2001;6:145–50. [PubMed] [Google Scholar]

50. Corpas FJ, Fernández-Ocaña A, Carreras A, Valderrama R, Luque F, Esteban FJ, et al. The expression of different superoxide dismutase forms is cell-type dependent in olive (Olea europaea L.) leaves. Plant Cell Physiol. 2006;47:984–94. [PubMed] [Google Scholar]

51. Cao X, Antonyuk SV, Seetharaman SV, Whitson LJ, Taylor AB, Holloway SP, et al. Structures of the G85R variant of SOD1 in familial amyotrophic lateral sclerosis. J Biol Chem. 2008;283:16169–77.[PMC free article] [PubMed] [Google Scholar]

52. Chelikani P, Fita I, Loewen PC. Diversity of structures and properties among catalases. Cell Mol Life Sci. 2004;61:192–208. [PubMed] [Google Scholar]

53. Gaetani G, Ferraris A, Rolfo M, Mangerini R, Arena S, Kirkman H. Predominant role of catalase in the disposal of hydrogen peroxide within human erythrocytes. Blood. 1996;87:1595–9. [PubMed] [Google Scholar]

54. Eisner T, Aneshansley DJ. Spray aiming in the bombardier beetle: Photographic evidence. Proc Natl Acad Sci USA. 1999;96:9705–9.[PMC free article] [PubMed] [Google Scholar]

55. Meister A, Anderson M. Glutathione. Annu Rev Biochem. 1983;52:711–60. [PubMed] [Google Scholar]

56. Brigelius-Flohe R. Tissue-specific functions of individual glutathione peroxidases. Free Radic Biol Med. 1999;27:951–65. [PubMed] [Google Scholar]

57. Hayes J, Flanagan J, Jowsey I. Glutathione transferases. Annu Rev Pharmacol Toxicol. 2005;45:51–88. [PubMed] [Google Scholar]

58. Smirnoff N. L-ascorbicacid biosynthesis. Vitam Horm. 2001;61:241–66. [PubMed] [Google Scholar]

59. Meister A. Glutathione-ascorbic acid antioxidant system in animals. J Biol Chem. 1994;269:9397–400. [PubMed] [Google Scholar]

60. Padayatty S, Katz A, Wang Y, Eck P, Kwon O, Lee J, et al. Vitamin C as an antioxidant: Evaluation of its role in disease prevention. J Am Coll Nutr. 2003;22:18–35. [PubMed] [Google Scholar]

61. Shigeoka S, Ishikawa T, Tamoi M, Miyagawa Y, Takeda T, Yabuta Y, et al. Regulation and function of ascorbate peroxidase isoenzymes. J Exp Bot. 2002;53:1305–19. [PubMed] [Google Scholar]

62. Meister A, Anderson A. Glutathione. Annu Rev Biochem. 1983;52:711–60. [PubMed] [Google Scholar]

63. Meister A. Glutathione metabolism and its selective modification. J Biol Chem. 1988;263:17205–8. [PubMed] [Google Scholar]

64. Fairlamb AH, Cerami A. Metabolism and functions of trypanothione in the Kinetoplastida. Annu Rev Microbiol. 1992;46:695–729. [PubMed] [Google Scholar]

65. Nassar E, Mulligan C, Taylor L, Kerksick C, Galbreath M, Greenwood M, et al. Effects of a single dose of N-Acetyl-5-methoxytryptamine (Melatonin) and resistance exercise on the growth hormone/IGF-1 axis in young males and females. J Int Soc Sports Nutr. 2007;4:14.[PMC free article] [PubMed] [Google Scholar]

66. Caniato R, Filippini R, Piovan A, Puricelli L, Borsarini A, Cappelletti E. Melatonin in plants. Adv Exp Med Biol. 2003;527:593–7. [PubMed] [Google Scholar]

67. Reiter RJ, Carneiro RC, Oh CS. Melatonin in relation to cellular antioxidative defense mechanisms. Horm Metab Res. 1997;29:363–72. [PubMed] [Google Scholar]

68. Tan DX, Manchester LC, Reiter RJ, Qi WB, Karbownik M, Calvo JR. Significance of melatonin in antioxidative defense system: Reactions and products. Biol Signals Recept. 2000;9:137–59. [PubMed] [Google Scholar]

69. Herrera E, Barbas C. Vitamin E: Action, metabolism and perspectives. J Physiol Biochem. 2001;57:43–56. [PubMed] [Google Scholar]

70. Brigelius-Flohe R, Traber M. Vitamin E: Function and metabolism. FASEB J. 1999;13:1145–55. [PubMed] [Google Scholar]

71. Traber MG, Atkinson J. Vitamin E, antioxidant and nothing more. Free Radic Biol Med. 2007;43:4–15.[PMC free article] [PubMed] [Google Scholar]

72. Wang X, Quinn P. Vitamin E and its function in membranes. Prog Lipid Res. 1999;38:309–36. [PubMed] [Google Scholar]

73. Jaeschke H, Gores GJ, Cederbaum AI, Hinson JA, Pessayre D, Lemasters JJ. Mechanisms of hepatotoxicity. Toxicol Sci. 2002;65:166–76. [PubMed] [Google Scholar]

74. Papas AM. Diet and antioxidant status. Food Chem Toxicol. 1999;37:999–1007. [PubMed] [Google Scholar]

75. Brown JE, Rice-Evan CA. Luteolin-rich Artichoke extract protects low density lipoprotein from oxidation in vitro. Free Radic Res. 1998;29:247–255. [PubMed] [Google Scholar]

76. Furuta S, Nishiba Y, Suda I. Fluorometric assay for screening antioxidative activities of vegetables. J Food Sci. 1997;62:526–8.[Google Scholar]

77. Wang H, Cao G, Prior RL. Total antioxidant capacity of fruits. J Agric Food Chem. 1996;44:701–5.[Google Scholar]

78. Lin JK, Lin CH, Ling YC, Lin-Shian SY, Juan IM. Survey of catechins, gallic acid and methylxantines in green, oolong, puerh and black teas. J Agric Food Chem. 1998;46:3635–42.[Google Scholar]

79. Devasagayam TP, Tilak JC, Boloor KK, Sane KS, Ghaskadbi SS, Lele RD. Free radicals and antioxidants in Human Health: Current status and future prospects. J Assoc Physicians India. 2004;52:794–803. [PubMed] [Google Scholar]

80. López-Varela S, González-Gross M, Marcos A. Functional foods and the immune system: A review. Eur J Clin Nutr. 2002;56:S29–33. [PubMed] [Google Scholar]

81. Roberfroid MB. What is beneficial for health? The concept of functional food. Food Chem Toxicol. 1999;37:1034–41. [PubMed] [Google Scholar]

82. Krishnaswamy K. Indian functional food: Role in prevention of cancer. Nutr Rev. 1996;54:127–31. [PubMed] [Google Scholar]

83. DeFelice SL. Nutraceuticals: Opportunities in an Emerging Market. Scrip Mag. 1992;9:14–5.[Google Scholar]

84. Dillard CJ, German JB. Phytochemicals: Nutraceuticals and human health. J Sci Food Agric. 2000;80:1744–56.[Google Scholar]

85. Tapas AR, Sakarkar DM, Kakde RB. Review article flavonoids as nutraceuticals: A review. Trop J Pharm Res. 2008;7:1089–99.[Google Scholar]

86. Vidya AD, Devasagayam TP. Current status of Herbal drug in India: An overview. J Clin Biochem Nutr. 2007;41:1–11.[PMC free article] [PubMed] [Google Scholar]

Источник: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3249911/

Online license activation

Offline (manual) license activation

To use Unity, you need an activated licence. For Unity Plus and Pro licenses, you need to get a serial number; for a Unity Personal license, you don’t need a serial number. To see what kinds of licences Unity offers, go to the Unity Store.

Tip: You should always sign into the Unity ID dashboard in order to access anything you purchased from the Asset StoreA growing library of free and commercial assets created by Unity and members of the community. Offers a wide variety of assets, from textures, models and animations to whole project examples, tutorials and Editor extensions. More info
See in Glossary, or any licenses your Organization granted you. If you don’t have a Unity ID yet, open the Unity ID dashboard to create one.

To activate a Unity Personal license, you must go to the Unity Hub and answer some questions about your situation and for what purposes you plan to use Unity. For more information, see Activating a licence in the Hub.

To activate a Unity Plus or Pro license, you need to provide the serial number for the machine you want to activate it on. For single licenses, Unity emails the license information and serial number to the purchaser.

If you don’t know your Unity license information, speak to the Owner of your license. Owners can assign a seat to you via the Organization, or you can contact Unity Customer Service.

You can activate your license either through the Unity Hub or the command line:

Activating a licence in the Hub

If you haven’t installed the Hub yet, you can download it from the Download Unity web page. Then follow these steps:

  1. Select the Settings button from the top right of the Hub. The Preferences window appears.

  2. Select License Management from the list on the left.

    Hub > Preference > License Management
  3. If you see a message at the bottom of the window warning you that you need to be logged in to manage your license, select the LOGIN button from the bottom right and then enter your Unity ID and password.

    Important: You can’t activate your license in the Unity Hub unless you are signed into your Unity ID.

  4. Select the ACTIVATE NEW LICENSE button. The New License Activation window appears.

    New licence activation window
  5. Select which license type you are activating:

    • If you select the Unity Personal option, you must select either of the options which best describes your current situation and click Done.
    • If you select the Unity Plus or Pro option, enter the Serial Number you received in an email when you purchased a single license. You need to enter the key in the exact format that it appears in the email. For example, .

    Note: To activate an educational license select the Unity Plus or Pro option.

  6. Click DONE. Unity displays a success message.

If Unity displays an error message that the license activation was not successful, check Activation issues to find a solution for your issue.

Activating a license from the command line

Before activating your Unity license on the command line, make sure that the license file folder exists and you have write permission.

Note: The command line procedure only works for Plus and Pro licenses because Personal licenses do not have serial numbers associated with them. Use the Unity Hub to activate Personal licenses.

On macOS, enter the following into the Terminal to launch Unity and activate your license:

Replace with the actual path to your Editor installer. Typically, this is usually something like on macOS.

Tip: If you use continuous integration (CI) tools like Jenkins to activate via the command line, add the flag to prevent a WindowServer error.

On Windows, enter the following into the Command Prompt to launch Unity and activate your license:

Replace with the actual path to your Editor installer. Typically, this is usually something like on Windows.

Wait a few seconds after running this command to give Unity enough time to communicate with the license server. If activation fails, you can open the Editor.log for information. For any activation errors, check Activation issues to find a solution for your issue.

Offline (manual) license activation

Источник: https://docs.unity3d.com/Manual/OnlineActivationGuide.html

Cloud tv pro activation code

1. Other services limit the amount of cloud DVR storage you can access, and some even upcharge for the privilege. 8. 2. Select your plan. This is what you’ll use to match up your Android Box and your web-based ScreenCloud account. It won't take long. x or above. Using APKPure App to upgrade Cloud TV Pro, fast, free and save your internet data. Je navigue jusqu'à la rubrique " Abonnements TV ". The XCIPTV Player, or the Xtream Codes Player, is a fully customizable m3u playlist and media player that is fully compatible with Android smartphones, Android TV, Android Tab and even Fire TV as well. Install. GRUNDIG TV ARGANTO 70 Service Menu Code. 1 Build 503 serial number maker. 7 inches. Discover and play over 265 million music tracks. 5 Day Course #12 in Popular Official Curriculum Next Date November 7th ( Virtual Classroom ) 5 Classroom Dates 21 Virtual Classroom Dates Course Code PWL015. in COUPON (4 days ago) May 31, 2021 · Adobe Acrobat Pro DC 2020 Activation Code/Serial Number; HMA VPN Pro Activation Code/ License key 2021; Sora Shaders 1. Create a Cloudflare account and add a website. The official site for HBO, discover full episodes of original series, movies, schedule information, exclusive video content, episode guides and more. Our senior tech experts are always ready to help with any of your needs or questions. The Official cCloud TV The Popcorn Time for Live TV is now an cloud based IPTV media links sharing system where the links are shared by the users and can be found anywhere on the internet. To set up a new Cloudflare account, enter your email address and a password in the provided fields, then log in to your Dashboard. A minimum of 10 Mbps Internet is required. As we move to this next chapter, we are closing registration to Project xCloud Aug 23, 2017 · Showing 1-10 of 576 answers. It’s the flexibility you need to build a network and manage it from anywhere. Issue 21 of IT Pro 20/20 looks at the newest innovations and projects shaping our interactions with the world Transfer to New Computer, to Windows 11, to Windows 10, to Cloud, to Azure - programs, profiles and files Connecting your PC to the TV the first time When selecting the Toshiba TV for the first time, a 4-digit or 8-digit code will appear on your TV screen. No tech knowledge or skills required. Where is my IMEI and S/N? Method 1 Find the code sticker on the back of device, or packaging box. Adfree including Latino stuff. Download the My Cloud mobile application from your smart devices app store; If this is the first time installing or using the application, you will need to log into your MyCloud. So you can easily access and manage your TV through the browser of your mobile phone, computer or tablet. Get more info on directv. 74238464 – Bobbyv123, Lots of streaming apps, and more. Download Avid Software. 01:03 her a free TV so on this pop up like. Method 2 Jul 21, 2021 · Apr 17, 2021 — Download and extract file New CloudTV archive zip 3. cloud tv pro username and password. Cloud Tv free download - Cloud System Booster, Online TV Player, Free Internet TVand many more programs Oct 22, 2021 · YouTube TV is one of the only streaming services to offer unlimited cloud storage space for its DVR. Activate the service and get 20GB common storage space for your company, to youtube by click patch download - Activators Patch and manage your files (BusinessCloud Storage) as well store backup version of your data (BusinessCloud BackUp). 9 hours ago. Recent Tech News. You can easily access coupons about "WPS Office Cloud Tv Pro Activation Code" by clicking on the most relevant deal below. Was this article helpful? yes / no. Onesafe pc cleaner license key free for you, while I’ll update the GitHub username, how is this “new” repository considered “new” if it is still the same repo version 1. Jun 08, 2021 · Search: Cloud tv free activation code. OR. 0. Mounting bracket specially engineered for Airstream roofs. If it isn't, you can download it from your device's app store: Apple® App Store® Google Play™: Smartphones; Then follow the steps below: Set up the Verizon Cloud mobile app. Included: unlimited cloud DVR storage space so you can record your favorites, and stream them wherever you go. DEVONagent 3. 0. FreeAir. We focus on the Television of the Future and intend to provide technologies for a Global Mobile Internet Platform to allow people to watch any TV content anywhere, anytime. 1. Activate your device! No device? No problems! You can use your computer! All your need is your device activation code. 2020 - Download cloud tv 2020 APK Android Game for free to your Android phone cloud tv. Y: Cloud Tv Pro Activation Code Coupons, Promo Codes 10-2021. Click Activation. They offer sports, international channels, a TV guide, music, radio, and on-demand options. Cloud Tv Pro Activation Code can offer you many choices to save money thanks to 19 active results. It lets you sort by channel, groups, and so on. Safari 4. eu Serialkey preview: VP9VV-VJW7Q-MHY Service Update: Telstra TV Maintenance. and watch them through various devices. Kodi is available for multiple operating-systems and hardware platforms, featuring a 10-foot user interface for use with televisions and remote controls. 3 streams and 6 accounts per household via Family Sharing. Reschedule or cancel an appointment online. On this screen, select ‘Business. Cloud TV Broadcast Package. Our roundup of the best www. THANK YOU May 05, 2021 · The final app on our list of the best IPTV and m3u Android apps is perhaps the most stylish looking one of them all. 4ft W* Bft 3 M » 1 114k. 35 Mbps - 21 Mbps download speed to my mac mini computer, and It works fine. Bypass iCloud Activation Lock in 3 simple steps. 0 here. The latest ones are on Mar 08, 2021 10 new Get Activation Code For Cloud Tv Pro results have been found in the last 90 days, which means that every 9, a new Get Activation Code For Cloud Tv Pro result is figured out. 5GHz CBRS Bands. Unlock within 3 Simple Steps. VidOn Membership also allows members to use the advanced features of the upgraded VidOn XBMC Pro across 5 devices simultaneously. Go back to the main menu and select the search icon. Jun 18, 2020 · Clear Choice – Premium App Installation Instructions. ALSALM ALYKOM i had okstar 50″ led andriod smart uhd tv with system (wisdom share smart cloud tv) 2020 model No. 9. 9 allows you to watch over 800 live TV channels from all over the world (UK, TV TOSHIBA 50L7333DF 127 cm, fiche technique détaillée, liste de prix, notes et avis utilisateurs, LCD Compare D: . Here you'll find information on the basics like product download, how to get up and running quickly, and activation, as well as multiple tutorials on each application featured in Creative Cloud. Search for “Downloader” and download the app. Aug 30, 2021 · Learn how to use a redemption code to activate an Adobe Creative Cloud membership or Acrobat DC subscription and more. Jul 02, 2018 · Download Cloud TV Pro APK [66 MB] ( Free) - CloudTV APK - Cloud TV Pro App - Latest Version. (If nothing happens, click here to download the latest version) For Windows 7, download CloudApp 6. You can manage the below information with this editor. smart cloud tv - how to setup smart iptv (cloud tv) on smart tv & free m3u cloud tv. Best Practices while using Plex Media Server on a My Cloud or My Passport Wireless Pro and SSD devic This article explains how to better use Plex Media Server on a My Cloud or My Passpo Using the My Cloud Mobile App's Auto Backup Feature avast internet security + keygen+crack - Crack Key For U Android VidOn membership allows access to VidOn XBMC Pro's awesome list of advanced features and can greatly enhance your home's entertainment experience by turning your living room into a private theater. Find a Comcast Service Center. Cloud application. If you are searching for Cloud tv free activation code, simply check out our information below : To install a TV Cloud app to your Fire Stick or Android device: Go to Settings->My Fire TV->Developer options and set “Apps from Unknown Sources” to “ON”. 6 and 10. 8 new Cloud Tv Pro Activation Code results have been found in the last 90 days, which means that every 11, a new Cloud Tv Pro Activation Code result is figured out. SS IPTV has become the first app of LG Smart World, which provided opportunity of IPTV viewing for its users. 264 encoding, compression, and multiplexing within broadcast and IPTV systems. Sva prava zadržana A leading U. Use the Adobe product code you received with your purchase from a retailer or reseller (or received as a gift card) and start using Adobe apps. Smart TVs. Screebl Pro (2. Advanced features 147 To register your TV to a DivX ® account: This TV allows you to play back files purchased or rented from DivX ® Video-On Demand (VOD) services. apk Jan 25, 2017 — Cloud TV and has one of the most popular channels on YouTube. Cloud From Amazon Fire TV or Roku ‎Cloud tv كلاود‎. By clicking the Get started button, you consent to the WD license agreement and privacy policy This introduction to Digital TV is a 5-day training course on DVB and MPEG-2, MPEG-4 / H. 0 or later cable, and have a TV that supports 4K, HDR, or both. Oct 22, 2021 · Give staff an easy, flexible and secure way to share files and folders. Date: 04/04/2019. You can subscribe to service and use your computer today. Download Free CloudTV 3. Information about Comcast Business services © TV Arena 2021. 9 for Mac on Mac Torrent Download. Monthly - Not available. Let's set up your personal cloud and create a My Cloud account. You can be the first one to see your new episode of your favorite TV series. If you are not founding for Cloud Tv Activation Code, simply look out our text below : cloudtv pro activation code Cloud TV yester- daywas reported safe and attempt- ing to finish the race. 7 Sends all search requests to Google and Bing using Dec 16, 2018 — CloudTV 3. Locate the 3-digit TV code for your TV. Instead of buying, owning, and maintaining physical data centers and servers, you can access technology services, such as computing power, storage, and databases, on an as-needed basis from a cloud provider like Amazon Web Services (AWS). Share public links shielded by passwords and expiration dates. I have my own phone that I didn't purchase from Verizon. An account only can be used on one device at same time. Key Pro Player 3. Last Name cloud tv pro activation code - nlpcoachingacademy. only 2 day and then start logo repeated 10 times and restart i found this NUMBERS SERIAL No. 00:39 so we're gonna go over to cloud TV. 9/5 Votes: 2. Mar 26, 2019 · Team Cloud. It's Wicked Fast! Puffin Web Browser shifts computing tasks from local devices to cloud servers. This service gets rid of the need to buy, install or maintain additional hardware. This means even the most resource-demanding web pages can load at incredible speeds, saving up to 90% bandwidth on regular browsing through our proprietary algorithm. Code for the web from a truly new one-window interface. Jul 27, 2015 · Installed Roku Applications. Simply follow the steps on your screen to complete activation and set up. tv is your personal IPTV service. When prompted, enter the activation code from your device. 2 Kodi app to the newest version on my M8S-II Android TV box. 11). 7 or above. Create an account in less than 1 minute. Leveraging this, an attacker on the local network can send a crafted request to broadcast a fake video. Puffin Web Browser. Item Details. 99*. May 19, 2020 · With these “codes” you can activate separate or shared applications. Use our handy guide to find the settings and features you need. PAC pro 12 streams live TV of countries like The USA, The UK, Canada, Vietnam, World. Amazon Web Services publishes our most up-to-the-minute information on service availability in the table below. First month is always for freeDigital "over IP" television administration, a content protection and analytics. The new EPG add-on is great, and I’ll definitely add that to this post. Cloud tv free activation code. Record your favorite show, just program what you like to watch. 1, 7 última versión. 2 APP Developer. Create your TV station, free and start broadcasting live, in just a few minutes. When you record a video in the PlayOn Cloud app, it starts a stream on a virtual PC in the cloud and the stream is recorded in real-time like a DVR records cable TV. Developer: Team Cloud - Package Name: com. 3 Press the p / q TROYPOINT recommends avoiding the following devices at all costs, Roku, Apple TV, Raspberry Pi, and other non-Android systems. Overview: watch your favorite Arabic channels with the best performance and reliability available. general entertainment network, ION’s Positively Entertaining lineup features major cable & broadcast shows, original series and special event programming. Are you looking for "WPS Office Cloud Tv Pro Activation Code"? We collect results from multiple sources and sorted by user interest. Smartphones & Tablets. The My Cloud mobile application is available for both iOS and Android. 2021 - La description de cloud tv Free. Get started. Je clique sur " Gérer mon abonnement " puis j'entre mon code d'achat (0000 par défaut). Features: -Live Arabic channels. 3 + activation code valid to 21-2-2021. Compatible with Android TV Box, Android Smart TV, Android Phone, Android Tablet. Continue Browsing TV ONE. x or above in Mac OS. 10 new Get Activation Code For Cloud Tv Pro results have been found in the last 90 My Book, WD Elements, EasyStore, Gaming Drive; My Passport Wireless, Pro, SSD; My. Aside from editing this basic information, it allows to do imports to add lists together (instead of just editing one list at a time). in DA: 21 PA: 37 MOZ Rank: 78. 4 pounds. With an elegant interface, easy to use, with Movies and TV categories in one application for you to enjoy the best content. Samsung 55NU7090 55-inch Ultra HD 4K Smart LED TV is a good TV from Samsung with Live TV, 55,000+ on-demand titles and 20 hours of cloud DVR storage. Get help activating your device. Subscribe a Packages via Alipay (支付宝) How can check the Version of CloudTV? Aug 16, 2021 · CLOUD TV tricks hints guides reviews promo codes easter eggs and more for android application. Offer 99. 5. Enjoy local and national live sports, breaking news, and must-see shows the moment they air. Press the space key then arrow keys to make a selection. Everyone. NO. First Name. Click the install file to finish up. Download cloud tv apk 2021. The post black TV pro v1. Sign in - Google Accounts 00:32 we are going to start with cloud TV. Updated. Latest Cracked APKs, Mod Apks, Mod of Game Apks, Go to AT&T TV or the AT&T TV app. Verizon Cloud may already be installed on your mobile device. lista IPTV premiere, space IPTV, IPTV sat, IPTV sat, teste IPTV, teste IPTV 12h, Lista IPTV gratis Maio, smart IPTV ativação gratis, IPTV Latino 2021, ok2, ok3, free IPTV m3u, codes, BestBuy IPTV, activate, activation codes, IPTV Free, orbit IPTV, IPTV smart tv, IPTV CloudTV is the best Cloud OTT TV Platform. Fully editable shapes, colors and scalable without losing it quality. To Install Clear Choice Premium App on a Firestick, follow the instructional video below, but rather than installing Cloud Television, install Clear Choice. 2. to download the APK file of the application and then you need to visit the official site of. ONELAN is a global leader in the development & management of digital signage media players and CMS solutions. By papercloudsfx. Internet Protocol television (IPTV) is the delivery of television content over Internet Protocol (IP) networks. SANSAT Pro IPTV, Best IPTV subscription for your Smart tv, Android, iPhone, Apple TV and MAG Box, subscribe to watch sport games and TV Shows; Need help? All help u need to unlock Icloud or bypass, also visit https://idevicetool. Chrome 15. 7 Key - Multilingual https://goo. Put the codes listed above in Downloader (just the 5 digit number). Cloud computing is the on-demand delivery of IT resources over the Internet with pay-as-you-go pricing. 99. More. Open the Verizon Cloud app on your device. GitHub Gist: instantly share code, notes, and snippets. Sa aplikacijom ArenaCloud možete da pratite sve kanale iz bogate ponude regionalnog televizijskok giganta Arena Channels Group, vrhunske sportske kanale ARENASPORT 1-5 sa ekskluzivnim sportskim sadržajem, sve popularnije gejming okršaje i sve aktuelnosti iz najbrže rastuće industrije na svetu putem Arena Esport specijalizovanog kanala, budite VIP gosti na premijeri spotova regionalnih 安博科技 - 安博盒子UPRO,安博電視盒子,4K電視盒子,高清電視盒子,ubox,安博平板,upad,機頂盒,電視直播盒子,招募代理商經銷商. More ways to shop: Visit an Apple Store, call 1-800-MY-APPLE, or find a reseller. Not a problem. Also available on smart TVs and streaming platforms. Starsat SR-T14 Extreme & Pricing studio - Free Activators SR-2020HD CloudTV is the best Cloud OTT TV Platform. Get IPTV subscription online effortlessly through us and pick your favorite device 2G/3G/4G/LTE antenna (5G Ready) 2 x MIMO LTE. ) 1 Month Package. If you are look for Cloud tv free activation code, simply look out our article below : Cloud tv activation code Cloud tv activation code Search: Cloud tv free activation code. Cloud Activation Code Tv. Additional Streams and increments of 50 Cloud DVR hours are available together at only /mo. Cloudflare offers four plans: Free, Pro, Business, and Enterprise. Cloud Tv Cracked Apk Sitesinstmank. S. We offer a number of services that fit your needs. In addition to its many TV channels, Kshaw IPTV also provides excellent picture quality, especially in HD, UHD 4K and 3D. 00 (Approx. Sign in to your Spectrum account for the easiest way to view and pay your bill, watch TV, manage your account and more. Cloud tv activation code Cloud tv activation code Sep 23, 2021 · Android TV has been slowly but steadily maturing as a platform since it was first introduced several years ago. STEP 2: Point your universal remote toward the component and press the button Component button (TV for TV, DVD for DVD, etc. If you have purchased a boxed ESET product, you can have it activated by filling out the form below. 8x8 helps businesses of all sizes empower employees and delight customers with a unified platform for contact center, business phone, video, and chat. It’s a top notch application that allows its users to stream Channels ,movies series. Plus, you’ll have peace of mind from enterprise-grade security, audit logging and user controls like SSO and custom permissions. gl/Csz2k3You to observe over 800 live tv channels from everywhere in the world, in addition to May 09, 2013 · Create a Cloud TV account. Easily display whatever's important to your business or organization – whether A keygen is made available by crack groups free to download. The media control for the device doesn't have any session management or authentication. 17779393 – Jo714 / JoAppReviews YouTuber, One of the Largest Lists of APK’s. 216, offers in-depth game coverage and highlights, real-time stats and scores, and the latest NBA and WNBA news 24 hours a day. What is cloud TV? Basically, Cloud TV is a cloud-based live TV streaming app or program in which you can view HD TV channels, movies, and TV shows. Descargar e instalar Cloud TV Pro para PC en Windows 10, 8. 97. gl/Csz2k3You to observe over 800 live tv channels from everywhere in the world, in addition to Sep 19, 2021 · 0. Download this app from Microsoft Store for Windows 10 Mobile, Windows Phone 8. cCloud has a EPG program guide and extra features to ensure channels stay HD! I have a TCL Roku 2016 Model TV, and I have a Samsung Blu Ray Remote Model # AK59-00149A From what I have read I need 2 digit codes that I enter while holding down the Power Button (No idea how long prior to entering the code I should hold it) So if anyone has any 2 digit codes that would help that would be just splendid! TiVo brings you live, recorded, and streaming TV together into one premium experience, whether you’re a cable fan or someone who has cut the cord – so you can spend less time searching and more time discovering, watching, and enjoying what you love. If you use a Microsoft service like Outlook. OKSTARA50HY-0311. Download. Then, I could watch my Kodi add-ons, YouTube, mobdro, cloud tv, and the rest of my apps but could get no sound on any of them. V8 golden, V9 Pro and V7 are cable tv box, V8 Angel is cable+cable dual system. Cloud TV APK is nothing more than an app, but it’s capable of delivering movies and TV shows for free. BOX-A-20120615 key generator. 2 Select Options > MediaShare Setup > DivX ® VOD and press the OK button. Ask a question or add answers, watch video tutorials & submit own opinion about this game/app. Dimensions: 10. Depending on the product, you can find your serial number: On the surface of your product. 00:59 free TV no premium child and 90 days go. NBA TV, found on Ch. Cloud tv pro apk 2021. To create a Cloud TV account: Using your TV remote, power on your Toshiba Cloud TV. 16. The TV Cloud - Stream Movies, TV Shows and more! - Instant Activations. Cloud Oct 23, 2021 · IPTV. Adobe. If you first connect to the My Cloud over the local network with the mobile device, you generally won’t need to enter in an activation code into the WD mobile app. 99 Offer19. Aug 17, 2020 · Kshaw IPTV + Activation Login. This makes PAC-12 pro as one of the reliable add-ons. Windows Vista and Windows 7. All rights reserved. Which give you real HD experience. For some reason in the eavening my stick loses connection. Pin: 9898. Use your Apple ID or create a new account to start using Apple services. To install a TV Cloud app to your Fire Stick or Android device: Go to Settings->My Fire TV->Developer options and set “Apps from Unknown Sources” to “ON”. The latest ones are on Oct 18, 2021. Activate your device. ) Open the CloudTV apps. The red light on the remote will blink twice and stay on. Best of all: all channels are in FULL HD and without crashes. You can get the best discount of up to 80% off. Names of these languages are Indian, English, Mandarin, French, Arabic, Japanese and Cantonese. 3. TV application BOX and SMARTPHONE World TV MALL. Mac OS ® X version 10. 00:50 idiom what you want to do. Installation. I have an amazon fire tv stick 4x. Watch here or on Apple devices. Kshaw IPTV is a very well known IPTV APK. Dynex DX-LCD32. 1 4 17 5 13 3. Welcome to My Cloud. Cloud Tv Apk Cracked Ipa. FastoCloud IT Pro 20/20: Using technology to create a better future. iptv, m3u8, m3u, iptv list, iptv usa, us iptv m3u8, m3u file, m3u playlist, m3u8 extinf, iptv stream, iptv address,m3u8 playlist, extinf playlist Best IPTV - 10,000 Channels from 82 Countries - No Buffering. We use cookies on this site for various purposes, including to enhance site performance, personalize your experience, and deliver interest-based ads. KeyEntertainment Entertainment. It will prompt you for Service ID, username and password. Cloud tv activation code Stay in touch. Most of the other Kodi live tv addons stream the contents from the same server as sports devil (sports devil framework). 4. Transform your business with innovative solutions; Whether your business is early in its journey or well on its way to digital transformation, Google Cloud Contact Apple support by phone or chat, set up a repair, or make a Genius Bar appointment for iPhone, iPad, Mac and more. Xtream codes 26-10-2021. 00 ( 3 ratings ) 5. No device. CLOUD TV APK WITH ACTIVATION CODE MIX COUNTRIES CHANNELS tv for your android and ios Cloud tv apk cloud tv apk 2017cloud tv apk cracked 2017 You'll use your Microsoft account for everything you do with Microsoft 365 or Office. Internet Protocol Television or IPTV gives you the ability to stream live TV channels on any device through the internet connection. Activation: Dial * 3 3 (four-digit code number) # Cancellation: Dial # 3 3 (four-digit code number) # The four-digit code number will be given to you by Cyta. 01:08 this and you got Vectric Aspire Free Downlod mechanic orders May 25, 2014 · STEP 1: Turn on the component you want to activate. Please enter verification code Invalid security code. Continue. 0P1. Adobe TV. cCloud has a EPG program guide and extra features to ensure channels stay HD! I have a TCL Roku 2016 Model TV, and I have a Samsung Blu Ray Remote Model # AK59-00149A From what I have read I need 2 digit codes that I enter while holding down the Power Button (No idea how long prior to entering the code I should hold it) So if anyone has any 2 digit codes that would help that would be just splendid! With our complete and large collection of TV channels, never miss your favorite sport games and TV shows. 00 Amazon Gift Card. CloudTV 3. 0 x 5. Add to Wishlist. Type SLUI 04, and then click OK to open the wizard. Jun 28, 2019 · This module exploits an unauthenticated remote file inclusion which exists in Supra Smart Cloud TV. Dans le menu " Mes bouquets TV ", je sélectionne " Disque dur numérique TV ". Once downloaded, launch the app on your TV and make a note of the pairing code that comes up. Say goodbye to slow VPN connections, unversioned documents attached to emails and shadow IT in public clouds of questionable security. The fire stick Nexrwork show Disconnected and I check the status and it switched between Scanning & Obtaining IP address by doesn Kodi is a free and open source media player application developed by the Kodi Foundation, a non-profit technology consortium. To let the user gets the most of it the cloud tv app is made available in eight languages. Next, enter the 3-digit TV code. CO 6 34 1714 U4 3 W 5. Get Activation Code For Cloud Tv Pro can offer you many choices to save money thanks to 20 active results. But you can transfer this account to other Android device after you untie it. 繁體中文. 5; Driver Easy Pro 5. ’. 2 MB) Versions. About Activation Tv Code Cloud. IPTV Editor is a program that lets you import M3U or CSV playlist files for IPTV channels. Related articles. Select “Cloud Setup Wizard” and follow the on-screen instructions to setup an account. Get your Activation Code instantly via email and bypass iCloud Activation Lock in no time. com account; A list of devices available to add to your My Cloud app will be displayed. Not only can you manage the files and APP applications on the TV, you can also push any type of Sep 13, 2019 · The cCloud Kodi addon is a free source for over 1000 live television channels. Collection of publicly available IPTV channels from all over the world. If unavailable, please continue with your activation now. ) + YuppFlix Movies + USD 0. tv service. 11 pm, 10 am, 4 am, no worries, we got your back and record on your schedule in the cloud. We recommend getting one stream for each member of your household. Supported Web Browsers: Internet Explorer® 9. Whenever coded barring is activated by an ISDN terminal, barring applies for both the pilot and for the slave number and therefore no device (analogue or digital) is capable of making calls to the ISDN phone. HAPPY TV APK CRACK. ) the Device ID (Serial Number) will show the bottom on screen. Welcome to Avid—thank you for your purchase. TT. Hybrid 2 comes with NO BUFFERING CLOUD TECHNOLOGY gives you no buffering and 100x ZAP technology. Cloud tv pro activation code. Nlpcoachingacademy. CloudTV was designed to enable operators to deliver a consistent, customized and refreshed TV experience to their installed based of QAM and IPTV set tops. 4 APK Download and Install. Safely involve contacts outside your organization with select documents. The TV Cloud is the fastest growing service around. Cloud Tv Apk Cracked Apps 5593a673d3 [Share on Facebook. See screenshots, read the latest customer reviews, and compare ratings for CloudTV. Enter your serial in the text box and click Activate. Nov 14, 2019 · Hi Bane! Thanks for your work on cCloud TV! Awesome stuff. Includes unlimited cloud DVR storage. IPTV Editor. Subscribe a Packages via Alipay (支付宝) How can check the Version of CloudTV? Nov 05, 2019 · YouTube TV is a subscription streaming service that lets you watch live TV from major broadcast and popular cable networks. iCloud Activation Bypass Tool Version 1. Instant Activation 82 Countries, 10,000 Channels XXX, VoD, Sports HD, PPV, TV Guide Unbeatable Price Support All Devices & Apps When you order a new phone from us, it will be ready to activate when you turn it on. -Works on all devices that support the Android system. Choose service that you want to use on your device Viaway offers a number of services that fit your needs. Oct 18, 2020 · Download Perfect Player IPTV MOD APK Perfect Player Pro is Nov 09, 2020 · Cloud TV App - Download Cloud TV APK 2020 For Android. Blu-ray Players. Cloud TV V4. Jun 16, 2021 · To enter an activation code: Open Kaspersky Endpoint Security Cloud Management Console. 0 X 5. Start today, with no ads or bandwidth limits. Robust and water resistant (IP68) antenna. Utilisez votre cloud tv sur ss iptv. 00:35 cloud TV is an IPTV so let's get started. Streaming Media Players. TAG’s MCM-900 chosen for EU’s 5G-Virtuosa project 2021-08-31T11:08:00+01:00 By Jo Ruddock CLOUDSKIPPER is a trademark and brand of Cloud TV, Inc. 4 + valid code {tested – working} Overview: WithCLOUD TV IPTV you have the possibility to watch several open and closed TV and Movies. We then combined the best features of every streaming service to provide a unique, enjoyable viewing experience

Источник: http://speaking-english.biz/hmqf

Microsoft Office 2019 Activation Key + Crack ISO Free Download

Microsoft Office 2019 Activation Key + Crack ISO Free Download

Microsoft Office 2019 Activation Key is a cross-platform that is comprehensive for everyone with smart tools for specific users, teams, and businesses. It can open your apps, documents anywhere on numerous products. It offers brand new compliance and deployment protection, giving companies more significant control of sensitive data and greater flexibility in deployment and management. Microsoft has expanded offices for iPad and Android pills. Updated Office features on Mac, iPhone, plus the web.

Additionally, brand new applications have been added to your family, a swipe and office lens. All things are made to keep work everywhere. We have concentrated on customizing Office for different platforms in the last 12 months. Office on the Windows desktop is Central to our strategy when you’ve seen just how.

Microsoft Office 2019 Crack is the latest version. It is the most demanded software ever. Everyone uses MS Office for their projects. Simply put, now you can’t do anything without Microsoft Office 2019 Crack. And one of the many difficulties that primary users can’t activate this version quickly. For sure, the user needs the Microsoft Office 2019 activation key for the activation of the software. So then you will solve your problem on this important platform if you’re certainly one of them. Right here, you’ll find working and activation that is valid for lifetime activation.

Microsoft Office 2019 Crack Download Full ISO

Microsoft Office 2019 Crack is utterly appropriate for its wide range – from students to office staff and personnel, along with business professionals. Due to the different enhancements, the essential functions, and the development of new people, document creation, processing, and handling are now doable and faster than previously. In addition, mathematical calculations and expressions are improved and generally are far more easily incorporated into practical use within PowerPoint 2016, Excel 2016, and Word 2016.

Microsoft Office 2019 Activation Key is useful for both Windows as well as a Mac users. It can open, edit, make, and save files regarding the cloud from your desktop. And a search that is new for commands is available in MS PowerPoint, Excel, and Outlook. Updated choices include its user software that supports Retina Display and uses ribbons as well. In addition, users can now share documents via social networks and email messages directly through the MS Office 2016 Crack toolbar.

Key Features of Microsoft Office 2019 Crack:

  • Better along with Windows 10– allow you to complete work, the papers in full fidelity across your Windows 10 products.
  • Better Control over Resource Scheduling– ensures that scarce resources are now being utilized effectively throughout the company.
  • You are permitted by the application font creator 12 - Free Activators enter everything you wish to do in your terms, then guide you on how best to do so – plus other resources.
  • Modern shapes benefit from newly redesigned forms in Visio professionals.
  • Real-time typing-view everyone’s edits and where these are typically into the document as you work simultaneously on the same document.
  • New chart types– present better visualization ability and enables you to identify the partnership that is statistical your computer data.
  • Improved data connectivity-connecting your diagram to Excel data is just one click away.
  • Themes – the latest themes give a fresh look and an amazing presentation.
  • Smart lookup checks your document and automatically teaches you the SERPs via your writing environment via the web.
  • One drive integration – enables you to store your details in one access and location other than anywhere, using any device.

What’s New in Microsoft Office 2019 Crack?

  • Powerpoint, one notes, Skype, PowerPoint, Access, Excel, Skype, and Outlook are the most popular and advanced applications.
  • Creates presentations and creates computer analysis records.
  • Tests the emails from Outlook.
  • It does not need VPN settings from anywhere to call.
  • In the layout parameter, it will also automatically customize the UI.
  • All papers, including PDF e-books, are very much sponsored.
  • Updates and edits the database and prints a selection of templates as well.
  • Use Excel to create tables and solve problems with mathematics.
  • It can also view and edit device-wide files.
  • Create amazing Microsoft PowerPoint 2019 presentations.
  • Besides, we can work on the same project with our team concurrently.
  • Get permission automatically and post the reports with one touch.
  • New charts for displaying nuanced details are also included.

Microsoft Office 2019 Activation Key

QZA3W-SE4XD-5CR6T-FV7BGY-8HUN9

J8BY7T-V6CR5-E4ZWS-EXD5CT-FVBG8H

UN9JIH-8UBYV7-CR65X-E4SZX-DCRTFV

BG8NH-UIJMK-OJIN9U-B8Y7TV-6C5RE

X4SDC-RTFVBG-8NHUI-JM0KO-M9NUB

8Y7TV6-DC5RE4-SXDCR-TFVBG-Y8NHU

Microsoft Office 2019 Key 2022

IJM9NH-8UBY7T-6DC5R-E4SEXD-C5RTF

VBY8NH-UIJ98N-HBGTFV6-E4SX-Z3WA

4SXDCE-TFV68N-HYU9IJ-MN8H-UTFV6

D5RE4S-ZW3E4-XTFV6B8-HYNU-J9IMN

HB8UTF-V6E4SX-ZW3AS-E4XTFV-6B8HY

NUJ8HB-YV7T5R-XE4SZA-W3ZSE-4XTFV

Microsoft Office 2019 Product key ISO

6B8YNH-U9B8Y-V7T6D5R-ESZWA-3SE4

XTFV68-NHUJ9-IH8BGF-DCXE4-SZ4XT

FV68NH-UBGFV6-DC5SX4-ZKWS-4XETF

V6BY8-NHU9IN-8YT76R-E54W-O3WAZ

Benefits of Using Microsoft Office 2019 Activation Key?

Also, several individuals will concurrently work on the paper or an Excel spreadsheet or PowerPoint. This functionality was updated by Microsoft and is gradually being marketed now. Not everybody will be compelled to go to the cloud, but there’s a lot of push with subscription providers. And one thing to do is, even now, the Office 365 Crack has such a feature as more, for example, or PowerPoint concept concepts, which are not routinely available for the Office 2016 Crack. It is a database adapted so that certain features bring users into the cloud. Office 2019 presents lots of new features and some of the coolest I’ll introduce. Many of them are not exactly new functions. But they were exclusive to office 365, so you can now get access to them if you do not want to pay the monthly fee.

Microsoft Office 2019 Keys

  • ZAQ3W-S4XED5-CRF6TV-B8HYN-UJ9IHU
  • 8BYV7T-6CD5RX-E4SZEX-TFV68N-HUIJM
  • K0OMIJ-98HUBVY-7TDC5-RESX4-SXTFV6
  • 8NHUIJ9-M0KJ9N-H8UTFV6-D5RXE-4STF
  • V6BG8H-UNIJM9-8NHBG-TFV6D-5RE4D5
  • XCRTFV-BGHUNIJ-9M09N-HBGY7-TFVDC
  • 5RXE4S-SX5DCR-TFV7BG-8HUN9IJ-MN8H
  • UTFV6D-5RESX4-X5DC6TF-V7B8H-UN9IJ
  • MNH8-UT6D5R-ESXDR-C6TFVGB-YHUNIJ

Microsoft Office 2019 Product Key

  • AZWSX-EDC5RF-V6TGY-BH87V-F6C5X
  • 4SZAW-SEXD5-CTFVG-YBHU8-FT6CD
  • 4ZA3W-Z4SEX-DCRTF-GBY8G-V7C6D
  • 5RZSE4-ZSE5D-RC6TF-V7BGY-H8VFC
  • 6D5XSZ-4AWE5-SXDCR-6GVY-BHUGV
  • FCD5R-SXEZ4A-WZ4E-SXDCT-FVGYB8
  • YFV6D-C5SXEA-4WZESX-DRCTF-VGYH

Installation Instructions for Office 2019 Crack:

  1. The download is given below to start.
  2. Extract zip file through WinZip.
  3. Click “setup” and proceed with the installation process.
  4. Complete configuration and close it.
  5. Open MS Toolkit located inside the crack directory.
  6. Click the button to the left of the Windows icon and go to the Service tab.
  7. Activate” Microsoft Office 2019.
  8. Done!
Источник: http://www.cci.ci/

Windows 10 Product Key Crack With Serial Number Free Download 2022

Windows 10 Crack Product Key 64/32 Bit Torrent + [100% Working]

Windows 10 Crack provides a product key for each user. A product key is the user’s century-old code for product authenticity created for each user. they may ask you to enter your product key. If your product is not available, you will need to crack Windows 10 to activate it. There are several Windows 10 hacks through which you can easily break windows to activate it.

Windows 10 64/32 Bit operating system has received an excellent set of tools for its flexible functions and wide choice. Windows 10 was unexpectedly released a long time ago in this already launched market. Users downloaded over a million copies on a single opening day. Windows has demonstrated satisfactory improvements in its runtime infrastructure since its inception. In this article, the smart and advanced features of Windows 10 are explained. Read through the article to understand the Windows preview and utilities.

Windows 10 Crack is a software program too. The product key you get together along with your model certifies that your reproduction became now no longer obtained in violation of Microsoft’s copyright. This is the software program (KMSpic, KMSauto) used to set off Windows 10 and it’s far free. Then we will get the KMSpico software program and set off our Windows 10. This article is ready Windows 10 product keys, what they are, and the way you may effortlessly get one whilst Windows asks for it. It’s so tough that even password-cracking gear can not locate one. However, that does not suggest you haven’t any hope. There are some hints and hints for activating Windows.

Note: Before installing Windows 10, make sure your computer meets the system requirements for Windows 10. It is also recommended to visit the PC manufacturer’s website for more information on updated drivers and hardware compatibility.

Windows 10 Key Features:

  • Disk analysis, clean up unnecessary files and improve disk storage space.
  • Troubleshoot all common system troubleshooting issues.
  • Security checkups to protect your system against all vulnerabilities that may be detrimental to your operating system.
  • Startup Manager to speed up the startup process and control task manager.
  • Clean up all the jack files to use the PC in a well-organized manner.
  • Improve your system hardware as well as the software resources to fast shut down your device.
  • Its Smart Installer allows all system unwanted or malicious programs to be removed.
  • Almost, it provides registry cleaners and detectors for the Windows operating system.
  • Increase the speed of copying files or moving files from one drive or folder to another.

Windows 10 Product Keys Method File List:

Windows 10 Home EditionKeys:

  • YTMG3-N6DKC-DKB77-7M9GH-8HVX7

Windows 10 Single Language Key:

  • BT79Q-G7N6G-PGBYW-4YWX6-6F4BT

Windows 10 Education:

  • YNMGQ-8RYV3-4PGQ3-C8XTP-7CFBY
  • NW6C2-QMPVW-D7KKK-3GKT6-VCFB2

Windows 10 Enterprise:

  • CKFK9-QNGF2-D34FM-99QX2-8XC4K
  • NPPR9-FWDCX-D2C8J-H872K-2YT43
  • PBHCJ-Q2NYD-2PX34-T2TD6-233PK

Windows 10 Pro Serial Key:

  • VK7JG-NPHTM-C97JM-9MPGT-3V66T
  • 6P99N-YF42M-TPGBG-9VMJP-YKHCF
  • 8N67H-M3CY9-QT7C4-2TR7M-TXYCV
  • W269N-WFGWX-YVC9B-4J6C9-T83GX

Windows 10 product key: Technical Preview for Consumer:

  • 334NH-RXG76-64THK-C7CKG-D3VPT

Windows 10 product number:

  • VK7JG-NPHTM-C97JM-9MPGT-3V66T
  • W269N-WFGWX-YVC9B-4J6C9-T83GX
  • T44CG-JDJH7-VJ2WF-DY4X9-HCFC6

Key for Every kindof edition:

  • 8DVY4-NV2MW-3CGTG-XCBDB-2PQFM
  • NKJFK-GPHP7-G8C3J-P6JXR-HQRJR

2021 product keys for Windows 10:

  • TX9XD-98N7V-6WMQ6-BX7FG-48Q99
  • TX9XD-98N7V-6WMQ6-BX7FG-H8Q99

How to Install and Use Windows 10 Crack?

  1. Download it by clicking the button below.
  2. Extract the file and start the installation.
  3. Copy the crack .exe file after extraction.
  4. When the registration is done immediately, copy and paste the cake file into the directory you installed.
  5. There you will see the option to ‘Activate Windows’.
  6. Then click Next and OK.
  7. That is, start using Windows 10 Crack.
  8. All Is Done.
  9. Enjoy!
  10.  The Latest Version 2022 Free Download.

Also Download:  Resolume Arena Crack.

Summary

Reviewer

Mia Paley

Review Date

Reviewed Item

Windows 10 Pro Product Key

Author Rating

Software Name

Windows 10 Crack

Software Name

Windows

Software Category

Windows

Источник: https://www.taalwinkel.nl/

Oral vaccination of piglets against Mycoplasma hyopneumoniae using silica SBA-15 as an adjuvant effectively reduced consolidation lung lesions at slaughter

Abstract

Mycoplasma (M.) hyopneumoniae is the main pathogen of porcine enzootic pneumonia (PEP). Its controlling is challenging, and requires alternative strategies. This study aimed to develop an oral vaccine against M. hyopneumoniae using a nanostructured mesoporous silica (SBA-15) as an adjuvant, and compare its effect with an intramuscular (IM) commercial vaccine (CV). Fifty 24 day-old M. hyopneumoniae-free piglets composed five equal groups for different immunization protocols, consisting of a CV and/or oral immunization (OI). Control piglets did not receive any form of immunization. All piglets were challenged with M. hyopneumoniae strain 232 on D49 by tracheal route. IgA antibody response in the respiratory tract, bacterial shedding and serum IgG were evaluated. The piglets were euthanized on 28 (D77) and 56 (D105) days post-infection. Lung lesions were macroscopically evaluated; lung fragments and bronchoalveolar fluid (BALF) were collected for estimation of bacterial loads by qPCR and/or histopathology examination. All immunization protocols induced reduction on Mycoplasma-like macroscopic lung lesions. IgA Ab responses anti-M. hyopneumoniae, undelete plus full - Crack Key For U expression of IL-4 cytokine and a lower expression of IL-8 were induced by CV and OI vaccines, while IgG was induced only by CV. Oral immunization using silica as a carrier-adjuvant can be viable in controlling M. hyopneumoniae infection.

Introduction

Mycoplasma hyopneumoniae (M. hyopneumoniae) is the main causative pathogen of porcine enzootic pneumonia (PEP), a chronic respiratory disease in pigs, and one of the main pathogens involved in the porcine respiratory disease complex (PRDC)1. The infections caused by this bacterium are highly prevalent worldwide and result in financial losses for the pig industry, mainly due to the costs of treatment and vaccination, decreased performance, and increased mortality from secondary infections2.

The microorganism's adhesion font creator 12 - Free Activators the respiratory epithelium, the stimulation of a prolonged inflammatory reaction, the suppression and modulation of innate and adaptive immune responses favoring the pathogen are recognized as important steps in the colonization and infection by this microorganism. As a result, infected animals become more susceptible to infections by other respiratory pathogens1. As in other animals, most porcine pathogens cross mucous surfaces when ingested or inhaled, due to contamination of food, environment and fecal matter. Systemic vaccination generally promotes little stimulation of mucosal associated lymphoid tissue (MALT) and, therefore, the host immune system can only fight against the pathogen after its entering into the body3,4.

In the mucosal lymphoid tissues, mature T cells and B cells are stimulated by antigen and induce IgA antibody response. These cells migrate from the submucosal lymphoid tissue by the bloodstream to the lamina propria, where B cells differentiate into plasma cells secreting dimeric IgA antibodies. Many of these cells return to the original mucosal surface, but others can be found at different mucosal surfaces, so that oral immunization can lead to a migration of IgA precursor B cells to the bronchi, which subsequently secreted IgA antibodies in the bronchial mucosa5.

Previous studies with other pathogens have demonstrated the feasibility of using oral immunization as a strategy for inducing protective immunity in the swine reproductive tract, reinforcing the interconnection between different mucosal sites6. The secretory IgA (SIgA) specific antibodies have been considered as a crucial factor in protecting pigs against infection by M. hyopneumoniae7, while local humoral immunity seems to play an important role in this infection. SIgA is the main effector of respiratory tract mucosa immunity, which can form a protective barrier to eliminate respiratory invading pathogens and prevent infection and active colonization8. Since mucosal immunity has the potential to control pathogens at their portal of entry, it would be advantageous to develop vaccines that trigger a mucosal and systemic immune response rather than simply stimulating the systemic immune system9.

Promising M. hyopneumoniae bacterin formulations have been identified based on their capacity to induce strong innate immune responses10. Limitations in the use of adjuvants for vaccine formulation can be found in the literature, such as toxicity, the ability to lead to an immune response against the agent and not against the adjuvant, induction of adverse reactions, among other factors11. Since the silica has the characteristic of incorporating and releasing molecules12, the mesoporous silica particles have gained attention due to its potential role as adjuvants. Its toxicity to respiratory cells has been described13,14 and depends on the physicochemical properties of particles, size and concentration15. On the other hand, its use may enhance antigen-specific cellular immune responses in dendritic and Langerhans cells15. In addition, it is able to stimulate the immune system in a similar or superior way than other adjuvants, such as the Incomplete Freund's Adjuvant16. Moreover, an improvement in the recruitment of defense cells was observed, which led to an increase in phagocytosis and processing by the gut antigen-presenting cells17,18,19,20.

Based on the importance of developing oral vaccine adjuvants that are efficient in presenting antigens to the cells of mucosal lymphoid tissues (MALT), the objectives of this study were (i) to develop an oral vaccine specific to M. hyopneumoniae by encapsulating a blend of proteins of this bacterium into the silica (SBA-15); (ii) to stimulate the immune responses of the respiratory mucosa; (iii) to evaluate the efficacy of the protection induced by this vaccine against experimental infection with a virulent strain of M. hyopneumoniae, compared to a commercial inactivated vaccine.

Results

Mycoplasma-like macroscopic and microscopic lung lesion score at slaughter

At slaughter, gross lesions were mainly located in the apical and cardiac lung lobes, cranio-ventral portions of diaphragmatic lobe, and in portions of the intermediate lobe. All immunized groups showed lower lung lesion scores when compared to the font creator 12 - Free Activators medians of macroscopic lung lesion scores at the first slaughter (28dpi), followed by the lowest and higher scores observed in each group, were: CV-4.8% (0–24.5%), OI-2.5% (2–9.8%), CV + OI-2.7% (0–13.5%), OI + OI-9.6% (0.5–10.9%) and CONT-32.0% (15.6–41.7%). At the second slaughter (56 dpi), the values obtained were: CV-0.0% (0–8.8%), OI-1.3% (0.8–2.9%), CV + OI-3.5% (0–14.3%), OI + OI-8.8% (2.6–12.9%) and CONT-21.3% (17.4–24.9%). All immunized groups showed significant differences (Dunn test, p-value = 0.0229) in the total Mycoplasma-like lung lesion area when compared with the percentage obtained in the control. Significant differences were observed between the immunized groups and the control, but not between the immunized groups at both post-infection time-points (Fig. 1A). A difference in consolidation lung lesion score of 85% in CV, 92% in OI, 91% in CV + OI, 70% in OI + OI was observed in the first slaughter, while in the second slaughter the difference was of 100% in CV, 94% in OI, 83% in CV + OI, and 59% in OI + OI. Representative photos of consolidation lung lesion from each group are shown below (Fig. 1B).

(A) Comparison of Mycoplasma-like macroscopic lung lesions extent observed at slaughter of piglets, 28- and 56-days post-infection with M. hyopneumoniae. Means followed by the same letter do not differ statistically from each other (Tukey test). (B) Representative photos of Mycoplasma-like macroscopic lung lesions extent observed at slaughter of piglets, 28- and 56-days post-infection with M. hyopneumoniae.

Full size image

Microscopically, all groups showed histological lesions score varying between 3 and 4, characterized as PEP-specific (Fig. 2A,B). No significant differences were found, neither between groups, nor between the two post-infection intervals. No significant correlations were observed between macro and microscopic analysis (Kruskall-Wallis, p-value = 0.341).

(A) Photomicrography of histological lung lesion characterized by (a) hyperplasia of lymphoid follicles (arrow) (100 ×); (b) inflammatory infiltrate predominantly compound by macrophages (400 ×); (c) amorphous and acidophilic material in addition to inflammatory infiltrate in the light of the alveoli (arrow) (100 ×); (d) inflammatory infiltrate in bronchioles (arrow) (400 ×); (e) light of the alveoli without noteworthy changes (arrow) (100 ×); (f) normal lymphoid follicle (arrow) (40 ×). (B) Percentage of animals showing different microscopic lung lesion score (0–4) according to each vaccination protocol. No significant differences were observed between groups.

Full size image

Detection of IgA and IgG anti-M. hyopneumoniae antibodies in nasal swabs, serum and BALF samples

All immunized groups showed IgA Ab response in nasal swabs at 14 days post immunization, while the control group only became positive for IgA Abs 28 days post infection (Fig. 3a). The mean values with the respective standard deviation of each group and day of sampling, as well as statistical differences, are shown in Supplementary Table S3. On D28, OI + OI was statistically different from the other groups (for p-values, see Supplementary Table S5 online). The IgA responses of CV and OI at D42 and D49 were significantly different from the others. At D56, CV was statistically different from the other groups, while OI and CV + OI were statistically similar to each other and different from CONT. At D70, only CV and CV + OI IgA Ab responses were significantly higher than other groups, and OI was different from CONT. From D91 onwards, no differences were found between experimental groups. All piglets from immunized groups were IgA positive before challenge. When comparing the moments of slaughter (28 dpi and 56 dpi), significant differences (Two Sample t-test) were found for CV group (p = 0.045), CV + OI group (p = 0.028) and for CONT (p = 0.0001). No significant correlation was observed for any experimental group between the IgA Ab levels and the macroscopic lung lesion scores either at 28 or 56 dpi intervals.

(a) Mucosal IgA antibody response related to different immunization protocols (D0) against M. hyopneumoniae along the experimental period. Dots represent the mean values of each group in each day of sampling. Positive S/P values > 0.4. (b) Serum IgG antibody response obtained in different immunization protocols (D0) against M. hyopneumoniae along experimental period. Piglets challenged with M. hyopneumoniae on D49 (red arrows). Dots represent the mean values of each group in each day of sampling. Positive S/P values > 0.3. Kruskall-Wallis test was used.

Full size image

The oral vaccine did not induce a serum IgG Ab response, whereas the commercial vaccine induced the highest levels of this antibody isotype (Fig. 3b). This result has to be further investigated by other experiments with larger concentration of antigens in SBA-15, providing no aggregation of proteins. It is important to point out that the concentration of antigen administered by the oral vaccine is much smaller than that the one used in the injected CV. The mean values with the respective standard deviation of each group and day of sampling, as well as statistical differences, are shown in Supplementary Table S4. All animals from the CV and CV + OI groups produced IgG anti-M. hyopneumoniae antibodies, and the Ab response started between D14 and D28, remaining with high levels until slaughter. From the moment that seroconversion was detected, the levels of IgG Ab were statistically similar for these two groups, but they differed from the others until the end of the experimental period (for p-values, see Supplementary Table S6 online). The animals from groups OI, OI + OI, and CONT seroconverted for IgG antibodies only four weeks after challenge with the pathogen (D70), and produced levels of IgG Ab statistically similar until the end of the experimental period. Comparing the differences between the moments of slaughter, significant differences (Two sample t-test) were found between the levels of IgG antibodies for CV (p = 0.008) and OI (p = 0.0003). No significant correlation was found for any experimental group between the IgG Ab levels and the macroscopic lung lesion scores, neither at 28 or 56 dpi intervals.

Regarding BALF, all groups showed positive responses to both IgA and Avast antivirus crack 2019 - Free Activators anti-M. hyopneumoniae Ab on 28 dpi and 56 dpi (Fig. 4). No statistical differences were found between groups for any of the evaluated immunoglobulins. All correlation analysis tested between variables are shown in Supplementary Table S7.

ELISA S/P (mean ± sd) results for BALF on Device Doctor Pro 5.3.521.0 Crack + License Key Free Download and 56 dpi regarding (a) IgA antibody response against M. hyopneumoniae; (b) IgG antibody font creator 12 - Free Activators against M. hyopneumoniae. Positive S/P values > 0.4.

Full size image

Detection and quantification of p102 gene of M. hyopneumoniae by qPCR

The samples of nasal swabs tested by qPCR indicated that at 7th dpi there were piglets already shedding the pathogen in all experimental groups in different proportions, and by the end of the evaluated period, most of the piglets from all groups were shedding intermittently M. hyopneumoniae (Table 1). No significant differences were found between groups along the experiment.

Full size table

Mycoplasma hyopneumoniae was detected and quantified in lungs and BALF of all piglets from all groups, and the respective estimate loads and standard deviation are shown in Table 2. No statistical differences (Two Sample t-test) were found for all groups in both sampled time-points (28 and 56 dpi). Considering the correlation between macroscopic lung lesion and M. hyopneumoniae estimate quantification in lungs, a lower quantification was found to be strongly correlated with a reduced macroscopic lung lesion in CV + OI at 28 dpi (Spearman’s correlation coefficient R = 1; p = 0.01667), with a trend on group CV (p = 0.0538, R = 0.87). A lower quantification was also correlated with a reduced M. hyopneumoniae quantification in the BALF (28dpi) of CV (R = 0.99, p = 0.0051), OI (R = 0.97, p = 0.0299), CV + OI (R = 0.96, p = 0.03), and on 56 dpi in OI + OI (R = 0.99, p = 0.0004).

Full size table

Cytokine coding gene’s expression and correlation with antibody levels and bacterial loads

Interleukin 8 was downregulated in immunized groups at 28 dpi compared to the control group, with a slight non-significant difference in the CV + OI group (Kruskall-Wallis test). Similarly, IFN-γ was less expressed in the immunized groups when compared to the control group, especially in CV, OI and CV + OI, with no significant differences between them. A significant and negative correlation was found between IFN-γ and IgA Ab response at 28 dpi (Pearson’s correlation coefficient R = − 0.43; p-value = 0.028). On the other hand, IL-4 expression was up regulated in all immunized groups, while it was less expressed in the control group. TGF-β expression was reported in all groups, with non-statistical differences between them (Fig. 5). TGF-β expression was positively correlated with IgA Ab response in the upper respiratory tract (nasal swabs) of piglets (Pearson’s correlation coefficient R = 0.95; p-value = 0.0401). All correlation analyses between variables are shown in Supplementary Table S7.

Bar graphs representing the fold change of cytokine gene expression (Fold Change mean ± sd) in lung lesion samples of five pigs per group, 28 days after experimental infection with M. hyopneumoniae strain 232, previously submitted to different immunization protocols. Target gene expression was normalized based on rpl-4 gene expression. No statistical differences were found between groups (Kruskall-Wallis test).

Full size image

Discussion

Oral vaccines are desirable in pig industry due to the relative ease of administration for large populations. In the present study, an oral vaccine for weaned piglets was developed by using the SBA-15 nanostructured mesoporous silica as a vehicle for a blend of M. hyopneumoniae proteins. Four immunization protocols were tested, followed by an experimental inoculation of M. hyopneumoniae strain 232, to evaluate the action of the oral and the intramuscular vaccines, alone or combined, and the results were compared with a non-immunized group. According to the manufacturer (MSD Animal Health), the commercial vaccine contains the proprietary dual adjuvant EMUNADE, capable of producing a rapid and prolonged immune response due to the aluminum hydroxide and an oil emulsion, respectively. As we have no information regarding the bacterial load present in M + PAC® and different routes of administration were performed, comparing its efficacy to the experimental oral vaccine is challenging. However, this IM vaccine was found to be an interesting option as a basis for comparison since it has been used worldwide and provided satisfactory results. In addition, most works with oral or aerosol vaccination against M. hyopneumoniae in piglets did not perform experimental challenge with this pathogen, which can limit the comparison of our results, such as bacterial shedding and quantification in the lugs, with other vaccines.

All immunization protocols showed reduced Mycoplasma-like lung lesion, and all groups presented a significant difference in lung lesion score when compared with the control group, being the highest differences observed in the medians of OI and CV + OI in the first slaughter (reduction of 92% and 91% of consolidated area, respectively), and in CV and OI (reduction of 100% and 94%, respectively) at the second slaughter. The oral and the commercial vaccines offered effective protection individually, but no combination effect with the two immunization ways were observed. These are promising findings, especially when compared to previous works in which no significant differences in lung lesions were found between vaccinated and non-vaccinated groups on the field with natural infection21, and with homologous and heterologous vaccines followed by experimental infection with M. hyopneumoniae22. On the other hand, microscopic lung lesions were statistically similar in all groups, vaccinated or not, with histological findings characteristics of M. hyopneumoniae infection, as also observed by Almeida23, after experimental infection using the same M. hyopneumoniae 232 virulent strain. An oral vaccine with recombinant E. rhusiopathiae strain expressing the M. hyopneumoniae P97 protein reduced the severity of pneumonic lung lesions caused by M. hyopneumoniae infection24. Similarly, a number of M. hyopneumoniae commercial inactivated vaccines administered by IM route provided such type of protection25,26,27.

Mycoplasma hyopneumoniae induces innate and adaptive immune responses, which is able to prevent significant systemic spread of the organisms. However, the immune system is unable to rapidly clear pulmonary airways infection, resulting in a prolonged localized inflammatory and cellular immune response, responsible for the majority of gross and microscopic lesions1. The vaccines and vaccination programs tested in the current study were able to induce IgG (commercial vaccine only) and IgA (commercial and experimental oral vaccines) antibodies against M. hyopneumoniae. Only the groups that received IM vaccination were able to produce serum IgG Ab detectable by ELISA test. It is well known that systemic anti-M. hyopneumoniae antibodies are considered to play a minor role in protection against PEP28,29. On the contrary, it is believed that specific locally secreted IgA may play a protective role by preventing the adhesion of the pathogen to the ciliate epithelium30. Independently on the presence of serum IgG Ab, all vaccinated animals showed a significant reduction of consolidation lung lesions, which allows to infer that mucosal IgA Ab probably participates in the protection and prevention against M. hyopneumoniae invasion and adherence, corroborating Martelli30. In this study, both oral and IM vaccines were capable of inducing IgA Ab production, which was found in the respiratory tract, as all immunized piglets were positive for this Ab at 14 days post-vaccination. Feng31 also observed the presence of IgA Ab in nasal cavities 14 days after administration of an aerosol vaccine against M. hyopneumoniae, although no challenge was performed by these authors.

Our results differ from previous ones that only detected significant antibody immune response after challenge infection24, and from studies using commercial vaccine which were not able to induce IgA Ab responses31,32. We have found that vaccinated pigs presented significantly higher antibody levels than the non-vaccinated ones, which may indicate a memory humoral immune response, either for IgA or IgG Abs. It is noteworthy that antibodies induced by IM vaccination may occur between 3 to 4 weeks after vaccination33, while seroconversion in natural M. hyopneumoniae infected pigs usually occurs around 8–24 weeks of age34,35,36. In the current study, seroconversion in the challenged piglets occurred 3 weeks post-infection. This early immune response, when compared to naturally infected ones, may be due to the differences in experimental and natural bacterial loads. The infecting dose under field conditions is expected to be lower, especially in farms with good management and biosecurity practices37. Determining the ideal time point to experimentally infect the piglets after vaccination is challenging, once vaccination and infections occur dynamically in field conditions. Furthermore, the primary or boosted immune response must be considered in the dynamics of M. hyopneumoniae infection. In the present study, the infectious challenge occurred two weeks after the booster vaccination, and the piglets from CV + OI and OI + OI could be still experiencing the effects of the immunization at that time. For this reason, further studies evaluating different moments of immunization in the field are necessary to a better understanding of this situation.

When considering the Ab immune response in BALF at slaughter (28 dpi and 56 dpi), all groups showed high S/P values for both IgA and IgG Ab, despite the statistically non-significance. As these samples were taken only at sony vegas pro 15 price - Crack Key For U, it was not possible to determine the contribution of each vaccination protocol in inducing the antibody immune response in BALF, once all piglets were challenged with the pathogen and an immune response was expected at 28 dpi. However, it is possible to observe that a substantial Ab detection occurred in piglets previously submitted to both IM and oral vaccination when compared to the control. It may be due not only to the capacity of vaccines to induce memory cells, but also to a previous local presence of these antibodies, as observed in the respiratory tract by nasal swabs. It was expected that vaccinated animals showed higher mucosal IgA Ab responses compared to the non-vaccinated ones after challenge28,38.

The number of organisms colonizing a pig possibly depends on cumulated infectious doses, capacity of the M. hyopneumoniae strain to multiply in the lungs, and time1. In the present study, a high dose of inoculum containing M. hyopneumoniae organisms was provided. M. hyopneumoniae replication in the lungs was lower in vaccinated pigs in both slaughter points compared to control, and quantification in lungs and BALF was lower at 56 dpi in vaccinated groups than in control, despite non-significant differences in bacterial load estimate quantification. The lower lung lesion score at 56 dpi was expected for the immunized groups, as previously observed39. However, it also indicates that vaccination alone does not significantly reduce the bacterial load in the lower respiratory tract of pigs, and would not eliminate infection with this pathogen from pig herds24,27, which may justify nasal shedding not differing between groups along the time37. In addition, several factors such as the challenge dose, the time post-infection, the strain virulence and individual immune responses of pigs may influence the number of M. hyopneumoniae organisms and its nasal shedding23,33.

The evaluation of cytokines production 30 days after challenge with M. hyopneumoniae could better illustrate the resistance status in the vaccinated pigs after infection40. Thus, in the current study, the expression of some cytokine genes at 28 dpi of vaccinated animals point out to a possible role of T cell response after pathogen exposure, so that the oral administration of M. hyopneumoniae antigens incorporated to the silica adjuvant may induce an activation of Treg lymphocytes, which can reduce part of the inflammatory response caused by this pathogen. In this context, it is known that mesenteric Treg lymphocytes of animals submitted to tolerance by the oral route secrete TGF-β with variable amounts of IL-441. Additionally, TGF-β expression was positively correlated with IgA Ab response in the upper respiratory tract, raising the hypothesis that the presence of TGF-β further enhances both secreted IgA and the number of IgA producing cells, as reported42.

T helper (Th) cells are essential to initiate the B-cell activation and generation of antibody responses, which will result in antibody production for T-dependent antigens5. The higher gene expression level of IL-4 in lungs of immunized piglets could also be associated with a positive regulation of a Th2-mediated immune response30, positively influencing the IgA Ab local secretion. Th lymphocytes activation is an important pathway for generating protective immune response against M. hyopneumoniae43.

Interferon-γ secretion is important for the control of several infectious diseases44, and it is usually evident between 4 to 8 weeks post vaccination against M. hyopneumoniae30,38. Although the piglets were tested only at 28 days post-infection, its lower expression in immunized groups may be involved with an immunosuppressed environment caused by the inhibition of Th1 differentiation, since IL-4 suppresses the production of IFN-γ by Th1 cells45. In addition, IFN-γ was negatively correlated with IgA Ab response in the upper respiratory tract. These findings may be justified either by the suppressive effect of IL-4 or the effect of TGF-β on IFN-γ secretion and the direct correlation of IL-4 and IgA Ab response. IL-8 was found to be less expressed in all immunized groups of this study, and its suppression at this time point may be similar to IFN-γ. This chemokine is mainly produced by tissue macrophages in response to infection, and it is an important neutrophil recruiter46. M. hyopneumoniae organisms stimulate the production of proinflammatory and immunoregulatory cytokines by the alveolar macrophages and lymphocytes, inducing lung inflammation and lymphoid hyperplasia. As colonization by M. hyopneumoniae progresses, there is an increase in cytokine secretion due to the increase in the number of inflammatory cells recruited1. In non-vaccinated challenged piglets23, IL-8 gene expression was positively correlated with M. hyopneumoniae bacterial loads, suggesting an intense inflammatory iobit driver booster pro - Activators Patch being required at lung lesion sites due to the presence of the pathogen. In our study, IL-8 and IFN-γ were found downregulated in vaccinated piglets, which may be due to modulation effect caused by the vaccines, preventing an intense inflammatory and immune responses in lungs, consequently leading to a lower consolidation lung lesion.

Under the conditions of this study, the immune response induced by IM and oral vaccination involved both humoral and likely T-cell immune responses. Thus, oral vaccination with a blend of M. hyopneumoniae antigens incorporated into the silica induced local humoral immunity in the gut, measured in this study as IgA anti-M. hyopneumoniae antibodies in respiratory secretions, which were comparable to those obtained by the intramuscular administration of the commercial inactivated vaccine with oil adjuvant. All vaccination protocols reduced the severity of macroscopic lung lesions in the challenged pigs. It suggests that mucosal antibodies and the inflammatory responses were involved in the mechanism of immune-protection, or by raising IL-4 and reducing the IL-8 expression, or even likely by down-regulating the expression of other pro-inflammatory cytokines in the vaccinated animals, which were not evaluated in the current study.

As the protection induced by M. hyopneumoniae vaccines evaluated here and conferred by others conventional vaccines do not prevent colonization and shedding of this microorganism, the immune-protection induced by these vaccines is often incomplete47. Considering that, better results may be achieved whether vaccination is combined with good management and biosecurity procedures in pig farms. Moreover, under field conditions, improved results should be expected for the vaccines tested in this study, once a lower infection challenge dose probably results in lower number of microorganisms in the respiratory tract21. Thus, future studies applying the oral vaccination against M. hyopneumoniae in pigs reared in field conditions could elucidate this point and bring more consistent results for the use of inactivated oral vaccines in the control of this important respiratory pathogen for pigs, combined with the advantage of being a needle-free strategy of M. hyopneumoniae control.

The oral vaccine developed in this research with the SBA-15 nanostructured silica proved to effectively reduce macroscopic lung lesions in challenged pigs and induce mucosal humoral immune response. Moreover, its efficacy was similar to the one conferred by the commercial vaccine parentally administered. The immunogenicity characterization determined in the current study provided useful data for the further development of this oral vaccine, which requires studies under field conditions to elucidate its potential for the effective control and prevention of Mycoplasma hyopneumoniae injuries in the pig production.

Methods

Oral vaccine preparation

Cultivation and preparation of M. hyopneumoniae for protein obtainment

A pure pathogenic strain of M. hyopneumoniae (232) was purchased from Iowa State University48, certified free of any other pathogens, and a small fraction was removed for cultivation in Friis medium. Initially, this fraction was inoculated into two sterile graduated vials (Corning®, USA) containing 5 ml of Friis medium, kept in a shaking oven at 37 °C, which color was observed daily until indicating bacterial font creator 12 - Free Activators (CCU). After approximately five days, 2 ml of the culture were used to inoculate each of two flasks containing 200 ml of Friis medium, which were maintained in the same incubation conditions and presented color changing about a week later. The determination of the concentration of M. hyopneumoniae was carried out by successive dilutions of the samples in Friis medium, varying from 10–1 to 10–8, which reached a concentration of 107, and the negative control remained unchanged. Sterility tests adobe acrobat pro dc 2021 patch performed for all flasks on blood agar and McConkey media, left in an oven at 37 °C for three days, proving the absence of contamination with other pathogens by the absence of colonies growth.

The contents were centrifuged in appropriate tubes, previously autoclaved, in an ultracentrifuge (Sorvall) at 13,700×g for 45 min. The bacterial cells were deposited at the bottom of the tubes, forming pellets, which were resuspended in 15 mL of Phosphate Buffered Saline 1X (PBS, Sigma-Aldrich, USA), pH 7.4, for washing. The tubes were centrifuged three times at 21,000×g for 10 min until a clean pellet was obtained, which was resuspended in 10 mL of 1X PBS and stored in sterile falcon tubes. The resuspended content underwent a sonication process, and before sonication, an aliquot of whole cell preparation was taken for Dynamic Light Scattering (DLS) evaluation, which will be further discussed. For sonication, the flask was kept on ice and sonicated for three consecutive times in a sonicator (Soni-tech Ultrasonic Cleaning) in 20 Hz frequency for 1 min, with one-minute breaks between processes. To determine the concentration of proteins in the cell lysate, the Bradford method was used (Thermofisher Scientific, USA) followed by spectrophotometer reading (NanoDrop One, Thermofisher Scientific, USA), which provided a concentration of 1048 µg/mL.

Characterization and development of the oral vaccine

A partnership was established with the Department of Applied Physics, of the Physics Institute of the University of São Paulo (USP-SP), Department of Chemistry, Federal University of São Paulo (UNIFESP/Diadema) and the Butantan Institute (São Paulo), which have been conducting researches with an innovative immunogenic complex for human oral vaccines. They are based on the use of ordered mesoporous silica (OMS, SBA-15 type) as protective vehicle of antigens. The SBA-15 sample was synthesized according to Cavalcante49. It is composed by a bi-dimensional matrix of hexagonally ordered mesopores (diameter around 10 nm), high specific surface (around 1240 m2 g−1) and pore volume 1.8 cm3 g−1 (see Supplementary Fig. S1 online), with amorphous silica walls and rod-like morphology, formed by aggregates of rods connected as rope-like domains (Fig. 6), capable of encapsulating different molecules into their macropores and mesopores. The immunogenic complex has to be able to cross the digestive tract in order to be absorbed by the intestinal mucosa.

Scanning electron microscopy images of SBA-15 (Santa Barbara Amorphous silica) before antigen adsorption in macro pores. (a) 1000x magnification; (b) 10,000x magnification.

Full size image

To protect the oral vaccine from the harsh stomach medium, the commercial polymer Eudragit® (Evonik Industries) was used for coating. This polymer is insoluble in acidic pH and dissolves in contact with the intestinal basic medium, providing slow release of the desired proteins12.

The preparation and characterization of the vaccine particles were carried out at the Crystallography Laboratory of the Department of Applied Physics at USP. The process of including the antigen in the pores of the SBA-15 was carried out considering the concentration of the obtained proteins. The 1:35 antigen-to-silica ratio was established (w/w), based on previous work with Hepatitis B encapsulation in SBA-1518,20. In the present study, the silica SBA-15, from a closed recipient (to avoid contamination and humidity) was weighed on a precision scale (± 1 μg), and immediately macerated with a glass stick. After that, the total amount of the silica was mixed with the flask containing the liquid antigen (concentration determined previously). The wet mixture was spread in a glass recipient and partially covered by a glass plate, and placed in an oven at 35 (± 2) °C for total drying. This drying procedure took 48 h. Then, the product was covered with Eudragit L30 D-55 (Evonik®), by mixing the polymer with the silica + antigen, using a glass spatula to homogenize the final product. The amount of Eudragit was equal (in weight) to the amount of silica added to the liquid antigen until a paste was formed, which was taken again to an oven at 35 (± 2) °C for drying, during 48 h. The dry contents were stored in a refrigerator to protect the antigen until use. Since the polymer has the ability to dissolve at a pH higher than 5.5, the compound was supplied to piglets in acidified water (pH 4.5).

Dynamic light scattering (DLS) evaluation of particle diameter and nitrogen adsorption/desorption isotherms of SBA-15:Antigen sample

Dynamic Light Scattering (DLS) evaluation was performed at the Physics Institute, USP-SP, using a DLS (DynaPro NanoStar, Wyatt) equipment at 90° scattering angle with a 100 mW He–Ne laser and wavelength of 658 nm. An aliquot of the whole-cells (concentration of 106 CCU/mL) dispersed in 25 mL of PBS (pH 7.4) was used to fill the sample holder. The same process was done to investigate by DLS the sonicated sample (cell lysate), having a concentration of 1048 µg/ml. The size distribution was obtained by the equipment software, calculated by the CONTIN method50. The antigen encapsulation, depending on its size, can occur in the silica mesopores, with an average diameter of ~ 10 nm, or in the morphological macrospores, with dimensions greater than 50 nm. The average diameter of the molecules present in the whole-cell preparation sample was 460 nm, while the protein preparation was 218 nm, which allowed inferring that cell lysis occurred, and the antigen would be adsorbed on the silica macro porosity, regarding the sizes of the antigen in solution compared with the mean diameter of the mesopores.

By nitrogen adsorption/desorption isotherms of SBA-15:Antigen sample (Supplementary Figs. S2 and S3 online) were evidenced the preservation of mesostructured SBA-15, which exhibited a type IV isotherm and type H1 hysteresis loops, according to the IUPAC classification51 that are characteristic of hexagonal cylindrical channel mesoporous such as SBA-15, with a pore size about 10 nm, as reported in the literature52. The reduction of specific surface area (336 m2 g−1) and pore volume (0.91 cm3 g−1) of SBA-15:Antigen, comparing to pure SBA-15, has evidenced antigen into the macropores of SBA-15.

Oral vaccine administration

After a pilot test, a concentration of 200 µg of protein antigen of M. hyopneumoniae per animal was established. The doses of the compound were weighed on a precision scale, considering the proportion between antigen, silica and polymer. The vaccine was provided by gavage using an esophageal tube with the aid of a laryngoscope, administered in individual doses diluted in 5 ml of filtered water plus acidifier (Selko pH, Trouw Nutrition). Each dose was diluted at the moment of administration.

Experimental design and sample collection

Experimental design

Fifty piglets were randomly separated into five groups (n = 10) to receive different vaccine protocols, consisting of a commercial vaccine (CV) or oral immunization (OI). Random numbers were generated using the standard = RAND() function in Microsoft Excel. Group 1 (CV) piglets received a single dose commercial vaccine at 24 days of age (D0). Group 2 (OI) piglets received a single dose of oral vaccine at D0. Group 3 (CV + OI) received a dose of the commercial vaccine at D0 and a booster with the oral vaccine at D28. Group 4 (OI + OI) received a dose of the oral vaccine on D0 and a booster with the same vaccine on D28; piglets of Group 5 (CONT) were the control group, which did not receive any form of immunization. The commercial vaccine chosen for this work was M+PAC (Merck Animal Health, USA), which promotes an effective protection against M. hyopneumoniae as it contains aluminum hydroxide, responsible for a rapid immune response, and an oil emulsion, which promotes a prolonged immune response by its slow release in the organism. At about 70 days of age (D49), all piglets were challenged by the tracheal route with 5 mL of Friis medium containing 106 CCU/mL of M. hyopneumoniae virulent strain 232, homologous to the vaccine.

Weekly, nasal swabs were collected since D0 for IgA measurement (ELISA), and, after the challenge, were used to evaluate M. hyopneumoniae shedding as well. Fortnightly, all piglets underwent blood collections to obtain serum for IgG measurement (ELISA). Half of the animals in each group were euthanized 28 days post-infection (D77), font creator 12 - Free Activators the other half was euthanized 56 days post-infection (D105). At slaughter, lungs of all animals were macroscopically evaluated following the European Pharmacopeia methodology, biological samples such as lung fragments were collected for qPCR and histopathology, and bronchoalveolar fluid (BALF) for qPCR. A schematic design is shown in the Supplementary Fig. S4 online.

Animal selection

Fifty 21-day-old piglets of commercial lineage (Landrace × Large White) and average weight between 6 and 7 kg were purchased from a certified M. hyopneumoniae-free commercial farm. The piglets were housed in the experimental barn of the Swine Medicine Laboratory of School of Agricultural and Veterinarian Sciences (FCAV), remaining in the nursery pens until 65 days of age (3 pigs/m2), and then transferred to fattening pens (1 pig/m2), where they remained until 130 days of age. Biosecurity standards were met to avoid cross-infections and minimize external interferences, such as shower-in/shower-out, specific and clean clothes for pig husbandry and no contact with any other pig during the experimental period. The animals received feed according to the production phase, free of antibiotics, and water ad libitum. Upon arrival, blood samples were taken from all piglets to obtain serum for measurement of antibodies, in addition to laryngeal swabs to confirm the absence of the pathogen. Piglets went through an adaptation period of three days before the beginning of the experiment.

All procedures described here were conducted in accordance with the Federal Council of Veterinary Medicine (Brazil), submitted for approval by the Ethics Committee on the Use of Animals (CEUA) of the School of Agricultural and Veterinarian Sciences, São Paulo State University—Campus Jaboticabal, being approved and registered under the protocol number 005174/18. The study was carried out in compliance with the ARRIVE guidelines53.

Blood serum and nasal swabs collection

The titers and duration of antibodies in serum and nasal swabs were assessed along the experimental period. The blood was collected every two weeks to obtain blood serum by puncture the jugular vein, using sterile disposable needles and syringes, deposited in tubes with clot activator and centrifuged at 1500×g for 10 min. The serum was aliquoted in duplicate and stored at − 20 °C until the time of analysis. Samples of nasal swabs were weekly collected to obtain quantitative data on the immune response induced by different protocols of immunization (ELISA), and on the dynamics of excretion of M. hyopneumoniae (qPCR). The animals were restrained, and the swab samples were collected from a light rubbing of the swab on the nasal mucosa and deposited in 2 mL graduated plastic microtubes (Kasvi, Brazil) containing 500 μL of 1 × PBS, and stored at − 80 °C until analysis.

Slaughtering of piglets, lung lesion scoring, BALF and lung fragments collection

Four and eight weeks after the challenge, half of the piglets of each group was euthanized with an intramuscular administration of a combination of ketamine and xylazine (6 mg/kg and 4 mg/kg, respectively), followed by intravenous administration of saturated potassium chloride solution (approved by Federal Council of Veterinary Medicine, Brazil). After evisceration, the respiratory set (trachea + lung) of each animal was separated to collect bronchoalveolar fluid (BALF), with the introduction of 20 mL of PBS 1 × in the cranial portion of the trachea. After pouring all the liquid, the lung was lightly massaged and the liquid was aspirated by the pipette, recovering an approximate volume of 10 mL. The aspirate was aliquoted in duplicate in 2 mL graduated microtubes, free of DNAses and RNAses (Kasvi, Brazil), while the remaining volume was deposited in sterile Falcon tubes (Kasvi, Brazil) and stored at – 20 °C until analysis by qPCR.

The lung was evaluated for the extent of lung lesions followed by photo documentation by a unique blinded investigator, and the extent of the lesions was quantified using the European Pharmacopeia method, in which the percentage of each lobe affected area is multiplied by the lobe relative weight and summed to provide the total weight percentage of affected lung54. The lung tissue fragments for the qPCR were collected with individual scalpel blades and sterile tweezers. The tweezers were kept in boiling water in the interval between collections, while each fragment was collected with a disposable scalpel blade. These fragments were collected in duplicate in the shortest possible time after the animal's death, and all samples were bathed in liquid nitrogen and later stored in a freezer at − 80 °C.

Histopathological evaluation

The histopathological analysis aimed at evaluating lung lesions caused by Mycoplasma hyopneumoniae in different groups, in order to classify qualitatively histological lesions as PEP-specific or non-specific. For that, during the necropsy, lung Movavi Video Crack with lesions caused by PEP resources were collected, mainly in the transition area between healthy and affected tissue. Samples of tissues apparently healthy were also collected for control. The fragments were collected with a thickness of approximately 5 mm and initially stored submerged in a 10% buffered formalin solution (pH 7.0) in an approximate ratio of 10: 1 formalin: tissue. After 24 h in formalin solution, the fragments were routinely processed for Hematoxylin/Eosin staining.

The slides were blind read under a light microscope and microscopic lesions on the tissues were classified into five different degrees55, in which: 0 = absence of lesion; 1 = lesions of interstitial pneumonia and/or purulent bronchopneumonia; 2 = light to moderate infiltrates of macrophages, lymphocytes and neutrophils into airways and alveoli; 3 = perivascular and peribronchiolar lymphoplasmacytic hyperplasia, type II pneumocyte hyperplasia, alveolar spaces with edema fluid, neutrophils, macrophages and plasma cells; 4 = lesions with characteristics of grade 3, together with peribronchial and perivascular lymphoid nodules. Grades 1 and 2 injuries were nonspecific, while injuries 3 and 4 were considered PEP-specific.

Detection and quantification of IgA and IgG antibodies by enzyme-linked immunosorbent assay

The detection and quantification of antibodies (IgA and IgG) in blood serum, nasal secretions and tracheobronchial lavage were carried out by enzyme-linked immunosorbent assay (ELISA). To detect serum IgG Ab, the commercial kit M.hyo Ab test (Idexx, USA) was used. For the detection of IgA Ab in nasal swab, and IgA and IgG Abs in BALF samples, standardization was performed using the sensitized plates and components of the kit mentioned above, with modifications. Initially, the plate was blocked with 1.5% ovalbumin in PBS, followed by incubation at 37 °C for 30 min. Then, a first procedure was performed to determine the ideal dilutions of the samples (positive and negative controls for anti-M. hyopneumoniae antibodies) and the anti-IgA or anti-IgG peroxidase conjugates. For standardization, samples of nasal swabs from animals known to be positive and negative for M. hyopneumoniae were used, and part of the negative and positive samples were homogenized in the form of a pool to compose the negative (CN) and positive (CP) controls, respectively.

To detect IgA Ab in nasal swabs, 100 μL of the sample's liquid fraction was used, as the swabs were deposited in 500 μL of PBS, which were quickly homogenized in a vortex and placed, without further dilution, in each microplate well. The same procedure was performed with the controls, which were tested in duplicate, followed by plate incubation for 60 min at room temperature. The conjugate from the kit was replaced by an immunoenzymatic conjugate of goat anti-Pig IgA Antibody HRP Conjugated (Bethyl Laboratories Inc., USA), at a dilution of 1:500 using the diluent provided by the kit, followed by incubation for 60 min at room temperature. The washing processes and all the following steps were performed according to the protocol of the kit M.hyo Ab test (Idexx, USA).

For BALF IgA Ab detection, the samples were diluted 1:10 using the diluent from the kit, and the same conjugate was used in the proportion of 1:800. For IgG detection in the BALF, the samples were diluted in the proportion of 1:2 and the conjugate (Pig IgG-Fc Fragment Antibody HRP, Bethyl Laboratories Inc., USA) in the proportion of 1:5000. In all microplates, the conjugate was tested in separate wells to determine its non-specific adsorption in the absence of samples. The plate was read in an absorbance microplate reader (iMark, Bio-Rad Laboratories Inc., USA), at a wavelength of 650 nm.

The mean optical densities (OD) for each of the test samples (ODs) were related with the OD found for the negative and positive controls (NC\(\overline{x }; PC\overline{x })\) in order to calculate the S/P values (sample/positive ratio) according to the formula: S/P = \(ODs- NC\overline{x }/PC\overline{x }- NC\overline{x }\). The threshold between positive and negative samples was calculated from the value of S/P \(NC\overline{x }\) + 2 × standard deviation. Serum samples were considered positive if S/P > 0.3; nasal swabs S/P > 0.4. BALF S/P > 0.4.

Detection and quantification of p102 gene fragment of M. hyopneumoniae by qPCR

DNA extraction from nasal swabs, lung and BALF samples

The DNA extraction was carried out by Tris–HCl protocol56. For nasal swabs and BALF samples, a centrifugation (Centrifuge 5804 R, Eppendorf, Germany) at 13,000×g at 4 °C for 20 min was performed previously to the DNA extraction protocol. For lung samples, 0.05 g of lung tissue were used. After DNA extraction, the samples were stored at − 20 °C until qPCR analysis. The measurement of the DNA concentration of the samples was made through spectrophotometry, with the aid of the Thermo Scientific NanoDrop 2000 Spectrophotometer (Thermo FisherScientific®, USA), having as exclusion factor the samples that did not reach the purity of 1.8 to 2.0 in the 260/280 ratio to perform the qPCR technique. To rule out the presence of inhibitors in the extracted DNA samples and the occurrence of false negatives in the qPCR for M. hyopneumoniae, all samples were submitted to a conventional PCR targeting the endogenous gene Glyceraldehyde-3-phosphate dehydrogenase (gapdh), and the conventional PCR technique57. The amplified products of gapdh gene with 437 bp were detected after horizontal electrophoresis on a 1% agarose gel stained with Ethidium Bromide (0.5 μL/mL) in TEB running buffer pH 8.0 at a current of 90 V/50 mA for 90 min.

qPCR assay

Absolute real-time quantitative PCR analysis (qPCR) was used to detect p102 gene fragment in nasal swab samples, and to detect and quantify it in lung fragments and bronchoalveolar fluids. For M. hyopneumoniae, the primers used in the reaction were based on the bacterium p102 adhesion protein gene sequence. All samples were tested in duplicate and the qPCR reaction was optimized from a previous published protocol58, adapted by Almeida23. The nucleotide sequences used were forward primer 5′-AAGGGTCAAAGTCAAAGTC-3′, reverse primer 5′-AAATTAAAAGCTGTTCAAATGC-3′ and hydrolysis probe 5′-FAM-AACCAGTTTCCACTTCATCGCC-§BHQ2-3′.

The results were only accepted for those with a standard deviation lesser than or equal to 0.5 cycle, and quantification data were used only if the efficiency obtained was between 90 and 105%57, otherwise, the samples were retested in triplicates. As a negative control in the qPCR reactions, sterile ultrapure water was used (Nuclease-Free Water, Promega®, Madison, Wisconsin, USA) q.s.p. Serial dilutions were made to determine the standard curve generated with different concentrations of synthetic DNA (GBlock®, IDT, USA) containing the target sequence (107 copies/μL to 101 copies/μL), that were also used as positive controls. The synthetic DNA was diluted according to the manufacturer's guidelines and maintained in a stock concentration of 107 molecules∕µL.

Quantification was performed using serial tenfold dilutions (starting at 107 until 101 copies∕ μL) of synthetic DNA (GBlock®, IDT, Iowa City, IA, USA) containing the 150 bp fragment amplified by the primer pair used in the qPCR. Quantification data based on the standard curve generated was only validated if the reaction efficiency was between 90 and 105%59. qPCR parameters are shown in Supplementary Table S1.

Cytokine coding gene expression in lung samples

RNA extraction and cDNA synthesis

Total RNA was extracted from 0.02 g of lung tissue samples collected on the first slaughter (28 dpi) using RNeasy Blood and Tissue Plus kit (Qiagen, USA), and 500 ng of extracted RNA were used per reaction to convert the extracted RNA into cDNA by Superscript IV First Strand Synthesis kit (Thermo Fisher, USA), both according to the manufacturer’s guidelines. RNA purity and integrity were assessed by Bioanalyzer® (Thermo Scientific, USA), and immediately stored at − 80 °C until use. Samples were only used for gene expression analysis if they had RNA Integrity Number (RIN) > 7.0. Oligo d(T)20 targeted the poly-A tail of mRNA for the synthesis of this molecule into cDNA, instead of other types of RNA. Reactions were all performed in a MyCycler thermocycler (Bio Rad, USA), and the cDNA was stored at − 20 °C until use for qPCR.

Detection and quantification of cytokine coding gene’s expression

The reference gene used to normalize the expression of target genes was standardized23, and the best result was obtained with Ribosomal protein L4 (rpl-4), which was also used in this study. Quantification of cytokine coding gene’s expression was performed using relative quantification of cDNA produced. Transcript levels of inflammatory (IL-8) and specific immunity regulation (IFN-γ IL-4 and TGF-β) cytokines evaluated to compare the immune response promoted by each type of immunization with the control group. Specific primers targeting the genes of IL-8 (CXCL-8) and IFN-γ were based on previous work23, while genes of IL-4 and TGF- β were designed based on the reference sequences deposited in GenBank and using software Primer360. To avoid genomic DNA interference, all primers were designed comprising the exon-exon span. The specific primers are shown in Table 3.

Full size table

The qPCR reactions were performed as previously described23, using the Quantitect® Sybr Green master mix (Qiagen, USA) and 1 μL of cDNA template, totalizing 10 μL of final volume per reaction, in a real time thermocycler CFX 96 (Bio Rad, USA). The dissociation curve was used to assess the specificity of the amplicons at the end of 40 cycles, with a maximum variation of ± 0.5 °C. Serial tenfold dilutions (107 copies∕μL until 101 copies∕μL) of positive controls (Gblock®, IDT, USA) of synthetic DNA containing the amplified fragment of each primer pair was used to assess the efficiency, which was only accepted between 90 and 105%59. In order to normalize the target gene expression, the 2−ΔΔCq calculation61 was performed, and, to attend this methodology, all qPCR reaction’s efficiency had to be close to 100% with a maximum difference of 5% between them. All parameters of cytokine gene expression qPCR are shown Supplementary Table S2.

Data analysis

The variables of each group for each moment were assessed for normality and homoscedasticity by the Shapiro–Wilk and Bartlet tests, respectively. The difference between the means was calculated using the Tukey test (p < 0.05). Variables that did not meet the assumptions were subjected to the Kruskal–Wallis non-parametric test (p < 0.05) and in cases which significance was observed, the Dunn test (Post hoc) was applied. Differences between the slaughter times for the quantitative variables were subjected to parametric analysis by the unpaired T-test, and the variables that did not meet the assumptions were subjected to the Wilcoxon–Mann–Whitney non-parametric test. The difference between the means (post hoc) was calculated by the last square mean adjusted by the Tukey method. For proportion analysis, the differences between groups by date were calculated using the Wilson score interval method. The correlation analysis of parametric data were subjected to Pearson's correlation test (p < 0.05), and non-parametric data, to Spearman's test. Correlation analysis to assess whether two variables can be considered dependent was performed using Kendall's nonparametric test. For this, the software R was used, with the kms activator download - Free Activators packages “agricolae”62; "LmerTest"63, "arm"64; "Emmeans"65; "Car"66; "Nortest"67; "MASS"68 with the software R69. Normalization of cytokine gene expression and the graphs were performed on software GraphPad Prism 6 (La Jolla, CA-USA).

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

References

  1. 1.

    Pieters, M. G. & Maes, D. Mycoplasmosis. In The Diseases of Swine (ed. Zimmermann, J. J.) (Wiley-Blackwell, 2019).

    Google Scholar

  2. 2.

    Holst, S., Yeske, P. & Pieters, M. Elimination of Mycoplasma hyopneumoniae from breed-to-wean farms: A review of current protocols with emphasis on herd closure and medication. J. Swine. Health. Prod.23, 6 (2015).

    Google Scholar

  3. 3.

    Murtaugh, M. P. Advances in swine immunology help move vaccine technology forward. Vet. Immunol. Immunopathol.159, 3–4. https://doi.org/10.1016/j.vetimm.2014.02.017 (2014).

    CASArticle Google Scholar

  4. 4.

    Pasternak, J. A., Ng, S. H. & Wilson, H. L. A single, low dose oral antigen exposure in newborn piglets primes mucosal immunity if administered with CpG oligodeoxynucleotides and polyphosphazene adjuvants. Vet. Immunol. Immunopathol.161, 3–4. https://doi.org/10.1016/j.vetimm.2014.08.006 (2014).

    CASArticle Google Scholar

  5. 5.

    Chase, C. & Lunney, J. K. Immune system. In The Diseases of Swine (ed. Zimmermann, J. J.) 264–291 (Wiley-Blackwell, 2019).

    Chapter Google Scholar

  6. 6.

    Hyland, K., Foss, D. L., Johnson, C. R. & Murtaugh, M. P. Oral immunization induces local and distant mucosal immunity in swine. Vet. Immunol. Pathol.102, 3. https://doi.org/10.1016/j.vetimm.2004.09.015 (2004).

    CASArticle Google Scholar

  7. 7.

    Lin, J. H., Weng, C. N., Liao, C. W., Yeh, K. S. & Pan, M. J. Protective effects of oral microencapsulated Mycoplasma hyopneumoniae vaccine prepared by co-spray drying method. J. Vet. Med. Sci.2, 7. https://doi.org/10.1292/jvms.65.69 (2003).

    Article Google Scholar

  8. 8.

    Hua, L. Z. et al. Comparative analysis of mucosal immunity to Mycoplasma hyopneumoniae in Jiangquhai porcine lean strain and DLY piglets. Genet. Mol. Res.13, 3. https://doi.org/10.4238/2014.July.7.13 (2014).

    CASArticle Google Scholar

  9. 9.

    Wilson, H. L. & Obradovic, M. R. Evidence for a common mucosal immune system in the pig. Mol. Immunol.66, 1. https://doi.org/10.1016/j.molimm.2014.09.004 (2015).

    CASArticle Google Scholar

  10. 10.

    Matthijs, A. M. et al. Systems immunology characterization of novel vaccine formulations for Mycoplasma hyopneumoniae bacterins. Front. Immunol.10, 1087. https://doi.org/10.3389/fimmu.2019.01087 (2019).

    CASArticlePubMedPubMed Central Google Scholar

  11. 11.

    Petrovsky, N. & Aguilar, J. C. Vaccine adjuvants: Current state and future trends. Immunol. Cell Biol.82, 5. https://doi.org/10.1111/j.0818-9641.2004.01272.x (2004).

    Article Google Scholar

  12. 12.

    Mariano-Neto, F. et al. Physical properties of ordered mesoporous SBA-15 silica as immunological adjuvant. J. Phys. D. Appl. Phys.47, 42 (2014).

    Article Google Scholar

  13. 13.

    Ko, J. W. et al. Silica dioxide nanoparticles aggravate airway inflammation in an asthmatic mouse model via NLRP3 inflammasome activation. Regul. Toxicol. Pharmacol.112, 104618. https://doi.org/10.1016/j.yrtph.2020.104618 (2020).

    CASArticlePubMed Google Scholar

  14. 14.

    Ravinayagam, V. & Jermy, B. R. Nanomaterials and their negative effects on human health. In Applications of Nanomaterials in Human Health (ed. Khan, F.) 249–273 (Springer, 2020).

    Chapter Google Scholar

  15. 15.

    Chen, L. et al. The toxicity of silica nanoparticles to the immune system. Nanomedicine13, 1939–1962. https://doi.org/10.2217/nnm-2018-0076 (2018).

    ArticlePubMed Google Scholar

  16. 16.

    Mercuri, L. P. et al. Ordered font creator 12 - Free Activators silica SBA-15: A new effective adjuvant to induce antibody response. Small2, 2. https://doi.org/10.1002/smll.200500274 (2006).

    ADSCASArticle Google Scholar

  17. 17.

    Carvalho, L. V. et al. Immunological parameters related to the adjuvant effect of the ordered mesoporous silica SBA-15. Vaccine.28, 50. https://doi.org/10.1016/j.vaccine.2010.09.087 (2010).

    CASArticle Google Scholar

  18. 18.

    Scaramuzzi, K. et al. Nanostructured SBA-15 silica: An effective protective vehicle to oral hepatitis B vaccine immunization. Nanomed. Nanotechnol.12, 8. https://doi.org/10.1016/j.nano.2016.06.003 (2016).

    CASArticle Google Scholar

  19. 19.

    Lopes, J. L. S. et al. Antigenic and physicochemical characterization of Hepatitis B surface protein under extreme temperature and pH conditions. Vaccine.37, 43. https://doi.org/10.1016/j.vaccine.2019.09.005 (2019).

    CASArticle Google Scholar

  20. 20.

    Rasmussen, M. K. et al. 3D visualisation of hepatitis B vaccine in the oral delivery vehicle SBA-15. Sci. Rep.9, 1. https://doi.org/10.1038/s41598-019-42645-5 (2019).

    CASArticle Google Scholar

  21. 21.

    Villarreal, I. et al. The effect of vaccination on the transmission of Mycoplasma hyopneumoniae in pigs under field conditions. Vet. J.188, 1. https://doi.org/10.1016/j.tvjl.2010.04.024 (2011).

    Article Google Scholar

  22. 22.

    Villarreal, I. et al. Effect of challenge of pigs previously immunised with inactivated vaccines containing homologous and heterologous Mycoplasma hyopneumoniae strains. Vet. Res.8, 1. https://doi.org/10.1186/1746-6148-8-2 (2012).

    CASArticle Google Scholar

  23. 23.

    Almeida, H. M. et al. Cytokine expression and Mycoplasma hyopneumoniae burden in the development of lung lesions in experimentally inoculated pigs. Vet. Microbiol1, 108647. https://doi.org/10.1016/j.vetmic.2020.108647 (2020).

    CASArticle Google Scholar

  24. 24.

    Ogawa, Y. et al. Oral vaccination against mycoplasmal pneumonia of kontakt 5 crack reddit using a live Erysipelothrix rhusiopathiae vaccine strain as a vector. Vaccine.27, 33. https://doi.org/10.1016/j.vaccine.2009.04.081 (2009).

    CASArticle Google Scholar

  25. 25.

    Dawson, A., Thevasagayam, S. J., Sherington, J., Harvey, R. E. & Peters, A. R. Studies of the field efficacy and safety of a single-dose Mycoplasma hyopneumoniae vaccine for pigs. Vet. Rec.151, 18. https://doi.org/10.1136/vr.151.18.535 (2002).

    Article Google Scholar

  26. 26.

    Moreau, I. A., Miller, G. Y. & Bahnson, P. B. Effects of Mycoplasma hyopneumoniae vaccine on pigs naturally infected with M. hyopneumoniae and porcine reproductive and respiratory syndrome virus. Vaccine.22, 17–18. https://doi.org/10.1016/j.vaccine.2003.10.041 (2004).

    CASArticle Google Scholar

  27. 27.

    Meyns, T. et al. Comparison of transmission of Mycoplasma hyopneumoniae in vaccinated and non-vaccinated populations. Vaccine.24, 49–50. https://doi.org/10.1016/j.vaccine.2006.07.004 (2006).

    CASArticle Google Scholar

  28. 28.

    Djordjevic, S. et al. Serum and mucosal antibody responses and protection in pigs vaccinated against Mycoplasma hyopneumoniae with vaccines containing a denatured membrane antigen pool and adjuvant. Austr. Veter. J.https://doi.org/10.1111/j.1751-0813.1997.tb14383 (1997).

    Article Google Scholar

  29. 29.

    Thacker, E. L., Thacker, B. J., Boettcher, T. B. & Jayappa, H. Comparison of antibody production, lymphocyte stimulation, and protection induced by four commercial Mycoplasma hyopneumoniae bacterins. J. Swine Health Prod.6, 107–112 (1998).

    Google Scholar

  30. 30.

    Martelli, P. et al. Systemic and local immune response in pigs intradermally and intramuscularly injected with inactivated Mycoplasma hyopneumoniae vaccines. Vet. Microbiol.168, 2–4. https://doi.org/10.1016/j.vetmic.2013.11.025 (2014).

    CASArticle Google Scholar

  31. 31.

    Feng, Z. X. et al. Development and validation of an attenuated Mycoplasma hyopneumoniae aerosol vaccine. Vet. Microbiol.167, 3–4. https://doi.org/10.1016/j.vetmic.2013.08.012 (2013).

    CASArticle Google Scholar

  32. 32.

    Bai, Y. et al. Application of a sIgA-ELISA method for differentiation of Mycoplasma hyopneumoniae infected from vaccinated pigs. Vet. Microbiol.https://doi.org/10.1016/j.vetmic.2018.07.023 (2018).

    ArticlePubMed Google Scholar

  33. 33.

    Sibila, M. et al. Chronological study of Mycoplasma hyopneumoniae infection, seroconversion and associated lung lesions in vaccinated and non-vaccinated pigs. Vet. Microbiol.122, 1–2. https://doi.org/10.1016/j.vetmic.2007.01.010 (2007).

    CASArticle Google Scholar

  34. 34.

    Andreasen, M., Nielsen, J. P., Bækbo, P., Willeberg, P. & Bøtner, A. A longitudinal study of serological patterns of respiratory infections in nine infected Danish swine herds. Prev. Vet. Med.45, 3. https://doi.org/10.1016/S0167-5877(00)00122-7 (2000).

    Article Google Scholar

  35. 35.

    Djordjevic, S. P., Eamens, G. J., Romalis, L. F. & Saunders, M. M. An improved enzyme linked immunosorbent assay (ELISA) for the detection of porcine serum antibodies against Mycoplasma hyopneumoniae. Vet. Microbiol.39, 3–4. https://doi.org/10.1016/0378-1135(94)90163-5 (1994).

    Article Google Scholar

  36. 36.

    Leon, E. A., Madec, F., Taylor, N. M. & Kobisch, M. Seroepidemiology of Mycoplasma hyopneumoniae in pigs from farrow-to-finish farms. Vet. Microbiol.78, 4. https://doi.org/10.1016/S0378-1135(00)00303-5 (2001).

    Article Google Scholar

  37. 37.

    Villarreal, I. et al. Infection with a low virulent Mycoplasma hyopneumoniae isolate does not protect piglets against subsequent infection with a highly virulent M. hyopneumoniae isolate. Vaccinehttps://doi.org/10.1016/j.vaccine.2008.12.005 (2009).

    ArticlePubMed

Источник: https://www.nature.com/articles/s41598-021-01883-2

Transition-metal-free intramolecular Friedel–Crafts reaction by alkene activation: A method for the synthesis of some novel xanthene derivatives

Our starting alkenes 4al are original and were synthesized in four steps involving coupling, Grignard, oxidation, and Wittig reactions. We synthesized the novel unactivated alkenes 4al containing three aryl groups as the starting materials. The synthesis of 4a is demonstrated in Scheme 1. First, 2-phenoxybenzaldehyde (1a) was synthesized by coupling reaction of phenol with commercial 2-fluorobenzaldehyde. This reaction was carried out with very high yield by refluxing the reactants in the presence of K2CO3 in DMF.

[1860-5397-17-142-i1]

2-Phenoxybenzaldehyde (1a) was converted into secondary alcohol derivative 2a by adding a phenyl group using a Grignard reaction. This reaction was carried out with high yield by adding the freshly prepared Grignard compound of phenyl bromide to 2-phenoxybenzaldehyde. As a result, phenoxy secondary alcohol 2a containing three aromatic rings was obtained. In the third step, 2a was oxidized and the ketone derivative 3a was obtained in high yield. The oxidation reaction was carried out using PCC in DCM at room temperature. In the fourth and final step, phenoxydiphenylalkene derivative 4a was prepared by a Wittig reaction, which was carried out using methyltriphenylphosphonium bromide with ketone 3a, in basic medium, at room temperature, and dry THF.

After the structures of the starting compounds were elucidated, the method development trials for the synthesis of xanthene derivatives were carried out. For this purpose, catalyst researches were carried out using compound 4a. An intramolecular Friedel–Crafts reaction was tried by activating the alkene with various organic Brønsted acids and Lewis acids (Table 1). In the reaction, iron(III) chloride hexahydrate, trifluoroacetic acid (TFA), N-trifylphosphoramide (NTPA), benzoic acid, diphenyl phosphate (DPP), malonic acid, chloroacetic acid, copper(II) triflate, acetic acid, and p-toluenesulfonic acid (p-TSA) were used as catalysts. TFA gave the best yield of these catalysts with 78% (Table 1, entry 2). The second-best yield was 65% when FeCl3·6H2O was used (Table 1, entry 1).

[Graphic 1]
BS Player Pro Free Download Entry hma pro vpn license key 2021 - Activators Patch Catalyst (10 mol %) Conv. (%)b
1 FeCl3·6H2O 65
2TFA78
3 font creator 12 - Free Activators NTPA 10
4 benzoic acid 0
5 DPP 2
6 malonic acid 0
7 chloroacetic acid 14
8 Cu(OTf)2 12
9 AcOH 0
font creator 12 - Free Activators 10 p-TSA 3

aConditions: 4a (0.1 mmol) and catalyst (10 mol %) in CHCl3 (1 mL) were stirred at room temperature for 24 hours. bConversions were determined with GC–MS.

Then the solvent was investigated. Toluene, methyl alcohol, ethyl acetate, THF, DMF, dichloromethane, chloroform, acetone, and acetonitrile were tested as solvents. As a result, it was determined that the best conversion was with dichloromethane (Table 2). Later, quantity and time experiments were performed (Table 2, entries 10–15) and, at the end of these trials, it was determined that the reaction was completed with >99% conversion in 6 hours with 10 mol % catalyst at room temperature (Table 2, entry 14).

[Graphic 2]
Entry Cat. amount (mol %) Solvent Time Conv.b
1 10 CHCl3 24 h 78
2 10 formz vs sketchup - Activators Patch acetone 24 h windows 8.1 pro activator 2019 - Free Activators 75
Password Depot 12.0.6 Crack - Crack Key For U 3 10 toluene 24 h 70 techtool pro 11 review - Free Activators 4 10 CH2Cl2 24 h >99
5 10 THF 24 h 45
6 macrium reflect 7.2 4473 crack - Free Activators 10 CH3CN 24 h 10
7 dolby atmos windows 7 crack - Free Activators 10 MeOH splash 2.0 premium crack - Crack Key For U 24 h 0
8 10 EtAc 24 h app builder 2019 - Free Activators 35
9 10 DMF 24 h 0
10 dvdfab passkey alternative - Free Activators 10 CH2Cl2 1 h 32
11 10 CH2Cl2 2 h 53
12 10 sketchbook pro 8.4.2 crack - Crack Key For U CH2Cl2 3 h 70
13 10 CH2Cl2 4 h 85
1410CH2Cl26 h>99
15 5 secure cleaning - Crack Key For U Acronis True Image 2021.25 Crack + Serial Key Free Download CH2Cl2 6 h 95

aConditions: 4a (0.1 mmol) and TFA in dry solvent (1 mL) were stirred at room temperature. bConversions were determined with GC–MS. total pdf converter - Activators Patch

After determining the most suitable conditions for the intramolecular Friedel–Crafts reaction with alkene activation, the synthesis of the new xanthene derivatives was performed according to this method. The synthesized xanthene derivatives with their isolated yields are shown in Figure 1. Compounds 5bl were synthesized for the first time in this study. The first synthesis of 5a was prepared by reduction of the corresponding xanthydrol [53] and VueScan 9.7.12 serial key - Crack Key For U also synthesized recently by a different method from our group [29].

[1860-5397-17-142-1]

Although in the reactions for FCA reagents and strong inorganic acids, such as AlCl3, H2SO4, or H3PO4, which have generally corrosive properties, were used, in this study, an intramolecular ring closure reaction was carried out under easy operating conditions with an organic Brønsted acid catalyst with high yields. So, the xanthene synthesis with alkene activation was performed for the first time using TFA. The reasonable mechanism of this reaction is delineated in Scheme 2.

[1860-5397-17-142-i2]

Despite this reaction occurring by the classical Friedel–Crafts mechanism, we believe that o-quinone methide is formed as an intermediate. Because of its Magoshare Data Recovery 4.8 Crack + License Code [2021] reactive structure, most of the xanthene synthesis is based on the o-quinone methide intermediate [54-58]. The carbocation formed by the activation of an alkene with acid turns into an intermediate o-quinone methide, resulting in a successful cyclization.

As seen in the mechanism, the acid catalyst adds to the vinyl group, allowing the formation of a tertiary carbocation. The carbocation is then transformed into the o-quinone methide intermediate, which undergoes cyclization to yield 9-methyl-9-arylxanthene by aromatization. When the yields of the synthesized compounds are examined, it is seen that the yields are high when there is no substituent at the ring to which the carbocation is attached or when there is an electron-donating group, such as a methoxy or methyl group (Figure 1). When there are electron-withdrawing groups, such as chlorine or cyano groups, in the ring, the yield is slightly reduced because they deactivate the ring in the transition state. The lowest yield was observed for compound 5j to which a cyclopentyl group is attached. Since the cyclopentyl group in this compound is directly attached to the carbon from which the carbocation is formed, byproducts are formed and the yield is reduced since conversions can occur.

Источник: https://www.beilstein-journals.org/bjoc/articles/17/142

Free radicals, antioxidants and functional foods: Impact on human health

1. Aruoma OI. Methodological consideration for characterization for potential antioxidant actions of bioactive components in plants foods. Mutat Res. 2003;532:9–20. [PubMed] [Google Scholar]

2. Mohammed AA, Ibrahim AA. Pathological roles of reactive oxygen species and their defence mechanism. Saudi Pharm J. 2004;12:1–18.[Google Scholar]

3. Bagchi K, Puri S. Free radicals and antioxidants in health and disease. East Mediterranean Health Jr. 1998;4:350–60.[Google Scholar]

4. Aruoma OI. Nutrition and health aspects of free radicals and antioxidants. Food Chem Toxicol. 1994;32:671–83. [PubMed] [Google Scholar]

5. Cheeseman KH, Slater TF. An introduction to free radicals chemistry. Br Med Bull. 1993;49:481–93. [PubMed] [Google Scholar]

6. Young IS, Woodside JV. Antioxidants in health and disease. J Clin Pathol. 2001;54:176–86.[PMC free article] [PubMed] [Google Scholar]

7. Liu T, Stern A, Roberts LJ. The isoprostanes: Novel prostanglandin-like products of the free radical catalyzed peroxidation of arachidonic acid. J Biomed Sci. 1999;6:226–35. [PubMed] [Google Scholar]

8. Ebadi M. Antioxidants and free radicals in health and disease: An introduction to reactive oxygen species, oxidative injury, neuronal cell death and therapy in neurodegenerative diseases. Arizona: Prominent Press; 2001. [Google Scholar]

9. Lea AJ. Dietary factors associated with death rates from certain neoplasms in man. Lancet. 1966;2:332–3. [PubMed] [Google Scholar]

10. Harman D. Role of free radicals in aging and disease. Ann N Y Acad Sci. 1992;673:126–41. [PubMed] [Google Scholar]

11. Sies H. Oxidative stress: Introductory remarks. In: Sies H, editor. Oxidative Stress. San Diego: Academic Press; 1985. pp. 1–7. [Google Scholar]

12. Docampo R. Antioxidant mechanisms. In: Marr J, Müller EZ Game Booster Pro For Windows, editors. Biochemistry and Molecular Biology of Parasites. London: Academic Press; 1995. pp. 147–60. [Google Scholar]

13. Rice-Evans CA, Gopinathan V. Oxygen toxicity, free radicals and antioxidants in human disease: Biochemical implications in atherosclerosis and the problems of premature neonates. Essays Biochem. 1995;29:39–63. [PubMed] [Google Scholar]

14. Rock CL, Jacob RA, Bowen PE. Update o biological characteristics of the antioxidant micronutrients- Vitamin C, Vitamin E and the carotenoids. J Am Diet Assoc. 1996;96:693–702. [PubMed] [Google Scholar]

15. Mc Cord JM. The evolution of free radicals and oxidative stress. Am J Med. 2000;108:652–9. [PubMed] [Google Scholar]

16. Rao AL, Bharani M, Pallavi V. Role of antioxidants and free radicals in health and disease. Adv Pharmacol Toxicol. 2006;7:29–38.[Google Scholar]

17. Stefanis L, Burke RE, Greene LA. Apoptosis in neurodegenerative disorders. Curr Opin Neurol. 1997;10:299–305. [PubMed] [Google Scholar]

18. Esterbauer H, Pubi H, Dieber-Rothender M. Effect of antioxidants on oxidative modification of LDL. Ann Med. 1991;23:573–81. [PubMed] [Google Scholar]

19. Neuzil J, Thomas SR, Stocker R. Requirement for promotion, or inhibition of α- tocopherol of radical induced initiation of plasma lipoprotein lipid peroxidation. Free Radic Biol Med. 1997;22:57–71. [PubMed] [Google Scholar]

20. Poppel GV, Golddbohm RA. Epidemiologic evidence for β – carotene and cancer prevention. Am J Clin Nutr. 1995;62:1393–5. [PubMed] [Google Scholar]

21. Glatthaar BE, Horing DH, Moser U. The role of ascorbic acid in carcinogenesis. Adv Exp Med Biol. 1986;206:357–77. [PubMed] [Google Scholar]

22. Sokol RJ. Vitamin E deficiency and neurologic diseses. Annu Rev Nutr. 1988;8:351–73. [PubMed] [Google Scholar]

23. Ashok BT, Ali R. The aging paradox: Free radical theory of aging. Exp Gerontol. 1999;34:293–303. [PubMed] [Google Scholar]

24. Sastre J, Pellardo FV, Vina J. Glutathione, oxidative stress and aging. Age. 1996;19:129–39.[Google Scholar]

25. Cantuti-Castelvetri I, Shukitt-Hale B, Joseph JA. Neurobehavioral aspects of antioxidants in aging. Int J Dev Neurosci. 2000;18:367–81. [PubMed] [Google Scholar]

26. Freeman BA, Crapo JD. Biology of disease: Free radicals and tissue injury. Lab Invest. 1982;47:412–26. [PubMed] [Google Scholar]

27. Lovell MA, Ehmann WD, Buffer BM, Markesberry WR. Elevated thiobarbituric acid reactive substances and antioxidant enzyme activity in the brain in Alzemers disease. Neurology. 1995;45:1594–601. [PubMed] [Google Scholar]

28. Woo RA, Melure KG, Lee PW. DNA dependent protein kinase acts upstream of p53 in response to DNA damage. Nature. 1998;394:700–4. [PubMed] [Google Scholar]

29. Hattori Y, Nishigori C, Tanaka T, Ushida K, Nikaido O, Osawa T. 8 Hydroxy-2-deoxyguanosine is increased in epidermal cells of hairless mice after chronic ultraviolet B exposure. J Invest Dermatol. 1997;89:10405–9. [PubMed] [Google Scholar]

30. Halliwell B. How to characterize an antioxidant- An update. Biochem Soc Symp. 1995;61:73–101. [PubMed] [Google Scholar]

31. Shi HL, Noguchi N, Niki N. Comparative study on dynamics of antioxidative action of α- tocopheryl hydroquinone, ubiquinol and α- tocopherol, against lipid peroxidation. Free Radic Biol Med. 1999;27:334–46. [PubMed] [Google Scholar]

32. Levine M, Ramsey SC, Daruwara R. Criteria and recommendation for Vitamin C intake. JAMA. 1991;281:1415–23. [PubMed] [Google Scholar]

33. Matill HA. Antioxidants. Annu Rev Biochem. 1947;16:177–92. [PubMed] [Google Scholar]

34. German J. Food processing and lipid oxidation. Adv Exp Med Biol. 1999;459:23–50. [PubMed] [Google Scholar]

35. Jacob R. Three eras of vitamin C discovery. Subcell Biochem. 1996;25:1–16. [PubMed] [Google Scholar]

36. Knight J. Free radicals: Their history and current status in aging and disease. Ann Clin Lab Sci. 1998;28:331–46. [PubMed] [Google Scholar]

37. Moreau, Dufraisse Comptes Rendus des Séances et Mémoires de la Société de Biologie. 1922;86:321.[Google Scholar]

38. Wolf G. The discovery of the antioxidant function of vitamin E: The contribution of Henry A. Mattill. J Nutr. 2005;135:363–6. [PubMed] [Google Scholar]

39. Frie B, Stocker R, Ames BN. Antioxidant defences and lipid peroxidation in human blood plasma. Proc Natl Acad Sci. 1988;37:569–71.[Google Scholar]

40. Rice-Evans CA, Diplock AT. Current status of antioxidant therapy. Free Radic Biol Med. 1993;15:77–96. [PubMed] [Google Scholar]

41. Krinsky NI. Mechanism of action of biological antioxidants. Proc Soc Exp Biol Med. 1992;200:248–54. [PubMed] [Google Scholar]

42. Niki E. Antioxidant defenses in eukaryotic cells. In: Poli G, Albano E, Dianzani MU, editors. Free radicals: From basic science to medicine. Basel, Switzerland: Birkhauser Verlag; 1993. pp. 365–73. [Google Scholar]

43. Sies H. Oxidative stress: Oxidants and antioxidants. Exp Physiol. 1997;82:291–5. [PubMed] [Google Scholar]

44. Magnenat JL, Garganoam M, Cao J. The nature of antioxidant defense mechanisms: A lesson from transgenic studies. Environ Health Perspect. 1998;106:1219–28.[PMC free article] [PubMed] [Google Scholar]

45. Zelko I, Mariani T, Folz R. Superoxide dismutase multigene family: A comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) gene structures, evolution, and expression. Free Radic Biol Med. 2002;33:337–49. [PubMed] [Google Scholar]

46. Banniste J, Bannister W, Rotilio G. Aspects of the structure, function, and applications of superoxide dismutase. CRC Crit Rev Biochem. 1987;22:111–80. [PubMed] [Google Scholar]

47. Johnson F, Giulivi C. Superoxide dismutases and their impact upon human health. Mol Aspects Med. 2005;26:340–52. [PubMed] [Google Scholar]

48. Wuerges J, Lee JW, Yim YI, Yim HS, Kang Winstep Nexus Ultimate Crack 20.13 (2021) + Serial Key Full Download, Djinovic Carugo K. Crystal structure of nickel-containing superoxide dismutase reveals another type of active site. Proc Natl Acad Sci. 2004;101:8569–74.[PMC free article] [PubMed] [Google Scholar]

49. Corpas FJ, Barroso JB, del Río LA. Peroxisomes as a source of reactive oxygen species and nitric oxide signal molecules in plant cells. Trends Plant Sci. 2001;6:145–50. [PubMed] [Google Scholar]

50. Corpas FJ, Fernández-Ocaña A, Carreras A, Valderrama R, Luque F, Esteban FJ, et al. The expression of different superoxide dismutase forms is cell-type dependent in olive (Olea europaea L.) leaves. Plant Cell Physiol. 2006;47:984–94. [PubMed] [Google Scholar]

51. Cao X, Antonyuk SV, Seetharaman SV, Whitson LJ, Taylor AB, Holloway SP, et al. Disk drill pro 3.5 crack - Crack Key For U of the G85R variant of SOD1 in familial amyotrophic lateral sclerosis. J Biol Chem. 2008;283:16169–77.[PMC free article] [PubMed] [Google Scholar]

52. Chelikani P, Fita I, Loewen PC. Diversity of structures and properties among catalases. Cell Mol Life Sci. 2004;61:192–208. [PubMed] [Google Scholar]

53. Gaetani G, Ferraris A, Rolfo M, Mangerini R, Arena S, Kirkman H. Predominant role of catalase in the disposal of hydrogen peroxide within human erythrocytes. Blood. 1996;87:1595–9. [PubMed] [Google Scholar]

54. Eisner T, Aneshansley DJ. Spray aiming in the bombardier beetle: Photographic evidence. Proc Natl Acad Sci USA. 1999;96:9705–9.[PMC free article] [PubMed] [Google Scholar]

55. Meister A, Anderson M. Glutathione. Annu Rev Biochem. 1983;52:711–60. [PubMed] [Google Scholar]

56. Brigelius-Flohe R. Tissue-specific functions of individual glutathione peroxidases. Free Radic Biol Med. 1999;27:951–65. [PubMed] [Google Scholar]

57. Hayes J, Flanagan J, Jowsey I. Glutathione transferases. Annu Rev Pharmacol Toxicol. 2005;45:51–88. [PubMed] [Google Scholar]

58. Smirnoff N. L-ascorbicacid biosynthesis. Vitam Horm. 2001;61:241–66. [PubMed] [Google Scholar]

59. Meister A. Glutathione-ascorbic acid antioxidant system in animals. J Biol Chem. 1994;269:9397–400. [PubMed] [Google Scholar]

60. Padayatty S, Katz A, Wang Y, Eck P, Kwon O, Lee J, et al. Vitamin C as an antioxidant: Evaluation of its role in disease prevention. J Am Coll Nutr. 2003;22:18–35. [PubMed] [Google Scholar]

61. Shigeoka S, Ishikawa T, Tamoi Dolby Atmos Crack For Windows [32bit + 64bit] Latest 2021 Free, Miyagawa Y, Takeda T, Yabuta Y, et al. Regulation and function of ascorbate peroxidase isoenzymes. J Exp Bot. 2002;53:1305–19. [PubMed] [Google Scholar]

62. Meister A, Anderson A. Glutathione. Annu Rev Biochem. 1983;52:711–60. [PubMed] [Google Scholar]

63. Meister A. Glutathione metabolism and its selective modification. J Biol Chem. 1988;263:17205–8. [PubMed] [Google Scholar]

64. Fairlamb AH, Cerami A. Metabolism and functions of trypanothione in the Kinetoplastida. Annu Rev Microbiol. 1992;46:695–729. [PubMed] [Google Scholar]

65. Nassar E, Mulligan C, Taylor L, Kerksick C, Galbreath M, Greenwood M, et al. Effects of a single dose of N-Acetyl-5-methoxytryptamine (Melatonin) and resistance exercise on the growth hormone/IGF-1 axis in young males and females. J Int Soc Sports Nutr. 2007;4:14.[PMC free article] [PubMed] [Google Scholar]

66. Caniato R, Filippini R, Piovan A, Puricelli L, Borsarini A, Cappelletti E. Melatonin in plants. Adv Exp Med Biol. 2003;527:593–7. [PubMed] [Google Scholar]

67. Reiter RJ, Carneiro RC, Oh CS. Melatonin in relation to cellular antioxidative defense mechanisms. Horm Metab Res. 1997;29:363–72. [PubMed] [Google Scholar]

68. Tan DX, Manchester LC, Reiter RJ, Qi WB, Karbownik M, Calvo JR. Significance of melatonin in antioxidative defense system: Reactions and products. Biol Signals Recept. 2000;9:137–59. [PubMed] [Google Scholar]

69. Herrera E, Barbas C. Vitamin E: Action, metabolism and perspectives. J Physiol Biochem. 2001;57:43–56. [PubMed] [Google Scholar]

70. Brigelius-Flohe R, Traber M. Vitamin E: Function and metabolism. FASEB J. 1999;13:1145–55. [PubMed] [Google Scholar]

71. Traber MG, Atkinson J. Vitamin E, antioxidant and nothing more. Free Radic Biol Med. 2007;43:4–15.[PMC free article] [PubMed] [Google Scholar]

72. Wang X, Quinn P. Vitamin E and its function in membranes. Prog Lipid Res. 1999;38:309–36. [PubMed] [Google Scholar]

73. Jaeschke H, Gores GJ, Cederbaum AI, Hinson JA, Pessayre D, Lemasters JJ. Mechanisms of hepatotoxicity. Toxicol Sci. 2002;65:166–76. [PubMed] [Google Scholar]

74. Papas AM. Diet and antioxidant status. Food Chem Toxicol. 1999;37:999–1007. [PubMed] [Google Scholar]

75. Brown JE, Rice-Evan CA. Luteolin-rich Artichoke extract protects low density lipoprotein from oxidation in vitro. Free Radic Res. 1998;29:247–255. [PubMed] [Google Scholar]

76. Furuta S, Nishiba Y, Suda I. Fluorometric assay for screening antioxidative activities of vegetables. J Food Sci. 1997;62:526–8.[Google Scholar]

77. Wang H, Cao G, Prior RL. Total antioxidant capacity of fruits. J Agric Food Chem. 1996;44:701–5.[Google Scholar]

78. Lin JK, Lin CH, Ling YC, Lin-Shian SY, Juan IM. Survey of catechins, gallic acid and methylxantines in green, oolong, puerh and black teas. J Agric Food Chem. 1998;46:3635–42.[Google Scholar]

79. Devasagayam TP, Tilak JC, Boloor KK, Sane KS, Ghaskadbi SS, Lele RD. Free radicals and antioxidants in Human Health: Current status and future prospects. J Assoc Physicians India. 2004;52:794–803. [PubMed] [Google Scholar]

80. López-Varela S, González-Gross M, Marcos A. Functional foods and the immune system: A review. Eur J Clin Nutr. 2002;56:S29–33. [PubMed] [Google Scholar]

81. Roberfroid MB. What is beneficial for health? The concept of functional food. Food Chem Toxicol. 1999;37:1034–41. [PubMed] [Google Scholar]

82. Krishnaswamy K. Indian functional food: Role in prevention of cancer. Nutr Rev. 1996;54:127–31. [PubMed] [Google Scholar]

83. DeFelice SL. Nutraceuticals: Opportunities in an Emerging Market. Scrip Mag. 1992;9:14–5.[Google Scholar]

84. Dillard CJ, German JB. Phytochemicals: Nutraceuticals and human health. J Sci Food Agric. 2000;80:1744–56.[Google Scholar]

85. Tapas AR, Sakarkar DM, Kakde RB. Review article flavonoids as nutraceuticals: A review. Trop J Pharm Res. 2008;7:1089–99.[Google Scholar]

86. Vidya AD, Devasagayam TP. Current status of Herbal drug in India: An overview. J Clin Biochem Nutr. 2007;41:1–11.[PMC free article] [PubMed] [Google Scholar]

Источник: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3249911/

Notice: Undefined variable: z_bot in /sites/homeover.us/free-activators/font-creator-12-free-activators.php on line 107

Notice: Undefined variable: z_empty in /sites/homeover.us/free-activators/font-creator-12-free-activators.php on line 107

4 Replies to “Font creator 12 - Free Activators”

  1. The true way of opening the pineal gland is through Jesus Christ, Jesus Christ was perfect in every way imaginable, he was the way, the truth, and the life, since the first second he was born, and in the bible it speaks about the third eye, it says "The light of the body is the eye, if thy eye be full of light, then you shall be full of light, but if thy eye be full of darkness, then you shall be full of darkness", then Jesus Christ says in the bible, "Truly, truly, I say to you, he who does not enter the sheep fold by the door but climbs in by another way, that man is a thief and a robber ", anyone who you see who is using kundalini and says they feel great will have their so called "enlightment" taken away either by the devil or demonic entity after their done using you for whatever they might want, or God will take it away from you, for you are a thief. It might not be this year or 10 years from now but it will be taken away, the sacred secretion or Christ within is the true way of communicated with god, that's why god created the pineal gland in the first place, and just as Jesus Christ said you cannot reach the father/god except through me, you have a choice to have that antenna connected to God or the devil, remember lucifer was a fallen angel he wanted authority over god/wanted to be god so God casted him out of heaven, lucifer knows about the pini

  2. Definitely enjoyed your idea more! The more experience you get I’m sure your ideas will come to light ❤️

Leave a Reply

Your email address will not be published. Required fields are marked *