There are three details that we highlight in the readings for this upcoming 4th Sunday of Easter
The first two details come from the first reading
The relevant apologetical topics are Peter the Rock (and thus the Papacy) and Jesus’ divinity
The third detail is the major theme of the Gospel—Jesus the Good Shepherd
The apologetical topics that come to fore with this theme is Jesus’ divinity and Peter’s role as the first pope
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a podcast where we reflect on the upcoming Sunday Mass readings and pick out the details that are relevant for explaining and defending our Catholic faith
staff apologist and speaker for Catholic Answers
There are three details that we’re going to highlight in the readings for this upcoming 4th Sunday of Easter
Here’s what Peter declares in Acts 4:8-12:
then all of you and all the people of Israel should know
that it was in the name of Jesus Christ the Nazorean
in his name this man stands before you healed
given to the human race by which we are to be saved.&#8221;
there are two details here that I want to focus on
The first is Peter’s application of Psalm 118:22 to the current situation: “[Jesus] is the stone rejected by you
Some Protestants appeal to this passage as evidence that Peter is not the “rock” upon which Christ promises to build his Church in Matthew 16:18
I’ve already addressed this objection in episode 46 of the Sunday Catholic Word
which was the episode for the 27th Sunday of Ordinary Time
I recommend that you listen to that episode if you want the details on how to respond to this objection
I also deal with it in my book Meeting the Protestant Challenge and Meeting the Protestant Response
The second detail worthy of note in this passage is Peter’s declaration
“There is no salvation through anyone else
no is there any other name under heaven given to the human race by which we are to be saved.” This is apologetically significant because Peter here quotes Joel 2:32
nor is there any other name under heaven given to the human race by which we are to be saved.”
How could Peter say such a thing unless he believed Jesus was Yahweh
the salvation that Joel speaks of is temporal salvation
Peter applies the text to spiritual salvation
which further drives home the point that he believes Jesus is God
Let’s now turn to the Gospel that grounds this upcoming Good Shepherd Sunday
A good shepherd lays down his life for the sheep
sees a wolf coming and leaves the sheep and runs away
This is because he works for pay and has no concern for the sheep
just as the Father knows me and I know the Father;
I have other sheep that do not belong to this fold
because I lay down my life in order to take it up again
This command I have received from my Father
The key detail here is Jesus’ declaration in verse 11
“I am the good shepherd.” There are couple of Old Testament prophecies that shed light on what Jesus is revealing here
where God describes himself as the shepherd gathering his flock unto himself
and judging “between one sheep and another
Jesus clearly sees himself fulfilling this prophecy in Matthew 25:46 when he identifies himself as the judge judging between sheep and goats at the final judgment
But he also indicates he is the fulfillment of the prophecy here in our Gospel reading when he says
Notice Jesus says he’s “the” good shepherd
Jesus speaks of some of his sheep that are scattered and how he will bring them into the fold: “I have other sheep
that are not of this fold; I must bring them also
Yahweh speaks of bringing in scattered sheep: “The lost I will search out
Jesus’ description of himself as the good shepherd
The second Old Testament prophecy that’s relevant to Jesus’ claim to be the good shepherd is Isaiah 40:9-11
Isaiah prophesies about “the gospel,” or the euangelion
euangelizomenos]; lift up your voice with strength
&#8220;Behold your God!&#8221;… the Lord GOD comes with might
and his arm rules….[11] He will feed his flock like a shepherd
Notice it’s God who is the shepherd feeding his flock
In light of this prophecy it becomes clear what Jesus is claiming about himself when he says
“I am THE good shepherd”: he’s saying he’s God
the good shepherd theme reveals Jesus’ self-understanding of being divine
But there’s another apologetical topic that this theme evokes: Peter’s role as the first pope
“I have other sheep that do not belong to this fold
one shepherd.” The “one shepherd” here ultimately refers to Jesus
The “other sheep” not of Jesus’ fold refers to the Gentiles
Jesus is prophesying of a time when both Jews and Gentiles will be within his fold guided by one shepherd
Who initially brought the Gentiles into Jesus’ fold
Jesus reveals to Peter that the Gentiles are allowed to be members of his flock
And this is visibly manifested by the Spirit being given to Cornelius
Peter goes to Jerusalem to describe to the circumcision party the things that had transpired with the Gentiles and we’re told they glorified God
“Then to the Gentiles also God has granted repentance unto life.”
Peter definitively declares that the Gentiles can be saved by God’s grace apart from circumcision
This shepherding role of Peter was initially promised to him in John 21:15-17:
do you love me more than these?” He said to him
Lord; you know that I love you.” He said to him
“Feed my lambs.” A second time he said to him
“Tend my sheep.” He said to him the third time
do you love me?” Peter was grieved because he said to him the third time
you know everything; you know that I love you.” Jesus said to him
the exclusive command to feed Jesus’ sheep clearly signals Peter’s unique role as leader/shepherd of Jesus’ Church
where Jesus singles out Peter and makes him the visible foundation of his Church
here Jesus singles out Peter again and makes him the shepherd of his flock—a universal charge that extends to both the young “lambs” (Greek
what’s interesting is that the second command
“shepherd my sheep.” The Greek word for “tend” is poimainō
which is a verb that means “to shepherd.” The Greek word for the one “shepherd” in John 21:16 is the related noun poimen
And given that Peter alone is given this exclusive command
thereby indicating that Peter is to shepherd them as well
we can read the “one shepherd” who tends the one flock of Christ in John 10:16
there is no shortage of apologetical material in this upcoming 4th Sunday of Easter
There are multiple details that relate to two important apologetical topics
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You might also want to check out the other great podcasts in our Catholic Answers podcast network: Cy Kellet’s Catholic Answers Focus
One last thing: if you’re interested in getting some cool mugs and stickers with my logo
I hope you have a blessed 4th Sunday of Easter
I had a Mary Magdalene resurrection morning experience two years ago when my husband and I visited Ireland
We decided to check out the Carrowkeel passage tombs in county Sligo
a series of rock-strewn mounds constructed before the pyramids
Each features a single passageway to a small burial chamber at the very center of each mound
We navigated an unpaved road and then a two-mile uphill walk where wind and sheep outnumbered signage and other humans
Photo of the Carrowkeel passage tombs in county Sligo
When we finally stood before one of these marvels
I was struck by the fact that the entrance to the passage was not sealed
Each structure was positioned so that on the morning of the Winter Solstice
rays of the rising sun cross the threshold
I found myself wanting to shout &ndash; to the wind and the sheep &ndash; something I realized deep in my spirit: Light wins
who built these passage tombs with a resilient hope that light would pierce the darkest of places on the darkest of days
Mary Magdalene oriented herself toward the Light
she is the one who remembers Jesus&rsquo; promise about the third day
She risks trekking to the tomb to bear witness to that promise
She encounters the exposed entrance and is filled with an energy that turns her into yeast
Paul invokes in his letter the Corinthians
for the otherwise hunkered down community of disciples
Her steadfast faith in the &ldquo;light that overcomes the darkness&rdquo; brought others to it.&nbsp;
by orienting ourselves to the light &ndash; to Jesus
the crucified and risen Christ &ndash; we join Mary Magdalene in participating in God&rsquo;s promise to refresh the face of the earth
Maureen H. O&rsquo;Connell is Associate Professor of Christian Ethics in the Department of Religion and Theology at La Salle University. She recently published <a href="http://www.beacon.org/Undoing-the-Knots-P1729.aspx">Undoing the Knots: Five Generations of American Catholic Anti-Blackness</a> with Beacon Press
O&rsquo;Connell es profesora asociada de &eacute;tica cristiana en el departamento de religi&oacute;n y teolog&iacute;a de la Universidad La Salle
Recientemente public&oacute; Undoing the Knots: Five Generations of American Catholic Anti-Blackness con Beacon Press.&nbsp;
Rejoice all the earth &ndash; Flooded with the New Light
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The Ignatian Solidarity Network (ISN) is a national social justice network inspired by the spirituality of St
ISN was founded in 2004 and is a lay-led 501(c)3 organization working in partnership with Jesuit universities
along with many other Catholic institutions and social justice partners
Tom Carson died thirty years ago this month
did evangelism in Montreal for a decade in the 1930s and ’40s
which I understand to be about seventy minutes from here
he returned to Ottawa as a translator for the Canadian government and began serving as an unpaid pastor
He died quietly and without fanfare on October 26
He was not well-known or celebrated in his day
In fact, his son, Don, as you may know, wrote a short book about him called Memoirs of an Ordinary Pastor. Today some of us remember Tom because of Don, and because of the book, but we remember Tom Carson for his blessed ordinariness. So, in his honor, I’ve entitled these two sessions tonight <a href="https://www.desiringgod.org/series/ordinary-elders">“Ordinary Elders.”</a> 
I would like for us to linger together in perhaps my favorite eldership passage of Scripture: 1 Peter 5:1–5
But before I read those verses and pray for our time together
which links this passage to chapter 4 and therefore to the hard times Peter and the elders knew
First Peter 4:12 mentions “fiery trials.” Verse 13
19: “suffer,” “suffers,” “suffer.” This is a word for elders who know hard times
as a fellow elder and a witness of the sufferings of Christ
as well as a partaker in the glory that is going to be revealed: shepherd the flock of God that is among you
as God would have you; not for shameful gain
but eagerly; not domineering over those in your charge
you will receive the unfading crown of glory
for “God opposes the proud but gives grace to the humble.”
One of the most precious promises in all the Bible for pastors in particular is Jesus’s words in Matthew 16:18: “I will build my church
and the gates of hell shall not prevail against it.” Jesus is the chief shepherd
and the Shepherd and Overseer of our souls (1 Peter 2:25; 5:4)
“the great shepherd of the sheep” (Hebrews 13:20)
And one of the ways Christ builds and governs his church
is by giving her the gift of leaders under him: “He gave the apostles
to equip the saints for the work of ministry
for building up the body of Christ” (Ephesians 4:11–12)
Faithful pastors and elders are a gift from Christ to guide and keep his church
This is a truth that may not be healthy to regularly preach to ourselves personally
but it can be good to have someone else preach to you from time to time
you are a gift from the risen Christ to your flock
No matter how flat it seems your last sermon fell
No matter what you hear whispered about leaders in society
not to mention the cynicism that isn’t whispered
No matter what that person posted online about your church — and you didn’t see it
“Did you see this?” No matter what has been said explicitly or implied
as you lean on Christ and remain faithful to his word
Have some who carry the name “pastor” made terrible mistakes
and harmed the very ones they were commissioned to protect
But such failures were not the fulfilling of the vision of what true Christian leadership is
such failures show — by contrast — what real leadership in the church should be
That’s our focus this evening: what Christ calls leaders in his church to be — especially the “lead office” or “teaching office” in the church
that of “pastor” or “elder” or “overseer,” three terms in the New Testament for the same lead office
I want us to give most of our focus to the three not-but pairs in verses 2–3
but first let me make three preliminary observations on the passage
One of the most important truths to rehearse about Christian ministry is that Christ means for it to be teamwork
so in every context in which local-church pastor-elders are mentioned in the New Testament
He is head (Ephesians 1:22; 5:23; Colossians 1:18)
The glory of singular leadership in the church is his alone
And he means for his under-shepherds to labor
The kind of pastors we long for in this age are good men with good friends — friends who love them enough to challenge their instincts
which is Peter’s charge to the elders: “shepherd the flock of God.” Shepherd
To shepherd is an image of what we might call “benign rule” (the opposite of “domineering,” as we’ll see)
in which the good of the shepherd is bound up with the good of the sheep
The concept of shepherding also has a rich Old Testament background
not just in the Patriarchs and the nation of Israel in Egypt and in the wilderness
the shepherd boy who became the nation’s greatest king
who anticipated the great Anointed One to come
had his own grave failures in shepherding the nation
the trend of the nation’s kings became worse and worse
the prophet Ezekiel condemned the nation’s leaders for “feeding themselves” rather than feeding the sheep:
shepherds of Israel who have been feeding yourselves
and with force and harshness you have ruled them
The leaders of Israel should have fed the people
but instead they have governed them “with force and harshness” — not benign rule but malignant rule
even as he protects them from their enemies
and he shall feed them: he shall feed them and be their shepherd” (Ezekiel 34:22–23)
Note the prominence of feeding in shepherding
The prophet Micah foretold that from Bethlehem
will “come a ruler who will shepherd my people Israel” (Micah 5:2; Mark 2:6)
Jesus himself says he is the good shepherd (John 10:11)
when Jesus asked Peter three times — this same Peter who wrote 1 Peter — if he loved him
“Feed my lambs,” “Tend my sheep,” and “Feed my sheep” (John 21:15–17)
Here “feeding” and “pastoring” are synonymous
and he will now pastor his sheep through Peter and other under-shepherds — not just apostles
as Paul says in Acts 20:28 to the elders in Ephesus: “Pay careful attention to yourselves and to all the flock [!]
in which the Holy Spirit has made you overseers
which he obtained with his own blood.” The elders are also overseers
“pastor the church of God” (elders = overseers = pastors)
“will guide them to springs of living water” (Revelation 7:17)
he will rule “with a rod of iron” (Revelation 2:27; 12:5; 19:15)
which doesn’t mean he is forceful or harsh with his people
but that he protects them from their enemies (with his rod)
The shepherd’s rod and staff are for protecting and guiding his flock: “your rod and your staff
So there’s just a taste of the richness in this shepherding image: centrally
feeding and watering (“green pastures” and “still waters,” Psalm 23:2)
and wielding the rod of protection toward various threats to the flock
more briefly: the verb that augments “shepherd” is “exercising oversight.” It’s a form of the noun “overseer” used in Acts 20:28
as well as four other New Testament texts (Philippians 1:1; 1 Timothy 3:2; Titus 1:7; 1 Peter 2:25)
“Oversee” in this context doesn’t mean only to watch and observe
but also to “see to it” that important observations about the flock
also become tangible initiatives and actions in the church
Which brings us to the heart of this passage where Peter gives us three “not-buts” — not this but that
Verses 2–3: “Shepherd the flock of God that is among you
We saw God’s condemnation for the leaders of Israel who ruled “with force and harshness.” Peter says “not domineering” — which is the same language we see elsewhere translated “not lording it over.” It’s built on a strong verb (katakurieuo) that can refer in other contexts to Jesus’s lordship (Romans 14:9; 1 Timothy 6:15); or the kind of lordship sin once had
14; 7:1); or the kind of lordship Christian leaders do not have over those in their charge (Luke 22:25)
The intensified form of the verb here in 1 Peter 5 is the same one Jesus uses in Mark 10:42: 
Those who are considered rulers of the Gentiles lord it over them
and their great ones exercise authority over them
Verse 43: “But whoever would be great among you must be your servant
and whoever would be first among you must be slave of all
For even the Son of Man came not to be served but to serve
and to give his life as a ransom for many.” So the opposite of “not lording it over” others is serving them
about his labors as an apostle: “Not that we lord it over your faith
but we work with you for your joy” (2 Corinthians 1:24)
“lord it over” implies the exercise of privilege
the seeking and obtaining of personal/private benefit; benefit from them (versus through or with them)
Paul’s vision of the opposite in leadership is “working with you for your joy.” The “we” here is Paul with his assistants Timothy and Silas (2 Corinthians 1:19)
expend energy; it is not just “overflow” but work
labor (as Jesus says in Matthew 9:37–38: “The harvest is plentiful
but the laborers are few; therefore pray earnestly to the Lord of the harvest to send out laborers into his harvest”)
as “overflow,” but then takes effort (sometimes great effort) to complete
Not only is there a “we” in the company of the leaders but it’s also “with you” — with the people
and invest energy — to work with us (which is vital to keep in mind in our discipling and counseling; we work with them
Joy that tastes of the next age even in this painful
blissful future in Christ is brought into the painful present — which means the frictions and sufferings of our present times do not preclude real joy but make us all the more desperate for real joy
as workers for their people’s joy (in the words of Paul) and examples to the flock (in the words of Peter): “not domineering over those in your charge
if you don’t want your life observed and imitated
or local hero?” Examples might sound so normal
Twice Peter says the elders are “among” the flock: “I exhort the elders among you 
: shepherd the flock of God that is among you” (1 Peter 5:1–2)
Pastors do not comprise a fundamentally different category of Christian
They need not be world-class in their intellect
as they lead and feed the flock through teaching God’s word
accompanied with wise collective governance
The hearts of good pastors swell to Jesus’s charge in Luke 10:20: “Do not rejoice in this
but rejoice that your names are written in heaven.” Their first and most fundamental joy is not what God does through them as pastors but what Christ has done (and does) for them as Christians
and consonant with our remembering Tom Carson as an ordinary pastor-elder
I can’t help but share quickly Bonhoeffer’s lightning strike against “celebrity” instincts in the church
This is at the end of chapter 4 in Life Together:
Jesus made authority in the fellowship dependent upon brotherly service
Every cult of personality that emphasizes the distinguished qualities
even though these be of an altogether spiritual nature
is worldly and has no place in the Christian community
One finds there [in the elder qualifications in 1 Timothy 3] nothing whatsoever with respect to worldly charm and the brilliant attributes of a spiritual personality
who rightly discharges his duties to the Church
The Church does not need brilliant personalities but faithful servants of Jesus and the brethren
is determined by the faithfulness with which a man serves Jesus Christ
never by the extraordinary talents which he possesses
Pastoral authority can be attained only by the servant of Jesus who seeks no power of his own
who himself is a brother among brothers submitted to the authority of the Word
Such is Bonhoeffer’s call for ordinary elders: “a brother among brothers,” present in the life of the church and accessible
They don’t presume to shepherd God’s flock in all the world through the Internet
but focus on the flock “that is among you” (verse 2) — those particular names and faces assigned to their charge — and they delight to be among those people
“Shameful gain” would be some benefit not commensurate with the work
or some gain that is against the gain of the flock
and the glory of Christ — whether money as the driving motivation
In terms of “eagerness,” the epistle to the Hebrews gives this important glimpse into the dynamic of Christian leadership as workers for the joy of the flock:
for they are keeping watch over your souls
Let them do this with joy and not with groaning
marriage-like vision of the complementary relationship between the church and its leaders
as we’ve seen; it is costly work) for the advantage — the profit
wants its leaders to work not only hard but happily
because the pastors’ joy in leading will lead to the church’s own benefit
The people want their leaders to labor with joy because they know their leaders are working for theirs
Christ gives leaders to his people for their joy
Pastors are glad workers for the gladness of their people in God
might the people be to submit to such a leader
The prospect of submitting to a leader drastically changes when you know he isn’t pursuing his own private advantage but genuinely seeking yours: what is best for you
what will give you the deepest and most enduring joy — when he finds his joy in yours
rather than apart from or instead of yours
The word “submission” has negative connotations today in many circles
But how might the charge to “submit” in Hebrews 13:17 and “be subject” in 1 Peter 5:5 change when we see it in the context of this vision of shepherding and oversight and pastoring as working for the joy of our people
There’s no charge to submit in verse 5 until verses 2–4 establish a context of “workers for your joy” who are willing
not themselves; they attend to the flock’s needs
not their own; they gain as the flock gains
It’s amazing to consider what actions and initiatives and care are presupposed (and commanded) in the New Testament
from husbands and fathers and governors and pastor-elders
When leaders in the church show ourselves to be workers for their joy
we walk in the steps of the great shepherd — the great worker for joy — the one who bore the greatest cost for others’ good
He found his joy in the joy of his Beloved
“For the joy that was set before him [he] endured the cross” (Hebrews 12:2)
as I just recently have been struck by in Isaiah 53:11
“Out of the anguish of his soul he shall see and be satisfied.”
we pastor-elders emphatically pursue gain — not shameful gain but the shameless gain that is our joy in the joy of the church
and joy in the coming shameless reward: “When the chief Shepherd appears
you will receive the unfading crown of glory” (1 Peter 5:4)
The kind of pastors our people want are pastors who want to do the work
They want pastors who serve “not under compulsion
God himself wants pastors who labor willingly
He wants us to aspire to the work (1 Timothy 3:1)
And not just “as God would have you” because he’s requiring something of us that is different than his own character and actions
But “as God would have you” meaning “as God himself is” and does — literally “according to God” (Greek: kata theon)
It says something about our God that he would have it this way
He is the infinitely happy “blessed God” (1 Timothy 1:11) who acts from the boundless
He wants pastors to work with joy because he works this way
He is a God most glorified not by heartless duty
and he himself cares for his people willingly
Let me close with just two practical manifestations of this vision for what it might mean for you
as a pastor-elder (or aspiring pastor-elder)
to be a worker with your people for their joy in Christ
late-night one (at least late-night for us
as we do our pastors’ meetings every other Thursday night after our kids’ bedtimes)
There are countless implications of this vision
What does it look like for me to pursue my joy in the joy of our people (to the glory of God)
my “first great and primary business to which I ought to attend every day” is “to have my soul happy in the Lord.” Don’t hear this as an obligation but an opportunity — not first and foremost a “have to” but a “get to.” To feed on God
“The first thing to be concerned about [is] not how much I might serve the Lord [what I might do for others’ joy] 
but how I might get my soul into a happy state
meditation on it” — oh the joys of unhurried
meditation on the words of God himself — “that thus my heart might be comforted
my heart might be brought into experiential communion with the Lord.”
How did he go about approaching God’s word
“searching as it were into every verse to get blessing out of it; not for the sake of public ministry of the word; not for the sake of preaching on what I had meditated upon; but for the sake of obtaining food for my soul.”
“Now what is the food for the inner man?” He answers “the word of God,” and adds
and applying it to our hearts” — in other words
“How different when the soul is refreshed and made happy early in the morning.”
but food for your own soul — is the well from which we draw in pastoring from joy
to lead through prayer and collective wisdom and decision-making for the church
do we find two (or more) options lying before us
What is our framework for the decisions of leadership
It can be easy to slip into a selfish mindset: what is easiest
what’s most convenient for those of us sitting around the table
how might our preferences and comforts shape this church
How might church life be more convenient for us
will be best for our people’s true joy in Christ
But beware: when you ask a question like this
you find that it’s often the path that is more costly to the pastors and elders
But this is the work to which we are called
If our team of pastors and elders trends toward the personal preferences and conveniences of the pastors and elders
We are not working with them for their joy
But when we are “workers for their joy” — knowing that Christ is most glorified in his church
when his church is most satisfied in him — then
we set aside our own convenience and personal preferences and together we labor for the joy of our people in Jesus
Roads can be dangerous places for cats of all sizes
Vehicle strikes can kill even the stealthiest and quickest of cats
wildlife crossings are one of the most effective ways to create a safe passage for species to move across landscapes that include roadways
Wildlife crossings are specific features that provide safe pathways for animals across barriers in their habitats
Some are recognizable as overpasses covered with native vegetation
while others are built into the landscape as culverts
Certain species have preferences for the type of wildlife crossings they use so developing a variety of different connectivity options is key to securing safe movement to avoid vehicular impacts
In the Appalachian Mountains, bobcats — as well as black bears, elk, deer and many more species — use wildlife crossings that Defenders and our partners are working to create in partnership with state agencies. As part of <a href="https://defenders.org/blog/2024/02/safe-passage-wildlife">the Safe Passage Coalition</a>
we are actively working to install five wildlife crossings within 20 priority areas
Like Florida panthers, vehicular strikes are the <a href="https://defenders.org/newsroom/tragic-loss-of-ocelot-may-still-bring-hope-future">leading cause of death for ocelots</a> in the United States
These unique cats prefer habitats with dense vegetation
which helps to camouflage the cat from both predators and prey
that camouflage also makes it difficult for oncoming traffic to see a cat planning a dash across a road
Within Laguna Atascosa National Wildlife Refuge in Southern Texas
ocelots have proven they will use a wildlife crossing when one is provided
Defenders is currently assisting with the planning and construction phase for numerous wildlife crossings in New Mexico
We are helping the state determine priority projects for wildlife crossings on major roadways using wildlife-vehicle collision hotspot data and ecological models
black bears and even wolves to benefit from these projects
As jaguars continue to reinhabit their historic lands and move upwards into New Mexico
it’s possible these crossings could be utilized by this elusive species
efforts to make Interstate 10 safer for wildlife to cross could help bolster and protect jaguar and ocelot populations
In addition to protecting animals from injury or death by vehicles
By allowing wildlife to move from one region to another
species can increase their genetic diversity
maintain proper population levels through predation and avoid intraspecies conflict for mates or territory
As more wildlife return to their historic ranges
and climate change and human development affect survivable existing habitats
the need for safe passage through wildlife movement corridors becomes more critical for the long-term survival and continuation of species
You can help stop animal deaths and vehicular damage with these simple tips:
Danielle has always had a passion for advocating for animals and education
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Many of the world’s species are on the move because of climate change
species are shifting their ranges north and to cooler places and higher elevations to adapt to a changing climate
species will be increasingly stressed and are projected to migrate as they adapt to changing conditions
mammals and plants are being pushed northward or up and around mountains and into cool riparian corridors by warming temperatures and changes in snow and rainfall patterns
and a safe passageway for non-migratory species is not guaranteed
There are fewer than five corridors in Nevada of passably connected mountains ranges and wet valley bottoms that fully allow species movement within a livable climate that can serve as stepping stones to a safe haven for Nevada's wildlife
As Nevada considers proposals for protecting 30 percent of land by 2030
it is imperative that we protect lands and waters that will be a refuge for plants and wildlife impacted by climate threats
We can help our ecosystems adapt to climate change by supporting meaningful and durable protections for climate adaptation passages
Monsoon Passage follows the Nevada-Utah border and is mostly in Nevada
extending from the confluence of the Muddy River and the Colorado River’s Lake Mead
Monsoon Passage includes an important migration flyway where bird watchers gather in the Goshute Mountains to view the raptor migration
The corridor is also at the western edge of the Baja monsoonal storms
The summer monsoon extends 60 miles west into the Great Basin from Utah
and encompasses the Las Vegas area in the Mojave Desert
The monsoonal footprint is critical because it provides increased moisture in an eastward direction
to buffer increasing temperatures in a changing climate
TNC in Nevada staff are working or have previously worked in multiple areas of Monsoon Passage: 
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Dam removals and fish passage construction have opened up new habitat in Maine&#039;s  Sheepscot River for sea-run fish
In 2019, we celebrated two habitat restoration milestones on Maine’s Sheepscot River, where <a href="https://www.fisheries.noaa.gov/insight/successful-fish-passage-efforts-across-nation">fish passage</a> barriers were removed at the two lowermost dams on the river
The Coopers Mills dam in Whitefield was fully removed in 2018
The Head Tide dam in Alna was partially removed and fish passage rebuilt in 2019
The dams were removed in partnership with the Atlantic Salmon Federation and the two towns where the dams were located
fish returning to the Sheepscot River to spawn have been delayed at the bottom of these dams
They’re often unable to get by to reach their historic spawning grounds upriver
Opening up river habitat can help increase the populations of these fish
many of which are prey for popular recreational fish like tuna and striped bass
The Sheepscot River is the southernmost river in the United States where endangered <a href="https://www.fisheries.noaa.gov/species/atlantic-salmon-protected#overview">Atlantic salmon</a>, a <a href="https://www.fisheries.noaa.gov/species/atlantic-salmon-protected#spotlight">NOAA Species in the Spotlight</a>, consistently spawn in the wild. The river also supports a longstanding commercial <a href="https://www.fisheries.noaa.gov/species/river-herring">alewife</a> fishery
where lobster fishermen regularly line up in the spring to purchase bait
It also provides spawning grounds for American shad
dozens gathered on the banks of the Sheepscot River to celebrate the removal of the Coopers Mills dam
It was a milestone nearly two decades in the making.&nbsp;
The dam removal restored access to more than 20 miles of river and 700 acres of pond habitat for sea-run fish to spawn and grow
The dam removal project included an innovative dry hydrant system that provides water for fire fighting and a small park with scenic overlook
Stone from the former dam was repurposed to create the overlook and pathways around historic mill foundations
the restoration community in Maine donned raincoats and carried umbrellas
and&nbsp;gathered to celebrate the breach of the Head Tide dam on the Sheepscot River
Fish passage after Head Tide dam partial removal
The project built on the success of the 2018 removal of the Coopers Mills dam upstream
The fish passage improvements at Head Tide dam included a partial removal of the dam
There were also improvements to an adjacent park
The historic mill site was gifted to the town of Alna with a stipulation that the dam not be removed
This project required some creative and collaborative work-arounds
The <a href="https://www.fisheries.noaa.gov/topic/habitat-conservation#how-we-restore">NOAA Restoration Center</a> oversaw the design
and construction of the fish passage improvements
The team used computer modeling to make sure that the speed of the water through the opening did not exceed the swimming capabilities of sea run fish
They also made it look and feel as natural as possible
biologists from the Maine Department of Marine Resources confirmed that adult salmon were freely swimming upstream of both the Head Tide and Coopers Mills dams
the Atlantic Salmon Federation and other partners are looking at the feasibility of other fish passage projects in the Sheepscot River watershed
We’re especially looking at historic alewife ponds to restore this important sea-run species to its native range.&nbsp;
Contact <a href="mailto:matthew.bernier@noaa.gov">Matt Bernier</a>
an expansive concept record based on George Orwell’s Animal Farm
the writer’s seminal critique of Stalinism when a farm is taken over by its overworked
Animals touched on class struggles experienced in 1970s Britain and was rife with various allusions to an underclass
‘Sheep’ is a creative reflection of the <a href="https://faroutmagazine.co.uk/rage-against-the-machine-75-favourite-revolutionary-books/" target="_blank" rel="noopener" title="From Dr Seuss to Karl Marx: Rage Against The Machine’s 75 favourite revolutionary books">Karl Marx quote </a>that “religion is the opiate of the masses”
religion is used as a vehicle to highlight the blind faith of the underclass
who are oblivious to their social standing underneath their masters
the sheep are blithely unaware they’re about to be led to the slaughterhouse
The biblical allusions flood in quickly with the line: “I’ve looked over Jordan
and I have seen / Things are not what they seem”
the river Jordan was crossed by the Israelites in order to escape slavery and enter the Promised Land
this place is described as “flowing with milk and honey,” a prosperous place that could deliver a brighter future
this could be a nod to the increasing industrialisation of Britain – something sworn to bring riches that
oppressed the working classes and threatened their livelihoods
who remain largely unaware they’re sacrificial pawns: “Hopelessly passing your time in the grassland away / Only dimly aware of a certain unease in the air / You better watch out / There may be dogs about”
The dark reinvention of the psalm comes with the verse: “The Lord is my shepherd
I shall not want / He makes me down to lie / Through pastures green / He leadeth me the silent waters by / With bright knives He releaseth my soul”
The lyrics are naturally the most significant to the song’s meanings
but its musicality adds another complex later
Its enduring guitar riff and heavy bassline reinforce the idea of oppression
and its grandiose synth offers a glimmer of hope and urgency
Discordant moments where the music fades are filled with “baa” sounds
in an unsettling nod to the fate of the flock who are being led to their own slaughter
the band’s use of the Bible on ‘Sheep’ is an effective metaphor for a ruling class
           Print                          A set of three wildlife crossings — meant to provide safe passage for bighorn sheep and other animals — has been added to the plans for a high-speed rail line project between Las Vegas and Southern California
The California Department of Fish and Wildlife
the state Department of Transportation  and rail builder Brightline West announced on Wednesday their agreement to design and build the crossings over the planned 218-mile rail line
slated to occupy the center divider of the heavily trafficked 15 Freeway
Construction of the rail line is slated to begin in the second half of 2023 with completion expected in as many as four years
“Roadways and rail lines must be designed to connect
“This project will not only protect the precious wildlife and habitat of the Mojave Desert region but will also get people between Las Vegas and Southern California safely and efficiently — preserving one of the most popular corridors in our state.”
The crossings, notably, stand to benefit <a class="link" href="https://www.latimes.com/environment/story/2021-09-16/naturalists-demand-wildlife-bridge-for-las-vegas-rail-line">bighorn sheep</a>.
               </a>         <a class="link" href="https://www.latimes.com/california">California</a> 
Facing long-standing allegations of “appalling” conditions inside the county’s jails and violent deputy “gangs” operating on its streets, Sheriff Robert Luna announced a new office designed to combat those problems.
When the project was proposed, conservationists voiced concern that the sheep’s movements would be restricted just as drought and heat sent them searching farther afield for food and water.
Crossings were not part of the initial plans for the rail.
The project was touted as a win for zero-emission travel, with Brightline also claiming that travel time between destinations would be 2¼ hours as trains reached speeds of 180 miles per hour.
On its website, Brightline claimed the rail line would reduce the number of vehicles traveling between the two regions by 3 million per year, saving 400,000 tons of carbon dioxide. 
Wildlife researchers noted in 2021 that three crossings in the Mojave Desert’s Soda, Cady and Clark mountains could help sustain the diversity in the desert.
Those are the areas that appear to be targeted with the crossings, which are planned for areas near Zzyzx and Rasor roads and the community of Mountain Pass.
“Giving wildlife freedom to roam despite the growing infrastructure needed to support California’s robust human population is a top priority for CDFW,” the agencies said in a joint news release Wednesday
the project will incorporate plans to maintain or improve hundreds of preexisting culverts and crossings under Interstate 15
Desert tortoise fencing and wildlife exclusionary fencing also will be restored or installed
The crossings are the latest wins for wildlife advocates who have been pushing for pathways for animals across the state
Last spring, construction began on a long-hoped-for crossing over the treacherous 101 Freeway in Agoura Hills <a class="link" href="https://www.latimes.com/california/story/2022-04-22/a-cougar-crossing-rises-over-a-deadly-l-a-freeway">that would benefit mountain lions</a>.
The $30-million Wallis Annenberg Wildlife Crossing is expected to be completed in 2025.
Christian Martinez is a former reporter for the Los Angeles Times. He previously wrote for the USA Today network of newspapers including the Ventura County Star, where he covered the Thomas and Woolsey wildfires and the Borderline mass shooting, the Spectrum &amp; Daily News in Utah and the Lansing State Journal in Michigan. He was born and raised in Southern California and attended Saint Mary’s College of California.
               </a>         <a class="link" href="https://www.latimes.com/environment">Climate &amp; Environment</a> 
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Nearly two years of monitoring wildlife crossings in western North Dakota revealed that a number of animals — from moose to mule deer to bighorn sheep to white-tailed deer and the occasional turkey — successfully navigated the underground passages hundreds of times out of harm’s way of passing traffic on U.S
“The number of animals that have successfully gone through the crossings so far is very encouraging and we realize that we’re probably going to see an even higher number of animals use them in the future,” said Bruce Kreft
North Dakota Game and Fish Department resource biologist
help fulfill their life cycles and allow them safe passage
these safe crossings translate to the possibility of hundreds of fewer wildlife-vehicle collisions.”
North Dakota’s first wildlife underpass was constructed on U.S
Highway 85 on the Lewis and Clark Wildlife Management Area near Williston
Highway 85 south of the Long X Bridge near the North Unit of Theodore Roosevelt National Park
was completed a few years later in 2021 to accommodate bighorn sheep movements
as well as the comings and goings of mule deer and other animals
Kreft said trail cameras were installed on both sides of the crossings
While cameras identified successful navigation through the structures
they also identified “deflections,” or animals that simply approached the crossings but didn’t fully commit
With increased traffic and the proposed widening of U.S
Kreft said there is a need to address human safety and economic losses attributed to vehicle-wildlife collisions by evaluating and designing crossings to accommodate the movement of large mammals and other critters
The 62-mile stretch of highway from Belfield to Watford City wanders through unique habitat types
badlands and the Little Missouri River valley
This corridor is home to a host of North Dakota’s big game species including bighorn sheep
“These species all rely on different habitat types to fulfill various portions of their life cycles
which requires crossing Highway 85 numerous times in a year to fulfill those requirements,” Kreft said
habitat connectivity is the important aspect to this
Many of these animals need different types of habitat during
and these structures provide them safe passage to those different habitats.”
This crossing experienced 386 and 989 mule deer approaching the crossing in 2021 and 2022 with a successful crossing rate of 91% and 93%
The crossing was only monitored for 143 days in 2021
resulting in fewer animals approaching compared to 2022
but had similar animals crossing per day of monitoring
Bighorn sheep movements resulted in 58 and 171 animals approaching the structure in 2021 and 2022
with successful crossing rates of 81% and 89%
Both mule deer and bighorn sheep experienced an increase in crossing success from 2021 to 2022
This likely is contributed to behavioral learning and hopefully the start of generational learning of fawns and lambs
“A lot of times with a new structure there’s generational learning that has to occur
that this structure is there for them and it’s a safe passageway for them to get to the other side,” Kreft said
it takes about 3 to 4 years to really see the full degree of crossing for those animals
The ewes have to teach their lambs and the does have to teach their fawns that it’s a safe way to get to the other side for breeding purposes
Some findings from the Lewis and Clark WMA crossing:
This crossing had 177 and 654 whitetail deer approach with only a 36% and 69% crossing rate in 2021 and 2022
The significant increase in 2022 likely attributed to flooding in the underpass in 2021
Moose had similar numbers approaching the structure in 2021 and 2022
resulting in crossing rates of 87% and 93%
Kreft noted that many of the animals caught on trail cameras at both crossings were the same animals photographed more than once as they came and went
As the North Dakota Department of Transportation continues its effort to widen U.S
Kreft said the Game and Fish Department and DOT are evaluating the construction of two wildlife crossings between Long X and U.S
Kreft said DOT will construct another wildlife underpass crossing near the Summit Campground
“Our current elk monitoring program with the GPS collars is indicating that elk want to cross just south of the badlands area on Highway 85,” he said
“There’s a predominant number of animals that are moving across the roadway there so we’re currently evaluating that.”
rather than one that runs under the highway
is being considered because research has shown that elk
While evaluation and construction of wildlife crossings south of Long X continues
what’s certain is that wildlife crossings work in North Dakota
“The implementation of wildlife crossings along Highway 85 has been exceptionally successful in reducing wildlife-vehicle collisions and providing habitat connectivity,” Kreft said
“Although we are unable to calculate the degree to which the crossings have reduced wildlife-vehicle collisions
they have successfully kept hundreds of animals off the road
creating a safer highway for the traveling public
As these animals become habituated to the crossings and generational learning of the fawns and lambs occur
the success of the crossings will be fully realized.”  
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Volume 9 - 2021 | <a class="ArticleLayoutHeader__info__doi" href="https://doi.org/10.3389/fcell.2021.785055">https://doi.org/10.3389/fcell.2021.785055</a>
Pluripotent stem cells (PSCs) have the potential to differentiate to all cell types of an adult individual and are useful for studying mammalian development
Establishing induced pluripotent stem cells (iPSCs) capable of expressing pluripotent genes and differentiating to three germ layers will not only help to explain the mechanisms underlying somatic reprogramming but also lay the foundation for the establishment of sheep embryonic stem cells (ESCs) in vitro
sheep somatic cells were reprogrammed in vitro into sheep iPSCs with stable morphology
delivered by piggyBac transposon system with eight doxycycline (DOX)-inducible exogenous reprogramming factors: bovine OCT4
Sheep iPSCs exhibited a chimeric contribution to the early blastocysts of sheep and mice and E6.5 mouse embryos in vitro
A transcriptome analysis revealed the pluripotent characteristics of somatic reprogramming and insights into sheep iPSCs
This study provides an ideal experimental material for further study of the construction of totipotent ESCs in sheep
Research in iPSCs provides novel insights into the developmental pathway of mammalian development and contributes to regenerative medicine
but there is no report of using the piggyBac transposable system to induce iPSCs in sheep
The purpose of this study was to establish sheep iPSCs (siPSCs) with greater developmental potential by expressing defined transcription factors (bovine OCT4
and human TERT) through the piggyBac transposon system and to provide experimental model for further study on the construction of sheep totipotent ESCs
The human TERT and SV40 large T antigen cDNA sequences were purchased from Addgene (pBABE-hygro-hTERT
The construction of PB-TRE-sLhT was based on PB-TRE-pNhL backbone transposons and was confirmed by sequencing
a quarter of the electroconversion product was seeded on STO feeders in M15 medium in 10-cm plates
M15 medium formulation was as follows: knockout DMEM
1&#x000d7; penicillin&#x02013;streptomycin
LIF1001); vitamin C (10&#x000a0;&#x003bc;g/ml
233-FB-025); and doxycycline (DOX) (1.0&#x000a0;&#x003bc;g/ml
The culture medium was changed on alternate days
and colonies were selected on days 7&#x02013;10 and maintained in M15 medium supplemented with DOX
Colonies with endogenous core pluripotent markers OCT4
and NANOG were detected by quantitative reverse transcription PCR (RT-qPCR) assay
The sheep iPSCs were maintained on STO feeder layers and enzymatically passaged every 2&#x02013;3&#x000a0;days using TrypLE&#x02122; Select (Gibco
The cells were dissociated and centrifuged (300&#x000d7;g for 3&#x000a0;min) in K10 medium
K10 medium composition was as follows: DMEM/F12 (Gibco
and 1&#x000d7; MEM non-essential amino acids
the sheep iPSCs were re-suspended and seeded in M15 medium
All cell cultures used in the study were carried out under conditions of 38.5&#x000b0;C and 5% CO2 unless otherwise noted
The sheep iPSCs were frozen once they were &#x0223c;80% confluent using cryopreservation medium
which contains 90% FBS and 10% (vol/vol) DMSO (Sigma
Small molecules and cytokines were supplemented as indicated at the following final concentrations: CHIR99021
10&#x02013;100&#x000a0;ng/ml; and Activin A
85,850) were used as basal medium for culture screening
The sheep iPSCs on feeder cells were switched to plates coated with 20&#x000a0;ug/ml fibronectin (Millipore
FC010) and maintained in M15 medium supplemented with DOX
The siPSCs were passaged every 2&#x02013;3&#x000a0;days and split at a ratio of 1:4
The siPSCs were collected in different passages for the detection of core pluripotent markers by RT-qPCR
Sheep iPSCs were detached from culture plates using TrypLE&#x02122; Select
and then seeded in a 96-U bottom well plates with ultra-low cell attachment (Corning
After 3&#x000a0;days in suspension culture
the embryoid bodies (EBs) were transferred and cultured in gelatin-coated plates for an additional 3&#x000a0;days to detect line gene expression by RT-qPCR and an additional 7&#x000a0;days for immunostaining
Gene expression was determined relative to GAPDH using the &#x00394;&#x00394;Ct method
The H2B-CAG-tdTomato plasmid was transfected into sheep iPSCs using Lipofectamine 2000 (Gibco
then screened with hygromycin (100&#x000a0;&#x003bc;g/ml
10687010) after transfection for 48&#x000a0;h
10&#x02013;15 tdTomato&#x0002b; siPSC-4 were injected into sheep blastocysts as described above to detect chimeric contribution in vitro and in vivo
Half of the injected sheep embryos were cultured in vitro in SOF medium and M15 with DOX mixture medium (1:1) at 38.5&#x000b0;C in a 5% CO2 atmosphere for 46&#x02013;48&#x000a0;h for the evaluation of chimerism
The other embryos were transferred to the uteri of pseudopregnant sheep at 7&#x000a0;days post coitus (dpc)
After the transplantation for 30&#x000a0;days
pregnancy was diagnosed by ultrasonography
The fetuses were isolated at embryonic stage of days 18&#x02013;60 to check chimeric contribution
10&#x02013;15 tdTomato&#x0002b; siPSC-4 were carefully injected into the early blastocysts of ICR mice using a piezo-assisted micromanipulator attached to an inverted microscope (Zeiss
One-third of the injected embryos were cultured in KSOM medium (Millipore
MR-020P-5F) and M15 medium combined with the DOX mixture medium (1:1) in vitro at 37&#x000b0;C for 16&#x000a0;h to check chimeric contribution in vitro
Other embryos were transplanted into the uterus of pseudopregnant ICR female mice at 2.5&#x000a0;dpc
Chimeric embryos were collected at E6.5 to check chimeric contribution in vivo
E6.5 mouse embryos (ICR) were dissected from the decidua under stereomicroscopy
10&#x02013;15 tdTomato&#x0002b; siPSC-4 were injected into the middle post-posterior of E6.5 mouse embryos (ICR) in the same way as above and then cultured in vitro in commercial rat serum and M15 with DOX mixture medium (1:1) at 37&#x000b0;C in a 5% CO2 atmosphere for 48&#x000a0;h for the evaluation of E8.5 chimerism
Total RNA was extracted from cells using TRIzol reagent (Invitrogen
United States) following the manufacturer&#x02019;s instructions
After quality control using NanoDrop ND-1000 (NanoDrop
1&#x02013;2&#x000a0;&#x000b5;g of RNA was used to extract mRNA according to the NEB Next Poly(A) mRNA Magnetic Isolation Module
The RNA sequencing libraries were then constructed according to the manufacturer&#x02019;s instructions for the Illumina NEBNext Ultra RNA Library Prep Kit (NEB)
The generated libraries were pooled and sequenced on an Illumina NovaSeq&#x02122; 6000 following the vendor&#x02019;s recommended protocol platforms with a 150-bp paired-end mode (sequenced by LC-Bio Technology Co.
Establishment of sheep induced pluripotent stem cell (iPSC) lines by expressing eight DOX-inducible exogenous reprogramming factors
(A) Schematic illustration of reprogramming SFs to iPSCs
PB-8F: bOMSK&#x000a0;&#x0002b;&#x000a0;pNhL&#x000a0;&#x0002b;&#x000a0;sLhT
pNhL (porcine NANOG and human LIN28 cDNAs)
and sLhT (SV40 large T and human TERT cDNAs)
and human TERT increased the number of reprogrammed colonies from 250,000 SFs along with four Yamanaka factors (n&#x000a0;&#x0003d;&#x000a0;3 independent experiments)
(C) The expression level of the eight exogenous reprogramming factors by RT-qPCR in siPSC-4 and siPSC-6
The error bars indicate three independent biological replicates (mean&#x000a0;&#x000b1;&#x000a0;SD)
&#x0002a;&#x0002a;p&#x000a0;&#x0003c;&#x000a0;0.001
&#x0002a;&#x0002a;&#x0002a;p&#x000a0;&#x0003c;&#x000a0;0.0001
(D) The morphology of siPSC-4 on feeder cells
(E) Alkaline phosphatase (AP) staining of siPSC-4 on feeder cells
(F) The karyotyping of siPSCs-4 at passage 25 revealed that 31 out of 40 (77.5%) metaphase spreads had a normal karyotype
(G) Relative expression of the core endogenous pluripotency genes OCT4
The relative expression was normalized to SFs and the GAPDH housekeeping gene
The results indicated that pluripotency in sheep iPSCs depended on the expression of the DOX-induced exogenous factors in the serum-containing medium and the feeder STO
Characterization of siPSCs and their culture conditions
(A) The relative expression of key endogenous pluripotency genes in siPSC-4 and siPSC-6
(C) The morphology of siPSC-4 without feeder cells
and NANOG in siPSC-4 in feeder-free condition
siPSC-4 for passage 14 were used in the analysis
(E) Quantitative reverse transcription PCR (RT-qPCR) analysis of pluripotency in sheep iPSCs under various culture conditions in the absence of DOX
Cells cultured in M15 media supplemented with DOX for passage 14 were used in the analysis
Differentiation of potency of siPSCs in vitro
(A) The morphology of embryoid bodies (EBs) derived from sheep iPSCs on day 3
(B) The relative expression of genes for the three germ layers in cells differentiated from sheep iPSCs by EB
The relative expression was normalized to sheep iPSCs and housekeeping gene GAPDH
(C) Immunostaining of cells differentiated in vitro from sheep iPSCs for GATA6 (endoderm)
indicating that sheep iPSCs were reprogrammed in a different state from ICM
Transcriptomic and epigenetic characteristics of siPSCs
(A) Volcano plot of differentially expressed genes for siPSC-4 and SFs
(B) Gene set enrichment analysis (GSEA) of siPSC-4 and SFs
The green line shows the enrichment profile
(C) Heatmap showing a comparison between na&#x000ef;ve and primed marker expression in SFs
(D,E) The expression of pluripotency genes
and siPSCs-6 (n&#x000a0;&#x0003d;&#x000a0;3)
the number of biologically independent samples
&#x0002a;p Val&#x000a0;&#x0003c;&#x000a0;0.01
(F) Principal component analysis (PCA) of global gene expression (RNA-seq) of sheep iPSCs (iPSC_4D and iPSC_6D) CTFR-sESCs (ESC_A and ESC_B)
Development potential of siPSCs in chimeras.(A) A schematic diagram of chimera experiments using siPSCs.(B) The contribution of siPSCs to the development of sheep preimplantation embryos (i
Injection of tdTomato&#x0002b; siPSC-4 (passage 19) into sheep early blastocysts (i)
The injected sheep early blastocysts cultured in SOF&#x000a0;&#x0002b;&#x000a0;M15 (1:1) medium for 46&#x000a0;h (ii)
(C) Some tdTomato&#x0002b; cells were positive for ICM-specific antibodies (OCT4)
(D) Contribution of siPSCs in mouse preimplantation embryo development (i
Injection of tdTomato&#x0002b; siPSC-4 (passage 19) in mouse blastocysts (i)
Injected mouse blastocysts cultured in KSOM&#x000a0;&#x0002b;&#x000a0;M15 (1:1) medium for 16&#x000a0;h (ii)
(E) Analysis of the contribution of siPSCs to E6.5 mouse embryos (i
Injection of tdTomato&#x0002b; siPSCs-4 to E6.5 mouse embryos (i)
Injected E6.5 mouse embryos cultured in commercial rat serum&#x000a0;&#x0002b;&#x000a0;M15 (1:1) medium for 48&#x000a0;h (ii)
(F) Detection of siPSCs-4 tdTomato&#x0002b; descendants in chimeric embryos at E8.5
tdTomato&#x0002b; siPSC-4 expressed markers of NESTIN (ectoderm) and SMA (mesoderm)
the differentiation data in vitro and our siPSCs showed a developmental potential in both sheep and mouse early embryos
but they could not synchronize with the developmental stages of recipient embryo after being transferred in vivo
our data in GSEA showed that sheep iPSCs significantly overrepresented genes in the WNT signaling
It will be interesting to test conditions including IWR-1 in sheep somatic cell reprogramming
According to the PCA of global gene expression (RNA-seq) of siPSCs
culture conditions with only WNT inhibitors may not be sufficient to maintain pluripotency in the absence of exogenous transgene expression
sheep iPSCs established in this study showed stable morphology
This study provides an ideal experimental protocol for further study on the construction of sheep totipotent ESCs and may provide a basis for sheep somatic reprogramming studies in the future
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: All the sequencing data were deposited in the NCBI, Sequence Read Archive (SRA) under the under the accession number PRJNA766237. Data of CTFR-sESCs and ICM are from previously published data (Accession: PRJNA609175) (<a href="#B51">Vilarino et al., 2020</a>)
The animal study was reviewed and approved by the special committee on scientific research and the academic ethics of Inner Mongolia Agricultural University
ML and LZ wrote the manuscript with help from all the authors
and embryo transfer and chimera experiment
and GY performed cryosectioning and immunofluorescence staining
ZY performed chimeric embryo immunofluorescence staining
and TM performed RT-qPCR and differentiation experiments
YZ performed bioinformatics analysis of this study
SB and YS provided technical support and result analysis
All authors contributed to the article and approved the submitted version
This work was supported by the Inner Mongolia Autonomous Region Science and Technology Plan of China
grant/award numbers: 2019GG241 and 2020ZD0003; the Inner Mongolia Autonomous Region Science and Technology Plan of China
subsidy project after evaluation of excellent Key Laboratory; and the National Natural Science Foundation of China
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations
Any product that may be evaluated in this article
or claim that may be made by its manufacturer
is not guaranteed or endorsed by the publisher
We thank Professor Pentao Liu for providing the plasmid
We also thank Inner Mongolia Saikexing Institute of Breeding and Reproductive for instrument support
The Supplementary Material for this article can be found online at: <a href="https://www.frontiersin.org/articles/10.3389/fcell.2021.785055/full#supplementary-material">https://www.frontiersin.org/articles/10.3389/fcell.2021.785055/full&#x00023;supplementary-material</a>
Supplementary Figure 1 &#x0007c; Reprogramming SFs to DOX-inducible iPSCs
(A) The morphology of primary sheep iPSC colonies
(B) The expression of endogenous OCT4 in stem cells by RT-qPCR
The error bars indicate three independent biological replicates (mean&#x000a0;&#x000b1;&#x000a0;SD); &#x0002a;p&#x000a0;&#x0003c;&#x000a0;0.05
(C) The expression of the eight exogenous reprogramming factors by nucleic acid electrophoresis in siPSC-4 and siPSC-6
Supplementary Figure 2 &#x0007c; Culture conditions and development potential in siPSCs chimeras
(A) The morphology of sheep iPSCs under several culture conditions in the absence of DOX
(B) Relative expression of the core endogenous pluripotency genes OCT4
and SOX2 in the labeled siPSCs and unlabeled siPSCs by RT-qPCR
Cells from passage 11 were used to transfect with tdTomato by Lipofectamine 2000
P19 tdTomato&#x0002b;: cells of 8 passages after transfection
and P23 tdTomato&#x0002b;: cells of 12 passages after transfection
(C) Detection of siPSC-4 tdTomato&#x0002b; by nucleic acid electrophoresis in embryos from chimaeras
The chimera embryos were harvested on day 59 after embryo transfer
1: negative control (sheep fibroblasts); 2: heart; 3: liver; 4: lung; 5: kidney; 6: stomach; 7: intestine; 8: limb; 9: skin; 10: positive control (plasmid of tdTomato); 11: H2O
(D) An analysis of the contribution of siPSCs to chimera embryos (i
The chimera embryos were harvested on day 11 after embryo transfer
i: embryos developed to day 18; ii: embryos developed to day 17 (conjecture); iii: embryo standstills on day 11 (predicted)
(E) Detection of siPSC-4 tdTomato&#x0002b; in post-implantation embryos (E6.5 chimaeras)
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Li X and Cao G (2021) Generation of Sheep Induced Pluripotent Stem Cells With Defined DOX-Inducible Transcription Factors via piggyBac Transposition
Received: 28 September 2021; Accepted: 12 November 2021;Published: 16 December 2021
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Foot-and-mouth disease (FMD) is an economically important contagious disease of livestock mainly cattle
There is limited data available on pathogenesis of foot and mouth disease in goats
the sheep and goats were infected experimentally with a serotype O foot-and-mouth disease virus by different challenge routes
The sheep and goats challenged by coronary band route and coronary band and intra-dermo-lingual route exhibited FMD clinical signs at 2&#x02013;5 days post challenge
Whereas intra-dermo-lingual challenged sheep and goats did not exhibit FMD clinical signs
Live virus could be isolated from blood of infected sheep and goats at 2&#x02013;5 days post challenge
Viral RNA could be detected from blood of infected sheep and goats at 1&#x02013;10 days post challenge
The neutralizing antibody titre was detected at 10 days post challenge and maintained up to 35 days post challenge in all infected sheep and goats
Non structural protein (NSP) antibodies were detected as early as 5&#x02013;10 days post challenge and remain positive up to 35 days post challenge in the infected sheep and goats
the pathogenesis of sheep and goats with serotype O foot and mouth disease virus by different challenge routes could be demonstrated
there is no detailed account of the pathogenesis of the disease in these small ruminants
This preliminary report describes pathogenesis of sheep and goats experimentally infected with type O foot and mouth disease virus using different challenge routes
Baby Hamster kidney (BHK) and primary bovine thyroid (BTY) cells were provided by the tissue culture laboratory at Research and Development Centre
BTY cells were grown using Hely cell growth medium supplemented with 10% adult bovine serum and antibiotics cocktail (penicillin
O/IND/R2/75 virus was received from the virus seed laboratory
Challenge virus O/IND/R2/75 was prepared and titrated by standard methods as described previously (<a href="#B15">15</a>)
One sheep and goat each were inoculated with O/IND/R2/75 cattle challenge virus by intra-dermo-lingual
coronary band and by both sites in 0.1 ml quantity in each site
The animals were monitored for 24&#x02013;72 h for signs of FMD (passage 1)
epithelial tissue collected from vesicles was triturated in 0.04 M phosphate buffer followed by centrifugation at 3000 xg
The clear supernatant was used to inoculate one sheep and goat each by intra-dermo-lingual
coronary band and by both sites in 0.1 ml quantity in each site respectively
The animals were monitored for 24&#x02013;72 h for signs of FMD
Two sheep and two goats was included as unchallenged control and maintained throughout the study period
Experiments were conducted in a bio-secure animal isolation unit at IIL
The studies involving animals were reviewed and approved by Institutional Animal Ethics Committee
Hyderabad and Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA)
Department of Animal Husbandry and Dairying
The sheep and goats were observed for clinical signs of disease and temperatures recorded daily. A subjective scoring system (<a href="#B16">16</a>) was used to evaluate the progression of disease in these animals with slight modification (<a href="#B8">8</a>)
Clotted blood for serology and NSP antibody was collected at days 0, 5, 10, 15, 21, 28, and 35 post-challenge. Heparinized blood was collected daily up to 10 dpc. Heparinized blood (200 &#x003BC;l) was mixed with 300 &#x003BC;l of lysis buffer (Roche Diagnostics, Germany) for analysis by real-time RT-PCR (qRT-PCR) and stored at &#x02212;70&#x000B0;C. Heparinized blood (1 ml) was used for virus isolation (VI) (<a href="#B15">15</a>)
Virus neutralization tests were performed for the sera in flatbottomed tissue culture grade micro titre plates (Nunclon&#x02122;, Denmark) as described previously (<a href="#B17">17</a>). Antibody titres were expressed as the reciprocal of the final dilution of serum in the serum/virus mixture which neutralized an estimated 100 TCID50 of virus at the 50% end-point (<a href="#B18">18</a>)
Antibodies to FMDV NSP 3ABC were tested using PrioCHECK&#x000AE;FMDV NS kit (Prionics Lelystad B.V., The Netherlands) (<a href="#B19">19</a>)
(A) Rectal temperature (&#x000B0;C) of challenged (intra-dermo-lingual (IDL)
coronary band (C) and by both route) and control sheep
Solid line indicates normal temperature of sheep
Rectal temperature (&#x000B0;C) of challenged (intra-dermo-lingual (IDL)
coronary band (C) and by both route) and control goats
Solid line indicates normal temperature of goats
(C) Virus neutralization titres of challenged and control sheep (Expressed as the log10 reciprocal antibody dilution required for 50% neutralization of 100 tissue culture infectious units)
Solid line indicates neutralizing antibody titre &#x0003E;1.2 log10SN50 is considered positive
(D) Virus neutralization titres of challenged and control goats (Expressed as the log10 reciprocal antibody dilution required for 50% neutralization of 100 tissue culture infectious units)
Lesion score of challenged and control sheep and goats
virus could not be isolated form challenged sheep and goats (S163
and G433) irrespective of challenge routes
infectious virus was isolated form intra-dermal-lingual route challenged sheep (S168) and goat (G31) at 3 dpc and coronary band challenged sheep (S164) and goat (G41) at 2&#x02013;5 dpc
Whereas in intra-dermal-lingual and coronary band route challenged sheep (S243) was positive for virus isolation at 4&#x02013;5 dpc
In the case of intra-dermal-lingual and coronary band route challenged goat (G65) virus was isolated on 2 and 5 dpc
Virus isolation and quantification of FMD viral RNA copy numbers (Log10 RNA copy numbers/ ml of blood) from blood of challenged and control sheep and goats
Four inoculated sheep (S163, S119, S108 and S243) were positive for NSP antibody on 10 dpc while other sheep NSP antibodies were observed on 15&#x02013;35 dpc. Three inoculated goats (G12, G31 and G433) were positive for NSP antibody on 5 dpc while in other goats NSP antibodies were observed on 15&#x02013;35 dpc. Both the unchallenged control sheep and goats were negative for NSP antibody up to 35 dpc (<a href="#T3">Table 3</a>)
FMDV NSP antibody responses of challenged and control sheep and goats
The neutralizing antibody titer was detected in all inoculated sheep and goats at 10 dpc (&#x0003E; 1.2 log10SN50). However, the highest neutralizing antibody titer was detected between 10 and 35 dpc (2.1 log10SN50) in sheep and goats inoculated by coronary and both by coronary and intra-dermo- lingual route. Both the unchallenged control sheep and goats had no serum neutralizing antibody titre up to 35 dpc (<a href="#F1">Figures 1C,D</a>)
The experiment described the preliminary results on pathogenesis and development of FMD in sheep and goats by inoculating the type O FMD virus in three different challenged routes
The development of clinical signs was observed
The viral RNA levels in blood were quantified
The incubation period of natural FMDV infection is normally between 3 and 8 days in sheep (<a href="#B23">23</a>), but can be as short as 24 h following experimental infection (<a href="#B23">23</a>, <a href="#B24">24</a>)
lesions were evident in three sheep and four goats on the 2nd day of challenge
type O virus and Indian breed of sheep and goats were used
This may be the reason for intra-dermo-lingual challenged sheep and goats did not show the clinical signs of FMD
Intra-dermo-lingual route of inoculation in cattle, dental pad/gum route of inoculation in buffalo (<a href="#B32">32</a>) and intra dermal inoculation in the heel bulb in pigs (<a href="#B33">33</a>) of FMDV resulted in generalized disease
Ryan et al. (<a href="#B27">27</a>) reported that all inoculated ewes developed viraemia at 1 dpi and viral RNA levels then peaked at 2 dpi. In the current study, viral RNA was detected as early as 1dpc, viral RNA level then peaked at 2&#x02013;5 dpc from the inoculated sheep and goats. Virus was isolated from blood of inoculated sheep and goats up to 2&#x02013;5 dpc as reported by Parida et al. (<a href="#B34">34</a>)
In the present study, neutralizing antibody titre was detected in all the inoculated sheep and goats at 10 dpc and the peak antibody titre was detected between 10 and 35 dpc. Dellers et al. (<a href="#B25">25</a>) reported neutralizing antibodies were first detected 60 h post inoculation and initial peak titers occurred by 10th day in inoculated sheep
In this study statistical analysis could not be carried out due to the small number of animals in each group (n = 2)
In coronary band and Intra-dermo-lingual challenge group of animals received double the dose of challenge virus (0.2 ml) against the Intra-dermo-lingual challenge group (0.1 ml) and coronary band challenge group (0.1 ml)
further study with increased number of animals and statistical analysis is warranted to confirm this result
The sheep and goats were infected experimentally with a serotype O foot-and-mouth disease virus by different challenge routes
NSP antibodies were detected as early as 5&#x02013;10 days post challenge and remain positive up to 35 days post challenge in the infected sheep and goats
All datasets presented in this study are included in the article/supplementary material
completed analysis and wrote the manuscript
and SV were employed by the company Indian Immunologicals Limited
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Response of sheep to experimental infection with foot-and-mouth disease virus
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Singanallur Balasubramanian N and Villuppanoor Alwar S (2020) Experimental Infection of Foot and Mouth Disease in Indian Sheep and Goats
Received: 06 April 2020; Accepted: 22 May 2020; Published: 25 June 2020
Copyright &#x000A9; 2020 Muthukrishnan, Singanallur Balasubramanian and Villuppanoor Alwar. This is an open-access article distributed under the terms of the <a rel="license" href="http://creativecommons.org/licenses/by/4.0/" target="_blank">Creative Commons Attribution License (CC BY)</a>
&#x0002A;Correspondence: Madhanmohan Muthukrishnan, <a id="encmail">bXV0aHVrcmlzaG5hbi5tYWRoYW5tb2hhbkBnbWFpbC5jb20=</a>
&#x02020;Present address: Nagendrakumar Singanallur Balasubramanian
Australian Centre for Disease Preparedness
Veterinary University Training and Diagnostic Centre
Tamil Nadu Veterinary and Animal Sciences University
We searched Facebook for the same Bible verse and found a seemingly endless wall of similar image posts that had no labels covering them
On Nov. 16, 2022, Facebook user Lib Grimmett <a href="https://www.facebook.com/lib.grimmett/posts/pfbid029K74sEZ4puBmoCabZtpfvJ8ADqBVhtoYL8NjbiRxRnQ1QYsH8GrjE2QYy7im9hpsl" target="_blank">posted</a> an image with words from a popular verse often shared by people of the Christian faith
and know that I am God," read the Bible verse from the book of Psalms
Grimmett's post was displayed with a label that said
This photo may show violent or graphic content." The label remained visible as of Nov
Another user attempted to post the image as well, <a href="https://www.facebook.com/kimberly.sinnett.0525/posts/pfbid0En2xfqLJECSKffaediAbHRA8HKMidfpUhA8iJLnJPbwSk3u3nfYQsD9ugx2nro7Ml" target="_blank">saying</a>
"Facebook has censored this Bible verse that says
'Be still and Know that I am God.' I am seriously considering deleting my account and figuring out another way to stay in touch with you all."
A "Learn More" button on the label added the context from Facebook and Meta
This photo doesn't go against our Community Standards
We use either technology or a review team to identify content that should be covered
We cover graphic content so people can choose whether to see it
The label disappeared from the posts shortly after we reached out to Facebook by email
It's likely that the label was human error or simply the wrong decision by the detection "technology" that the "Learn More" message mentioned. We note, for example, that a search of Facebook for the same Bible verse showed <a href="https://www.facebook.com/search/photos/?q=%22Be%20still%2C%20and%20know%20that%20I%20am%20God.%22" target="_blank">a massive wall of posts</a> that had no similar "sensitive content" label added over them
We sometimes receive reader inquiries asking if Facebook "censors" various kinds of posts
such as those with political or religious themes
the image showed a "sensitive content" label
this appeared to be an anomaly when compared to other posts of the same content
"Psalm 46:10 - New International Version." Bible Gateway
https://www.biblegateway.com/passage/?search=Psalm%2046%3A10&version=NIV
2022: This story was updated to note that the label disappeared from the image posts shortly after we reached out to Facebook for comment
Jordan Liles is a Senior Reporter who has been with Snopes since 2016
This material may not be reproduced without permission
Snopes and the Snopes.com logo are registered service marks of Snopes.com
FILE PHOTO: Livestock carrier Bahijah berthed at North Quay
in the inner harbour of the Port of Fremantle
The Australian government said on Monday it had refused a request by a livestock exporter to send a ship carrying around 14,000 sheep and 1,500 cattle on a month-long voyage around Africa to Israel
The animals have been on board the vessel for a month
prompting outcry from animal rights advocates who have likened their treatment to torture
The MV Bahijah sailed from Australia for Israel on Jan
5 but abandoned a passage through the Red Sea due to threat of attack by Yemen&#39;s Houthi militia and was ordered home by the Australian government
The ship has been waiting off Western Australia for a week for the government to decide if it can head back to sea
Several hundred cattle were offloaded in recent days but Australia&#39;s biosecurity rules mean any animals that disembark must be quarantined
The agriculture ministry said it was not satisfied that the exporter&#39;s application met Australian or Israeli regulatory requirements or that the animals&#39; transportation would ensure their health and welfare
It did not give further details on the decision but said the animals were still in good health
&quot;The next steps for the livestock onboard the vessel are commercial decisions for the exporter to make,&quot; it said
&quot;The department (ministry) stands ready to assess any future application.&quot;
Australia is a major exporter of live animals
shipping more than half a million sheep and half a million cattle last year
The government plans to ban live sheep exports in the coming years
Another livestock vessel carrying around 60,000 animals left Australia last week for the Jordanian Red Sea port of Aqaba
Reuters was unable to contact Bassem Dabbah
the exporter of the animals on the Bahijah
The Israeli military said it carried out airstrikes against Yemen&#39;s Hodeidah Port on Monday
An international NGO that intends to deliver humanitarian aid to Gaza by sea said on Sunday it was in talks with Malta&#39;s… 
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is associated to the existence of prion strains
which are different pathogenic prion protein (PrPSc) conformations with distinct pathobiological properties
To faithfully study scrapie strain variability in natural sheep isolates
transgenic mice expressing sheep cellular prion protein (PrPC) are used
we used two of such models to bioassay 20 scrapie isolates from the Spain-France-Andorra transboundary territory
Animals were intracerebrally inoculated and survival periods
proteinase K-resistant PrP (PrPres) banding patterns
lesion profiles and PrPSc distribution were studied
Inocula showed a remarkable homogeneity on banding patterns
a number of isolates caused accumulation of 21-kDa PrPres in TgShp XI
A different subgroup of isolates caused long survival periods and presence of 21-kDa PrPres in Tg338 mice
It seemed that one major 19-kDa prion isoform and two distinct 21-kDa variants coexisted in source inocula
and that they could be separated by bioassay in each transgenic model
The reason why each model favours a specific component of the mixture is unknown
although PrPC expression level may play a role
Our results indicate that coinfection with more than one substrain is more frequent than infection with a single component
This progressive degeneration of the central nervous system manifests as a set of neurological signs appearing after long incubation periods
and thus only a number of prion strains will induce its misfolding while others will not be able to template its conversion into a disease-associated conformation
the degree of overlapping between the Prnp gene sequence of host and donor influences the capability of an isolate to transmit the disease
which provides a molecular explanation for the transmission barrier phenomenon
studying scrapie strain variability by means of bioassay in a model expressing a non-ovine PrPC may alter the original portfolio of prion variants to the point that it keeps little if any resemblance with the original sheep scrapie strain range
its economic and sanitary impact is meaningful
Characterizing and holding control of the variety of enzootic scrapie strains present in the small ruminant population through the Spain-France-Andorra transboundary territories is crucial for scrapie control and eradication purposes and for public health
Both nervous (medulla oblongata) and lymphoid tissues (mesenteric lymph node) of sheep were subjected to biochemical analyses prior to bioassay
In contrast, medulla oblongata of sheep #8 and #10 were negative on Western blot (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-020-61977-1#Fig1">1A</a>)
Since this is likely to be associated with reduced infective titres
Survival periods and attack rates of first and second-passage TgShp XI and Tg338 mice and banding patterns of second-passage spinal cord pools are presented in Table <a data-track="click" data-track-label="link" data-track-action="table anchor" href="/articles/s41598-020-61977-1#Tab2">2</a>
Banding patterns on first passage were coherent with those of second passage
This suggests the presence of minor quantities of a 21-kDa prion conformer
that is preferentially amplified by the TgShp XI line
Long survival periods on first passage reflect low infectivity titres in lymph node-derived isolates
while on second passage remarkable reductions were observed
Five of these inocula (1 L, 3 L, 7 L, 9 L and 10 L) transmitted to Tg338 with second-passage survival periods ranging from 163 to 249 dpi. Presence of 19-kDa PrPres was noted in spinal cords of both passages (Fig. <a data-track="click" data-track-label="link" data-track-action="figure anchor" href="/articles/s41598-020-61977-1#Fig3">3B</a>)
The characteristics of these isolates in Tg338 agreed with those of brain-sourced isolates
suggesting that they contain the same type of agent
which provides further evidence for the propagation of a different conformer in this mice
According to transmission patterns and biochemical signatures of the accumulated PrPres
the presence of three PrPSc variants can be envisioned: (i) a 19-kDa variant
present as the major component of the majority of sheep-sourced inocula
which we termed “19K”; (ii) a 21-kDa conformer that appears as the major isoform in inoculum 7 L and as a minor component in other three isolates
and that causes disease in TgShp XI with transmission patterns similar to the 19-kDa variant
hereafter termed “21K-TgShp XI”; and (iii) a 21-kDa conformer
that seems to block the propagation of the 19-kDa major component exclusively in Tg338 mice
triggering reduced PrPSc accumulation in spinal cord and protracting clinical disease
The characteristics of spongiform change in both transgenic models were fairly similar
ventral mesencephalon and zona incerta and ventrolateral nuclei of diencephalon showed invariably the most severe spongiosis
vacuoles in cerebellar cortex were usually absent or very scarce
Cortex of superior colliculus and central regions of thalamus suffered a milder vacuolization
the degree of spongiosis was highly variable among infected mice groups
frontal and temporo-parietal cortices and hippocampal formation were poorly affected
Lesion profiles were drawn for each inoculum and murine line
curves fitted to a general profile characterized by high scores at brainstem and subcortical structures
moderate scores in cerebral cortices and hippocampus
a number of isolates triggered lesion profiles with significantly lower vacuolization severity in all areas
</a>Lesion profiles of second-passage TgShp XI mice
Both inocula associated to the 19K phenotype (A) and inocula associated to the 21K-TgShp XI phenotype (B) provoked similarly shaped lesion profiles in TgShp XI mice (C)
most of them characterized by peak scores at mesencephalon and thalamus
The brains of all mice dying after the onset of clinical signs in each challenged group were analyzed to plot lesions profiles (usually 6 and never less than 3 animals per group)
</a>Lesion profiles of second-passage Tg338 mice
Inocula associated to the 19K phenotype provoked similar lesion profiles in Tg338 mice
mostly characterized by peaks at medulla oblongata and thalamus (A)
isolates linked to the 21K-Tg338 phenotype triggered lower and flatter profiles (B,C)
</a>PrPSc distribution in representative brain sections from TgShp XI mice infected with inocula 2 N (A)
Two different trends of PrPSc accumulation were observed on PET-blots: a generalized low-intensity immunostaining (A,B,C)
usually associated with the alveus of hippocampus (B
and which correlated with coarse/coalescing plaque-like deposits on immunostochemistry (E)
</a>PrPSc distribution in representative brain sections from Tg338 mice infected with inocula 1 L (A)
A general pattern arose in all examined brain samples consisting of intense staining of brainstem and subcortical areas
conspicuous immunostaining was observed in specific structures
including the cingulate gyrus (A,B,E; thick orange arrows)
and the stratum lacunosum-moleculare and the alveus of hippocampus (B,E; thin green arrows)
High-intensity staining of the medial amygdaloid nuclei (D,E; blue triangles) was observed in a subset of samples obtained from animals challenged with isolates causing prolonged survival periods and low vacuolization scores
which were associated to long survival periods and low average spongiosis
florid (D to F) and amorphous perivascular plaques (G to L) in brain of second-passage Tg338 mice infected with inocula 6 L and 8 L
immunohistochemistry with anti-PrP mAb SAF84 (B,E,H,K) and Congo red staining that proves the amyloid nature of the plaques (C,F,I,L)
PrPres in isolates prepared from mesenteric lymph nodes reproduced the same pattern in almost all cases; the only exception was isolate 7 L
whose NG band had a molecular weight of 21 kDa
most isolates showed survival periods always shorter than 330 dpi on second passage
6 L and 8 L) triggered very protracted survival periods (longer than 481 dpi) and absence of evident reductions on second passage in the Tg388 line
Regardless of whether this difference was caused by reduced accumulation of infectivity in spinal cord of first-passage mice or because they have intrinsically longer survival periods
it suggests the existence of a different strain in this group of samples
non-equivalent results were found between TgShp XI and Tg338 lines
TgShp XI mice accumulated high (21-kDa) PrPres in spinal cord after the inoculation of isolates 3 N
although only one of these (inoculum 7 L) displayed this high-molecular weight banding pattern
The prion variant propagated in these cases has been termed “21K-TgShp XI” and did not correlate with other differential phenotypic features such as lesion profiles
whose properties resemble those of Italian scrapie (O
demonstrated zoonotic potential as it was able to infect transgenic mice expressing human PrPC43
Studies are underway to determine if the coincidence of phenotypic traits is enough to endorse our inocula with zoonotic capability
Whether such a mechanism may participate in the divergence observed in our study needs to be evaluated
A possibility is that the ability of each transgenic line to amplify distinct prion conformers is due to their different rates of PrPC expression (4–8-fold vs
differences in the sequence of the PrPC they bear
our results indicate that in natural scrapie cases
sheep can be infected with more than one strain
These distinct variants of the agent can be present in different organs (we assessed nervous and lymphoid tissues)
or even coexist in the nervous system as prion mixtures with a dominant conformer and one or more subdominant isoforms
which can be detected by bioassay in sensitive rodent models or other in vitro techniques
the coinfection seems to be more frequent that the infection with a single strain
All these results are in agreement with ours and drag the risk of sheep getting infected with multiple scrapie strains into the spotlight
which raises doubts about the current classification of human prion strains and the actual responsible of each type of clinical profile
Characterization and monitoring of prion strains in small ruminant populations in Europe is crucial for control and eradication purposes
Shedding light on the actual variability of scrapie prions and its implications for inter-species transmission and
on the zoonotic potential of scrapie field isolates is necessary to update communitarian public health policies and to prevent a putative reemergence of prion diseases as a public hazard
Animals were sacrificed by intravenous injection of sodium pentobarbital followed by necropsy and systematic sampling
Samples from brain and mesenteric lymph nodes were divided into two halves; one half was fixed in a solution of 10% formalin for further histological studies
while the other was immediately immerged in liquid nitrogen and later conserved at −80 °C for biochemical analyses and the preparation of inocula
the presence of PrPSc was confirmed for both the terminal and the preclinical/early clinical group in both nervous and lymphoid tissues using immunohistochemistry with monoclonal anti-PrP antibody L42 (R-Biopharm)
whose epitope spans amino acids 145–163 of ovine PrP
the Prnp genotype was determined through sequencing of genomic DNA obtained from whole blood samples
First-passage inocula were prepared from nervous (N) and lymphoid tissues (L) of the aforementioned sheep
Inocula 1 N to 10 N corresponded to inocula prepared from medulla oblongata (obex)
while inocula 1 L to 10 L were those prepared from mesenteric lymph nodes of the animals
Inocula 1 N to 6 N and 1 L to 6 L derived from tissues of terminal sheep
while inocula 7 N to 10 N and 7 L to 10 L sourced from preclinical / early clinical sheep
Second-passage inocula were prepared from pools of spinal cords harvested from diseased first-passage mice
All inocula consisted of 10% (w/v) tissue homogenates in physiological saline and were subjected to microbiological analysis to ensure sterility prior to intracerebral inoculation
Germany) and were brought to our facilities to be inoculated after the appropriate adaptation period
The level of expression of this line is approximately 8-fold that of sheep brain
France) and was maintained and bred in our facilities
All experimental procedures in this study were approved by the Ethics Committee for Animal Testing of the University of Zaragoza (permit number PI19/14) and performed in accordance with the recommendations for the care and use of experimental animals and in agreement with Spanish law (RD 1201/05)
A dose of 20 µl/animal of each inoculum was administered by the intracerebral route to groups of six mice using a precision syringe and under general anaesthesia
Animals were provided adequate analgesia after the procedure
consisting of buprenorphine at a dose of 0.01 mg/kg bodyweight by the subcutaneous route
Animals were monitored three times per week for clinical signs of prion disease
animals were sacrificed by cervical dislocation under heavy anaesthesia
Brain and spinal cord were harvested and stored in a 10% formalin solution and at −80 °C for histopathological and biochemical analyses
Western blotting was performed following a protocol based on TeSeE Western Blot kit (Bio-Rad)
medulla oblongata and mesenteric lymph node samples from sheep and spinal cord pools from diseased mice were thawed and homogenised in a detergent-containing solution
A volume of 200 µl was submitted to proteinase K digestion for 10 min
which was stopped using a β-mercaptoethanol-containing stop buffer
clarification and resuspension of the remaining PrPres in 30 µl of Laemli loading buffer
It was then subjected to SDS-PAGE electrophoresis using commercial 12% Bis-Tris gels (Bio-Rad)
followed by transference to a PVDF membrane with 0.20-µm pore diameter (Bio-Rad)
Immunoblot was performed by sequentially immerging the membrane in a blocking solution (0.2% BSA in PBS+Tween 0.1%)
primary anti-PrP antibody Sha31 (whose epitope spans amino acids 148–155) diluted 1:8,000 in PBS+Tween 0.1%
and HRP-conjugated secondary antibody diluted 1:5,000 in PBS+Tween 0.1%
membranes were developed by incubation with a luminol-based substrate (SuperSignal West Pico Chemiluminescent Substrate)
Tissues were then embedded in paraffin wax and mounted in histological cassettes
Four µm-thick sections were obtained using a microtome and mounted on glass slides for subsequent histological procedures
Haematoxylin and eosin (H&amp;E) staining of the sections was performed following a standard protocol
dewaxing and rehydration was performed by sequentially passaging the preparations in xylene and graded alcohols
followed by incubation in a haematoxylin solution
preparations were subjected to incubation in acid alcohol (1% acetic acid in a 70% ethanol solution)
followed by immersion in an eosin solution
preparations were dehydrated and mounted prior to visualization under light microscope
Immunohistochemistry was applied to brain and lymph node samples from sheep and brain sections from mice
three different pre-treatments for antigen retrieval were performed sequentially: immersion in 98% formic acid for 15 min
treatment with 4 µg/ml proteinase K for 15 min at 37 °C
and hydrated autoclaving in citrate buffer at 96 °C for 20 min
endogen peroxidase activity was blocked using a commercial blocking solution
followed by 1-hour incubation with primary anti-PrP antibody L42 (1:500
R.Biopharm) for sheep tissues or 6H4 (epitope aa 147–155
The EnVision+ System (Agilent Dako) was used as the secondary antibody
based on the use of 3,3’-diaminobenzidine (DAB) as chromogen
PET-blot was performed as described elsewhere<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 77" title="Schulz-Schaeffer, W. J. et al. The paraffin-embedded tissue blot detects PrP(Sc) early in the incubation time in prion diseases. Am. J. Pathol. 156, 51–56 (2000)." href="/articles/s41598-020-61977-1#ref-CR77" id="ref-link-section-d414727225e3879">77</a> on mice brain samples
4-µm paraffin-embedded brain sections were collected onto a nitrocellulose membrane and dried at 37 °C for 24 hours
Membranes were then subjected to dewaxing and rehydration and incubated for 2 hours in a solution of proteinase K (250 µg/ml) at 56 °C to completely digest PrPC
Denaturation of the remaining PrPres was achieved by incubating the membranes in a solution of guanidine thiocyanate 3 M
After blocking the membrane with 0.2% BSA to avoid cross-reactivity
detection was carried out through sequential incubation with the anti-PrP antibody Sha31 (1:8,000
SPI-Bio) and a secondary alkaline phosphatase (AP)-conjugated antibody (1:500
followed by development with NBT/BCIP (Thermo Scientific)
Membranes were then washed and dried for 24 hours at room temperature
The areas were: (1) dorsal nuclei of medulla oblongata
(3) superior colliculus of the mesencephalon
(8) cerebral cortex at the level of the thalamus and (9) frontal cortex
Semiquantitative scores from 0 (absence of vacuolization or PrPSc accumulation) to 5 (very abundant and confluent vacuoles or PrPSc deposits) were given to each area
Mean values at each area were plotted to trace the curves for each inoculum
Visual comparison was performed between curves corresponding to each inocula
as well as between lesion profiles and or PrPSc distribution curves
the statistical correlation between both parameters was computed through the Spearman’s correlation coefficient for each transgenic line
Novel proteinaceous infectious particles cause scrapie
A cellular gene encodes scrapie PrP 27-30 protein
Scrapie prion protein contains a phosphatidylinositol glycolipid
Neuropathology of scrapie: a study of the distribution patterns of brain lesions in 222 cases of natural scrapie in sheep
Distinction of scrapie phenotypes in sheep by lesion profiling
Astrocyte gene expression in experimental mouse scrapie
Microglia and the pathogenesis of spongiform encephalopathies
Differences in the activation of the GFAP gene promoter by prion and viral infections
Neuropathological diagnostic criteria for Creutzfeldt-Jakob disease (CJD) and other human spongiform encephalopathies (prion diseases)
Scrapie produced experimentally in goats with special reference to the clinical syndrome
Further observations on the experimental transmission of scrapie from sheep and goats to laboratory mice
The sequential development of the brain lesion of scrapie in three strains of mice
Host-genotype and agent effects in scrapie incubation: change in allelic interaction with different strains of agent
Agent-strain differences in the distribution and intensity of grey matter vacuolation
Effects of agent strain and host genotype on PrP accumulation in the brain of sheep naturally and experimentally affected with scrapie
Distinct profiles of PrP(d) immunoreactivity in the brain of scrapie- and BSE-infected sheep: implications for differential cell targeting and PrP processing
Molecular and transmission characteristics of primary-passaged ovine scrapie isolates in conventional and ovine PrP transgenic mice
Emergence of multiple prion strains from single isolates of ovine scrapie
Identification of two biologically distinct strains of transmissible mink encephalopathy in hamsters
Distinct PrP properties suggest the molecular basis of strain variation in transmissible mink encephalopathy
Evidence for the conformation of the pathologic isoform of the prion protein enciphering and propagating prion diversity
A general model of prion strains and their pathogenicity
Strain characterization of natural sheep scrapie and comparison with BSE
Propagation of ovine prions from “poor” transmitter scrapie isolates in ovine PrP transgenic mice
A newly identified type of scrapie agent can naturally infect sheep with resistant PrP genotypes
Atypical/Nor98 scrapie: properties of the agent
Prion protein and species barriers in the transmissible spongiform encephalopathies
Darwinian evolution of prions in cell culture
Markedly increased susceptibility to natural sheep scrapie of transgenic mice expressing ovine prp
Scrapie strain transmission studies in ovine PrP transgenic mice reveal dissimilar susceptibility
Classic scrapie in sheep with the ARR/ARR prion genotype in Germany and France
Characterization of atypical scrapie cases from Great Britain in transgenic ovine PrP mice
Amino acid sequence and prion strain specific effects on the in vitro and in vivo convertibility of ovine/murine and bovine/murine prion protein chimeras
New in vivo and ex vivo models for the experimental study of sheep scrapie: development and perspectives
A descriptive study of the prevalence of atypical and classical scrapie in sheep in 20 European countries
The prevalence of atypical scrapie in sheep from positive flocks is not higher than in the general sheep population in 11 European countries
Molecular analysis of ovine prion protein identifies similarities between BSE and an experimental isolate of natural scrapie
Molecular behaviors of “CH1641-like” sheep scrapie isolates in ovine transgenic mice (TgOvPrP4)
Histopathological studies of “CH1641-like” scrapie sources versus classical scrapie and BSE transmitted to ovine transgenic mice (TgOvPrP4)
Evidence for zoonotic potential of ovine scrapie prions
Molecular screening of sheep for bovine spongiform encephalopathy
In vitro amplification of ovine prions from scrapie-infected sheep from Great Britain reveals distinct patterns of propagation
Cell-associated variants of disease-specific prion protein immunolabelling are found in different sources of sheep transmissible spongiform encephalopathy
Ovine infection with the agents of scrapie (CH1641 isolate) and bovine spongiform encephalopathy: immunochemical similarities can be resolved by immunohistochemistry
Evidence for co-infection of ovine prion strains in classical scrapie isolates
Epidemic of transmissible spongiform encephalopathy in sheep and goats in Italy
Evidence for the transmission of scrapie to sheep and goats from a vaccine against Mycoplasma agalactiae
Molecular analysis of iatrogenic scrapie in Italy
Isolation from cattle of a prion strain distinct from that causing bovine spongiform encephalopathy
Stability of murine scrapie strain 87V after passage in sheep and comparison with the CH1641 ovine strain
Phenotype shift from atypical scrapie to CH1641 following experimental transmission in sheep
Divergent prion strain evolution driven by PrP(C) expression level in transgenic mice
Species-barrier-independent prion replication in apparently resistant species
Anchorless prion protein results in infectious amyloid disease without clinical scrapie
Accumulation of prion protein in the brain that is not associated with transmissible disease
Host PrP glycosylation: a major factor determining the outcome of prion infection
Early accumulation of PrP(Sc) in gut-associated lymphoid and nervous tissues of susceptible sheep from a Romanov flock with natural scrapie
Distribution of prion protein in the ileal Peyer’s patch of scrapie-free lambs and lambs naturally and experimentally exposed to the scrapie agent
Facilitated cross-species transmission of prions in extraneural tissue
Prominent and persistent extraneural infection in human PrP transgenic mice infected with variant CJD
Sialylation of prion protein controls the rate of prion amplification
the ratio of PrPSc glycoform and prion infectivity
Use of murine bioassay to resolve ovine transmissible spongiform encephalopathy cases showing a bovine spongiform encephalopathy molecular profile
Selection of distinct strain phenotypes in mice infected by ovine natural scrapie isolates similar to CH1641 experimental scrapie
Intraspecies prion transmission results in selection of sheep scrapie strains
Coexistence of multiple PrPSc types in individuals with Creutzfeldt-Jakob disease
Detection of type 1 prion protein in variant Creutzfeldt-Jakob disease
An assessment of the efficiency of PrPsc detection in rectal mucosa and third-eyelid biopsies from animals infected with scrapie
The paraffin-embedded tissue blot detects PrP(Sc) early in the incubation time in prion diseases
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This work was supported by grants from the “Ministerio de Educación
Cultura y Deporte” (FPU 14/04348) and the “Ministerio de Economía y Competitividad” of the Spanish Government (AGL2015–65560-R)
and from the Spain-France-Andorra Cooperation Program (POCTEFA)
co-funded by the European Regional Development Fund (ERDF) (EFA 148/16 REDPRION)
Centro de Encefalopatías y Enfermedades Transmisibles Emergentes
Instituto Agroalimentario de Aragón - IA2 (Universidad de Zaragoza - CITA)
Centre de Recerca en Sanitat Animal (CReSA)
UMR Virologie Immunologie Moléculaires (VIM-UR892)
Institute of Novel and Emerging Infectious Diseases
reviewed the results and the discussion and commented on the final manuscript
The authors declare no competing interests
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations
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by  <a href="/authors/jeffrey-parkin">Jeffrey Parkin</a>
LinkImage: Massive Monster/Devolver Digital via Polygon<a class="_1ec3ypo6" href="/authors/jeffrey-parkin">Jeffrey Parkin</a> (he/him) has been writing video game guides for Polygon for almost seven years. He has learned to love just about every genre of game that exists.There’s a lighthouse in <a href="/guides/23296988/cult-of-the-lamb-beginners-guide-tips-tricks-followers-crusade-farm-difficulty">Cult of the Lamb</a>’s Pilgrim’s Passage
Once you fuel up the lighthouse with wood to complete the Light in the Darkness quest
The cultists in the lighthouse will ask you for 25 Crystals
we’ll explain where to find those Crystals
Each of Cult of the Lamb’s four regions give you a different resource
Darkwood is where you’ll gather Camellia flowers to heal your cultists
Anura gets you Menticide Mushroom Spores to brainwash them
and Silk Cradle is where you’ll find Spider Silk
On the far left side of the world selection screen, you’ll find the door to Anchordeep — the bishop Kallamar’s area. To get the door opened, you’ll need <a href="/guides/23296990/cult-of-the-lamb-follower-hunger-sickness-faith-farm-cooking-heal-sermon">at least 9 followers</a>.
Once you’ve hit that threshold, just approach the door to unlock it.
With the door to Anchordeep open, you just need to crusade for a bit. You’ll pick up Crystals like any other loot — especially from chests when you clear a room, but also from just destroying the environment.
Once you have 25 Crystals, head back to the lighthouse is Pilgrim’s Passage to receive a new follower form that looks like the head of the lighthouse cult (an axolotl, I think).
The best of Polygon in your inbox, every Friday.
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Viral interference is a common occurrence that has been reported in cell culture in many cases
viral interference between two capripox viruses (sheeppox SPPV and lumpy skin disease virus LSDV in cattle) with Rift Valley fever virus (RVFV) was investigated in vitro and in their natural hosts
A combination of SPPV/RVFV and LSDV/RVFV was used to co-infect susceptible cells and animals to detect potential competition
In-vitro interference was evaluated by estimating viral infectivity and copies of viral RNA by a qPCR during three serial passages in cell cultures
whereas in-vivo interference was assessed through antibody responses to vaccination
When lamb testis primary cells were infected with the mixture of capripox and RVFV
the replication of both SPPV and LSDV was inhibited by RVFV
SPPV/RVFV or LSDV/RVFV combinations inhibited the replication SPPV and LSDV and the antibody response following vaccination
The combined SPPV/RVFV did not protect sheep after challenging with the virulent strain of SPPV and the LSDV/RVFV did not induce interferon Gamma to LSDV
while immunological response to RVFV remain unaffected
Our goal was to assess this interference response to RVFV/capripoxviruses’ coinfection in order to develop effective combined live-attenuated vaccines as a control strategy for RVF and SPP/LSD diseases
Our findings indicated that this approach was not suitable for developing a combined SPPV/LSDV/RVFV vaccine candidate because of interference of replication and the immune response among these viruses
Combining multiple antigens has produced safe and efficacious vaccines
interference among antigens can be an obstacle for the development of such combined vaccines
Combining the RVFV vaccine with other routinely used vaccines such as LSD of cattle or SGP of small ruminant may ensure regular vaccination and build herd immunity against RVFV infection for the new epidemics
we combined SPPV/RVFV and LSDV/RVFV to perform coinfection of susceptible cells and animals
to investigate the potential for interference of the replication of these viruses
In-vitro interference was evaluated based on estimates of infectivity determined the co-titration of SPPV/RVFV and LSDV/RVFV after three passages in lamb testis cell cultures and in-vivo interference was assessed based on the immunological response after co-injection of sheep and cattle with combination of these viruses
Vero cells (African green monkey kidney) were used for growth and titration of RVFV
Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) for growth and 1% FBS was used with the medium for preparing virus to inoculate the animals
The susceptibility of LT and Vero cells to SPPV and RVFV was compared by infection using multiplicity of infection (MOI) 0.001
then titrated after harvest based on the development of generalized cytopathic effect (CPE)
Coinfection was then performed by inoculating mixtures of SPPV/RVFV and LSDV/RVFV onto LT cells as following: for the first passage (P1)
growth medium was removed and cells propagated in 25 cm2 flask were inoculated with LSDV or SPPV at fixed MOI then adsorbed for 45 min
RVFV was inoculated onto cells at different MOI with media and without adsorption
The cell and virus suspensions were thawed at room temperature and the virus suspension was used for the next passage
infection of cells was performed with the P1 harvest without MOI calculation
The infectivity titer and qPCR (Ct) were determined after each of the three passages
Lamb testis cells were inoculated with RVFV and capripoxviruses and incubated for 4 days at 37° C under 5% CO2
and the cells were fixed with methanol for 15 min at room temperature
cells were covered with Giemsa stain (4% v/v Giemsa-stain in double-distilled water) for 10 min
was used to determine viral genetic loads in clinical samples
Tests were performed in 96-well Optical Reaction Plates (Applied Biosystems)
contained 10 μl Luna Universal probe QPCR Master Mix
4 μl of template and nuclease free water to 20 μl
The reactions were run on the Quant Studio1 System (Applied Biosystems) using the following amplification program: 95 °C for 10 min; 45 cycles of 95 °C for 15 s and 60 °C for 1 min
The results were generated by the determination of the threshold cycle (Ct)
that contained 10 μl SensiFAST Probe Lo-ROX one step Master Mix
Reactions were run on the Quant Studio1 System (Applied Biosystems) using the following amplification program: reverse transcription at 45 °C for 15 min
45 cycles with 95 °C for 15 s and 60 °C for 30 s
Fluorescence was read at the combined annealing-extension step at 60 °C
Animal experiments were carried out in accordance with the international guidelines for the care and handling of experimental animals described in chapter 7.8 of the Terrestrial Animal Health Code and Directive 2010/63/UE of the European commission
The protocol was approved by the Internal Ethic Committee for animal experiment: Multi-chemical industry: MCI santé animale (Project number: 1M1604)
Animals were separately housed into an insect proof BSL3 animal facility
sample size and statistical analyses of the in vivo experiments in the manuscript followed the recommendations in the ARRIVE guidelines
were were vaccinated as follow: one group was inoculated with SPPV (4.0 (Tissue Culture Infectious Dose 50%) TCID50/dose)
one group (n = 10) with RVFV (4.5 TCID50/dose) and one group (n = 10) with a mixture RVFV/SPPV (4.0 and 4.5 TCID50/dose
6 to 8 months old were divided into five groups
Group 1 (n = 20) was inoculated with LSDV (103.5 TCID50/dose)
Group 2 with LSDV (n = 20) (104.5 TCID50/dose)
Group 3 with RVFV (n = 20) (104.5 TCID50/dose)
with Group 4 (n = 20) LSDV/RVFV (103.5 and 104.5 TCID50/dose respectively) and Group 5 (n = 20) co-infected with LSDV/RVFV (104.5 and 104.5 TCID50/dose respectively)
Animals were observed daily for sign of illness and sera were collected weekly for serological analysis
Sera were screened for RVFV antibodies with a commercial competitive ELISA kit (RVF-ID Screen) from IDvet
Testing was performed according to the manufacturer’s instructions and results were read at a wavelength of 450 nm
Interferon gamma (IFNG) test (Bovigam TB kit
France) was used to evaluate the IFNG levels
Animal blood samples were incubated overnight with positive control
Samples were then tested for IFNG using a sandwich ELISA according to manufacturer’s instructions
The control animals were euthanized when severe generalized signs of illness were observed
All results were calculated and presented as the means ± standard error of the mean obtained from triplicate tests
The statistical significance was determined by one-way or two-way analysis of variance 
P values of &lt; 0.05 were considered as statistical significance
SPPV and LSDV viruses for coinfection of LT cell culture (× 400)
(B) RVFV cytopathic effect: RVFV necrotic foci (red arrows)
(E) SPPV/RVFV 0.01/0.01 coinfection of LT cells and (F) LSDV/RVFV coinfection
Arrows showing capripox virus-induced vacuoles in cells and intracytoplasmic eosinophilic inclusion bodies (Arrowheads)
</a>Infectivity titers of SPPV and RVFV co-cultured in Vero cells (A) and in LT cells (B) at different MOI 1
Data shown are means of at least 3 independent experiments
</a>Infectivity titration (TCID50/ml) and viral genome copies by qPCR (threshold cycles: Ct) of SPPV (A) and RVFV (B) after coinfection in three successive passages (P1, P2 and P3). Infection was performed in (LT cells with different MOIs (RVFV/SPPV: 0.01/0.01; 0.01/0.1; 0.01/0.25; 0.01/0.5; 0.01/1). Data shown are means of at least three independent experiments.
</a>Infectivity titration (TCID50/ml) of LSDV (A) and RVFV (B) alone and after coinfection involving three successive passages (P1
LSDV and RVFV alone at MOI = 0.01; and coinfection at different MOIs: RVFV/LSDV:0.01/0.01; 0.01/0.1 and 0.01/0.5
Data shown are means of at least three independent experiments (n = 3)
</a>Antibody titers response of sheep injected with SPPV alone
The antibody response was determined from from D0 to 3 months pv
(A) Viral neutralizing antibody titers of sheep injected with RVFV and combined SPPV/RVFV (B) Viral neutralizing antibody titers for SPPV Monovalent and SPPV/RVFV co-infected sheep
Data shown are means of at least three neutralization experiments
</a>Percentage of antibody positive sheep (%) (n = 10) injected with RVFV alone
(B) SPPV antibody positive sheep in n = 10 each of tested groups
</a>Challenged vaccinated sheep showing hypersensitivity reaction and no local inflammations on site of inoculation with10−1 to 10−6 dilutions (left to right) of virulent SPPV
(A) Figure of challenged unvaccinated sheep showing local inflammation on site of inoculation
(B) Sheep vaccinated with SPPV/RVFV and challenged with SPPV virulent strain (C) sheep vaccinated with SPPV alone and challenged with SPPV virulent strain
Black arrow shows inflammation or hypersensitivity reaction on inoculation site
Red arrow shows absence of the hypersensitivity reaction
</a>Antibody titers response in cattle vaccinated with LSDV and with combined LSDV/RVFV monitored from D0 to 3 months pv
(A) LSDV neutralization antibody titers in log10
(B) Percentage of cattle positive for LSDV injected with two different doses for LSDV (103.5 and 104.5 TCID50/dose) and RVFV (103.5 and 104.5 TCID50/dose)
</a>Antibody titers response in cattle vaccinated with RVFV alone
Cattle are monitored from D0 to 3 months pv
(B) Percentage of cattle thar seroconverted to RVFV injected with a dose of 104.5 TCID50/dose and with a dose of LSDV/RVFV 104.5 TCID50/dose
Interferon Gamma using Bovigam assay was used to assess cellular immunity response to LSDV. Table <a data-track="click" data-track-label="link" data-track-action="table anchor" href="/articles/s41598-021-91926-5#Tab1">1</a> showed that 85% of animals (17 out of 20 cattle) had a positive IFNG reaction to LSDV vs 30% (6 out of 20) in co-infected LSDV/RVFV group
Understanding interactions between viral pathogens is essential for the development of effective combined vaccines
This study is the first work that illustrates interference between RVFV and capripoxviruses attenuated strains in cell culture and in target animal species during the testing of these combined viruses
cytolytic viruses could rapidly deplete cellular resources and induce cell death as we observed for RVFV clone 13 T and SPPV coinfection of cells
if coinfecting viruses vary significantly in the length of their replication cycle
the one with the shorter replication cycle will persist because the second virus with the longer replication cycle will be prematurely terminated
and 0.5) of SPPV or LSDV was increased as compared to and MOI of 0.01 for RVFV MOI for infecting LT cells
the purpose of this experiment was to be able to correct the respective titers when both viruses are combined
this study opens new insights toward studying the reason(s) behind immunity inhibition of capripoxviruses attenuated strains in the presence of RVFV lacking NS gene (clone 13)
It was clearly shown that injection of SPPV and LSDV in the presence of RVFV clone 13 T was counteracted
in this study we showed that sheep were not protected when co-injected with SPPV/RVFV and neither LSDV immunity (humoral nor cellular) was induced in cattle when co-injected with LSDV/RVFV
Co-inoculation with RVFV and SPPV/LSDV on susceptible cell culture revealed interference between RVFV and SPPV and/or LSDV
Further investigations should be done in order to elucidate and comprehend the mechanism behind this “interference” in sheep
cattle and in goats using goatpox virus GTPV (Gorgan strain)
where there is also a clear interference between RVFV and GTPV when challenged with Vietnamien virulent strain (data not shown)
Two strategies could be suggested a) to increase the SPPV titer and decrease RVFV infectivity titer/dose for combined SPPV/RVFV infection
to evaluate the immune response of sheep co-injected with 104.5 TCID50/dose for SPPV and 103.0 TCID50/dose for RVFV
to increase the LSDV titer (105.5 TCID50/dose) and decrease RVFV to (103.5 TCID50/dose)
b) Since capripoxviruses use lymphatic route of infection and RVFV spread through blood
two separate injections of the SPPV alone in one side and the monovalent RVFV in another site in sheep should be considered in order to avoid this interference
Rift Valley fever virus (Bunyaviridae: Phlebovirus): An update on pathogenesis
Characterization of sheep pox virus vaccine for cattle against lumpy skin disease virus
Diallo, A. &amp; Viljoen, G. J. Genus Capripoxvirus. In Poxviruses. Birkhäuser Advances in Infectious Diseases (eds Mercer, A. A., Schmidt, A. &amp; Weber, O.) (Birkhäuser, Basel, 2007). <a href="https://doi.org/10.1007/978-3-7643-7557-7_8" data-track="click_references" data-track-action="external reference" data-track-value="external reference" data-track-label="10.1007/978-3-7643-7557-7_8">https://doi.org/10.1007/978-3-7643-7557-7_8</a>
Economic impact of lumpy skin disease and cost effectiveness of vaccination for the control of outbreaks in Ethiopia
Development and field application of a new combined vaccine against Peste des Petits Ruminants and Sheep Pox
Evaluation of the efficacy and duration of immunity of a canine combination vaccine against virulent parvovirus
and distemper virus experimental challenges
Ngoshe, Y. B., Avenant, A., Rostal, M. K., et al. Patterns of Rift Valley fever virus seropositivity in domestic ruminants in central South Africa four years after a large outbreak. Sci. Rep. 10, 5489. <a href="https://doi.org/10.1038/s41598-020-62453-6" data-track="click_references" data-track-action="external reference" data-track-value="external reference" data-track-label="10.1038/s41598-020-62453-6">https://doi.org/10.1038/s41598-020-62453-6</a> (2020)
Cell culture for biochemists (Elsevier/North-Holland Biomedical Press
A simple method of estimating fifty per cent endpoints
The genomes of Sheeppox and Goatpox viruses
Experimental infection of young adult european breed sheep with Rift Valley fever virus field isolates
Etude experimentale de l’immunite anticlaveleuse post-vaccinale
Review article: Sociocultural and economic dimensions of Rift Valley Fever
Review: Lumpy skin disease: An emerging threat to Europe
Review: Capripoxvirus diseases: current status and opportunities for control
Lauer, K. B., Borrow, R. &amp; Blanchard, T. J. Multivalent and multipathogen viral vector vaccines. Clin. Vaccine Immunol. 24(1), e00298-16. <a href="https://doi.org/10.1128/CVI.00298-16" data-track="click_references" data-track-action="external reference" data-track-value="external reference" data-track-label="10.1128/CVI.00298-16">https://doi.org/10.1128/CVI.00298-16</a> (2017)
Protection of sheep against Rift Valley fever virus and sheep poxvirus with a recombinant capripoxvirus vaccine
Protection of cattle elicited using a bivalent Lumpy skin disease virus-vectored recombinant Rift Valley fever vaccine
Salas-Benito, J. S. &amp; De Nova-Ocampo, M. Viral interference and persistence in mosquito-borne Flaviviruses. J. Immunol. Res. 2015, 873404 (2015). <a href="https://doi.org/10.1155/2015/873404" data-track="click_references" data-track-action="external reference" data-track-value="external reference" data-track-label="10.1155/2015/873404">https://doi.org/10.1155/2015/873404</a>
Fields virology (Wolters Kluwer Health/Lippincott Williams &amp; Wilkins
Study of cultural characteristics and interference of Peste des Petit ruminants virus and sheep pox virus in co-culture
A Sheeppox virus outbreak in central Turkey in 2003: isolation and identification of Capripoxvirus ovis
Kumar, N., Sharma, S., Barua, S., Tripathi, B. N. &amp; Rouse, B. T. Virological and immunological outcomes of coinfections. Clin. Microbiol. Rev. 31(4):e00111-17 <a href="https://doi.org/10.1128/CMR.00111-17" data-track="click_references" data-track-action="external reference" data-track-value="external reference" data-track-label="10.1128/CMR.00111-17">https://doi.org/10.1128/CMR.00111-17</a> (2018)
Rift Valley fever virus NSs mRNA is transcribed from an incoming anti-viral-sense S RNA segment
Cytoplasmic organization of Poxvirus DNA replication
Multiple mechanisms for the inhibition of entry and uncoating of superinfecting Semliki Forest virus
Superinfection exclusion of vaccinia virus in virus-infected cell cultures
Sensitivity of lateral flow technique for evaluation of inactivated Rift Valley fever virus vaccine in comparison with serum neutralization test
Present position of immunization against poliomyelitis with live virus vaccines
Absence of circulating interferon in patients with infectious and serum Hepatitis
In vivo interference by Newcastle disease virus in chickens
Whitaker-Dowling, P. &amp; Youngner, J. S. Viral interference-dominance of mutant viruses over wild-type virus in mixed infections. Microbiol. Rev. 51(2),  179–191. <a href="https://doi.org/10.1128/mr.51.2.179-191.1987" data-track="click_references" data-track-action="external reference" data-track-value="external reference" data-track-label="10.1128/mr.51.2.179-191.1987">https://doi.org/10.1128/mr.51.2.179-191.1987</a> (1987)
Evaluating viral interference between infectious bronchitis virus and newcastle disease virus vaccine strains using quantitative reverse transcription-polymerase chain reaction
Viral interference: Some considerations of basic mechanisms and their potential relationship to host resistance
Population dynamics of an RNA virus and its defective interfering particles in passage cultures
Continuous Influenza virus production in cell culture shows a periodic accumulation of defective interfering particles
Defective interfering viruses and their potential as antiviral agents
Effects of defective interfering viruses on virus replication and pathogenesis in vitro and in vivo
Persistent infection of mammalian cells by Rift Valley fever virus
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The authors declare that this study received funding through MCI Santé Animale from the Livestock Vaccine Innovation Fund
LVIF is supported by the Bill &amp; Melinda Gates Foundation (BMGF)
and Canada's International Development Research Centre (IDRC)
The funder was not involved in the study design
the writing of this article or the decision to submit it for publication
&amp; M.E.: wrote the main manuscript text &amp; prepared figures
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations
<a data-test="citation-link" data-track="click" data-track-action="download article citation" data-track-label="link" data-track-external="" rel="nofollow" href="https://citation-needed.springer.com/v2/references/10.1038/s41598-021-91926-5?format=refman&amp;flavour=citation">Download citation</a>
DOI: https://doi.org/10.1038/s41598-021-91926-5
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by  <a href="/authors/ryan-gilliam">Ryan Gilliam</a>
but one quest in particular focuses on a mysterious character: the evil
we’ll tell you everything you need to know about this diabolical figure
and show you where to find them in each of the game’s four main areas
The evil fox NPC won’t ever give you their name
and they only appears at night in certain areas
There is a moon-shaped icon hidden in Pilgrim’s Passage
you’ll have the option to “Peer into the Darkness,” which will summon the cloaked figure
the fox will give you a piece of a Holy Talisman after each interaction
Once you have four pieces of a Holy Talisman
you’ll be able to purchase a new Fleece from your chapel
and have a variety of unique effects that will impact your Crusades
You’ll unlock the Pilgrim’s Passage area during your adventure through Darkwood
the first Crusade location in Cult of the Lamb
Go to the Pilgrim’s Passage at night and walk to the end of the dock
Hit the “Peer Into the Darkness” prompt to summon the fox
They’ll talk to you for a bit and then ask for a fish
You’ll unlock the Spore Grotto area while adventuring through Anura
the second Crusade location in Cult of the Lamb
Go to the Spore Grotto at night and walk up to the vendor on the left
“Peer Into the Darkness” to summon the fox again
They’ll ask for one of your followers this time
Oblige them and you’ll get another Holy Talisman piece
As usual, we <a href="/guides/23296988/cult-of-the-lamb-beginners-guide-tips-tricks-followers-crusade-farm-difficulty">recommend giving over an elderly follower if possible</a>, as they’re no longer of much use to the cult.
You’ll unlock the Smuggler’s Sanctuary area while adventuring through Anchordeep, the third Crusade location in Cult of the Lamb.
Go to the Smuggler’s Sanctuary at night and walk up to the card vendor carpet on the right. Walk past the vendor onto the dock. “Peer Into the Darkness” to summon the fox for the third time. This time they’ll ask for two followers. Give them over and you’ll get yet another piece of a Holy Talisman.
Midas’s Cave is the final location in Cult of the Lamb, and you’ll unlock it while adventuring through Silk Cradle, the fourth Crusade location.
Go to Midas’s Cave at night and walk to the back. Turn right at where you usually sacrifice followers to Midas, and walk to the edge of the map, just under the piles of gold. Interact with the final moon emblem and “Peer Into the Darkness” to summon the fox.
[Ed’s note: The following contains a light spoiler for some Cult of the Lamb plot.]
This time, the fox will ask for Ratau, your mentor who first taught you to build a cult for The One Who Waits. Losing Ratau doesn’t actually affect your cult at all, and is more of an emotional hit than a practical one. However, you’ll lose the ability to play Knucklebones with Ratau, so make sure you do that before sacrificing him — you’ll unlock a Tarot card after beating him for the first time.
Once the fox takes Ratau, he’ll offer another Holy Talisman piece and give you a new follower form. He’ll then say goodbye, and disappear into the darkness forever.
Note: If you complete this quest before taking on the game’s final boss, they’ll remark on your ruthlessness and ability to succeed where Ratau failed.
either observed and verified firsthand by the reporter
or reported and verified from knowledgeable sources
Translations may contain inaccuracies—please refer to the original content
Billions of users rely on <a href="https://www.newsweek.com/topic/facebook" data-sys="1" class="multivariate">Facebook</a> as a means for online connection to chat freely with friends and family
Private technology companies like Facebook hold a lot of power, which was demonstrated in their recent decision to join other platforms such as <a href="https://www.newsweek.com/topic/twitter" data-sys="1" class="multivariate">Twitter</a> in banning former President <a href="https://www.newsweek.com/topic/donald-trump" data-sys="1" class="multivariate">Donald Trump</a>
differing online opinions have emerged regarding censorship alongside fears that religious content is being banned on Facebook
British YouTuber and writer Paul Joseph Watson posted a video titled Facebook bans Bible passages to YouTube on Monday that has been viewed almost 50,000 times
he claims that a recent Facebook post was censored and banned when a user tried to share two links to Biblehub.com and an excerpt of a Bible passage
Watson shows a screenshot of the user receiving a "Your post couldn't be shared
because this link goes against our Community Standards" message from Facebook for the post in his video at the 1:19 minute mark
Watson repeated his claim on several social platforms such as Twitter
where he wrote "Facebook was the main platform for planning the Capitol riot
but at least it's cracking down on Bible posts," and shared the image of a Facebook user allegedly being censored with over thousands of likes and retweets
what next?" with a screenshot of him attempting to post a link to Biblehub.com and receiving a "Your post couldn't be shared
because this link goes against our Community Standards."
Facebook was the main platform for planning the Capitol riot, but at least it&#39;s cracking down on Bible posts. <a href="https://t.co/LgUXia0Hmf">pic.twitter.com/LgUXia0Hmf</a>
Facebook is no stranger to being accused of censoring and banning religious content
In response to the allegations that it censors and bans Bible passages
This has been debunked several times in the past."
When Newsweek asked Facebook if it specifically banned posts with links from Biblehub.com
the company said it was looking into the matter
Biblehub.com is an online Bible study site with passages available in multiple languages
"This site is a great way to link any verse on your site to an instant menu of 25 versions!"
The title of Watson's video asserts that all Bible passages are banned by Facebook
but he later states in the video that the exact reason as to why the user's post was banned is unknown
"It could be that the Bible passage is in question or that the text associated with this post because it had the word 'blood' in it which again is another paraphrase from a Bible quote
or it could be that the website 'Biblehub' is censored
blocked on Facebook," Watson said in his widely seen and shared clip
it confirmed to Newsweek on Wednesday night that links to Biblehub.com were banned
"The Web site BibleHub.com links were flagged as low-quality content in error
which can restrict sharing capabilities," the Facebook spokesperson said in an email
and links to Biblehub.com can now be shared after Newsweek's inquiry
"We apologize for this error," the Facebook spokesperson said
Watson is deemed as "far right" by multiple online sources and was banned from Facebook and <a href="https://www.newsweek.com/topic/instagram" data-sys="1" class="multivariate">Instagram</a> in May of 2019
but he was correct about links from Biblehub.com being banned
Facebook does not censor Bible passages or posts with religious content
links to Biblehub.com were banned and prohibited from being posted
After Newsweek reached out about the issue
Facebook stated it was in error and corrected it
Links to Biblehub.com can now be shared in Facebook posts
Other posts containing Bible passages appear publicly on Facebook and have not been banned or censored
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Although Bovine Spongiform Encephalopathy (BSE) is the cause of variant Creutzfeldt Jakob disease (vCJD) in humans
the zoonotic potential of scrapie prions remains unknown
Mice genetically engineered to overexpress the human prion protein (tgHu) have emerged as highly relevant models for gauging the capacity of prions to transmit to humans
These models can propagate human prions without any apparent transmission barrier and have been used used to confirm the zoonotic ability of BSE
Here we show that a panel of sheep scrapie prions transmit to several tgHu mice models with an efficiency comparable to that of cattle BSE
The serial transmission of different scrapie isolates in these mice led to the propagation of prions that are phenotypically identical to those causing sporadic CJD (sCJD) in humans
These results demonstrate that scrapie prions have a zoonotic potential and raise new questions about the possible link between animal and human prions
The occurrence of vCJD has provided significant evidence to show that the transmission barrier does not constitute an absolute protection against the zoonotic risk of prions that circulate in animal populations
This suggests that the need for an in-depth assessment of the permeability of the human species barrier to animal TSE agents including those responsible for scrapie in sheep
Despite converging evidence that scrapie is caused by a variety of prion strains
there is at this stage no comprehensive description of their diversity
we inoculated tgHu mice transgenic for expression of human PrP codon 129 variants intracerebrally with a panel of (i) biologically distinct ovine scrapie isolates
(ii) human prions and (iii) cattle BSE prions
The serial transmission of different scrapie isolates in tgHu mice led to the propagation of prions that were phenotypically identical to those that cause sporadic CJD (sCJD) in humans
</a>Brain homogenate dilutions (neat or 1/4, 1/8, 1/16 diluted in Laemmli’s buffer) were analysed by SDS–PAGE and western blot using 12% acrylamide gel. Normal PrP was detected using the monoclonal antibody 3F4 (0.8 μg ml−1). (a) tgMet129, tgMet/Val129 and tgVal129 samples. (b) tgVal129 and human control brain samples.
</a>(a) The PrPres western blot pattern of six different natural sheep scrapie isolates (MF17
PS42 and PS310) was established using a monoclonal antibody Sha31
This panel of scrapie isolates was inoculated in transgenic mice (n=6 per group) that express the murine (tg20-grey circle)
the ovine A136R154Q171 (tgshpXI-triangle up) and the ovine V136R154Q171 (tg338-triangle down) PrP variants
For each isolate two serial passages were carried out: (b) the incubation period (mean±s.d.) as days post inoculation (dpi) of the second passage in the different mouse lines and (c–f) the PrPres western Blot profiles (10% brain homogenate) observed after two passages in each of the mouse lines
PrPres detection was carried out by SDS–PAGE and western blot with an anti-PrP monoclonal antibody Sha31
The data also support the contention that the relative permeability of the human species barrier (as modelled here by the intracerebral transmission in tgHu mice) to cattle BSE and scrapie is not fundamentally different
These observations support the contention that the adaptation of ovine scrapie prions to the bovine PrP amino-acid sequence increases their capacity to propagate in tgHu mice
PK-resistant PrP detection was carried out by PET blot using an anti-PrP monoclonal antibody Sha31
</a>Transgenic mice (tgMet129/tg650) that overexpress the methionine variant at codon 129 of human PrP were inoculated with different natural sheep scrapie isolates (ov)
human (hu) vCJD and sCJD samples (MM1 and VV2)
Brains from first and/or second passage tg650 mice were tested for the presence of PrPres by (a) western blot (Sha31anti-PrP antibody) and (b) histo Blot (3F4 anti-PrP antibody)
these observations raise some concerns with regards to the relevance of primates as a model of human species barrier
An unexpected result of this study was the finding that transmission of sheep scrapie isolates in tgHu mice resulted in the emergence of prions with a similar phenotype to those associated with sCJD
As this result was obtained in two different tgHu mouse lines
we consider that our data support the view that passage of sheep scrapie prions across a human transmission barrier can lead to the emergence of sCJD prions
This suggests that mice that express physiological levels of PrP might not be the most appropriate animal model to assess the permeability of the human species barrier to animal prions
it will be a challenge to combine epidemiological data collected contemporarily in animal populations and humans to investigate the existence of a causative link between prion disease occurrence in these different hosts
it is crucial to bear in mind that sporadic sCJD in humans is a rare disease (1–2 individuals per million of the population per year) and that scrapie has been circulating in small ruminants populations used for food purposes for centuries
it is our opinion that even if a causative link was established between sheep scrapie exposure and the occurrence of certain sCJD cases
it would be wrong to consider small ruminant TSE agents as a new major threat for public health
it remains clear that our data provide a new impetus to establish the true zoonotic potential of sheep scrapie prions
All these sheep scrapie cases were confirmed positive by histopathology (identification of vacuolar changes in the brainstem)
154 and 171 of the sheep PrP gene (ARQ/ARQ)
154 and 171 of the sheep PrP gene (VRQ/VRQ)
The O100 scrapie isolate (inoculated in tg650 mice) was provided by the National French reference laboratory for scrapie diagnosis (ANSES
France) and had been collected from a VRQ/VRQ sheep of unknown age
Both tg340 and tg361 are homozygous for human PRNP gene and tgMet/Val129 mice used in our experiments were obtained by mating tg340 and tg361 mice (F1 generation)
All animal experiments were performed in compliance with our institutional and French national guidelines in accordance with the European Community Council Directive 86/609/EEC
The experimental protocol was approved by the INRA Toulouse/ENVT ethics committee
Six- to ten-week-old female mice were anaesthetized and inoculated with 2 mg of brain equivalent (20 μl of a 10% brain homogenate) in the right parietal lobe using a 25-gauge disposable hypodermic needle
Mice were observed daily and their neurological status was assessed weekly
When clinically progressive TSE disease was evident
animals were killed and their brains collected
Half of the isolated brain from those animals that had displayed TSE clinical signs was fixed by immersion in 10% formol and the other half was frozen at −20 °C
Tissues from found dead animals were frozen (no formalin fixation)
Spleens from animals were not systematically collected (in particular in found dead animals) and data related to PrPSc accumulation in this tissue are not available
Survival time was expressed as the mean of the survival days post inoculation (d.p.i.) of all the mice scored positive for PrPres
In cages where no clinical signs were observed
mice were killed at the end of their natural lifespan (650 to 800 days)
incubation periods reported in the table (&gt;650 to 750 d.p.i.) corresponded to the survival time observed in at least three out of the six mice
Brain homogenates from PrPres positive mice
When all mice were scored negative for PrPres on primary passage
PrPres-negative brain homogenates of animals that survived for more than 500 d.p.i
Mortality from Creutzfeldt-Jakob disease and related disorders in Europe
The epidemiology of Creutzfeldt-Jakob disease: conclusion of a 15-year investigation in France and review of the world literature
Case-control study of risk factors of Creutzfeldt-Jakob disease in Europe during 1993-95
European Union (EU) Collaborative Study Group of Creutzfeldt-Jakob disease (CJD)
Further experimental observations on scrapie
A bovine prion acquires an epidemic bovine spongiform encephalopathy strain-like phenotype on interspecies transmission
Mouse-adapted ovine scrapie prion strains are characterized by different conformers of PrPSc
The physical relationship between infectivity and prion protein aggregates is strain-dependent
A protease-resistant protein is a structural component of the scrapie prion
Long-term subclinical carrier state precedes scrapie replication and adaptation in a resistant species: analogies to bovine spongiform encephalopathy and variant creutzfeldt-jakob disease in humans
Biochemical and physical properties of the prion protein from two strains of the transmissible mink encephalopathy agent
Evidence that the transmission of one source of scrapie agent to hamsters involves separation of agent strains from a mixture
Transgenic mice expressing hamster prion protein produce species- specific scrapie infectivity and amyloid plaques
Experimental subacute spongiform virus encephalopathies in primates and other laboratory animals
Transmissions to mice indicate that 'new variant' CJD is caused by the BSE agent [see comments]
Sheep and goat BSE propagate more efficiently than cattle BSE in human PrP transgenic mice
Joint Scientific Opinion on any possible epidemiological or molecular association between TSEs in animals and humans.
Genetic basis of Creutzfeldt-Jakob disease in the United Kingdom: a systematic analysis of predisposing mutations and allelic variation in the PRNP gene
Genetic risk factors for variant Creutzfeldt-Jakob disease: a genome-wide association study
BSE prions propagate as either variant CJD-like or sporadic CJD-like prion strains in transgenic mice expressing human prion protein
Transmission of atypical bovine prions to mice transgenic for human prion protein
Evaluation of the human transmission risk of an atypical bovine spongiform encephalopathy prion strain
Second passage of a US scrapie agent in cattle
Intracerebral transmission of scrapie to cattle
Different prion disease phenotypes result from inoculation of cattle with two temporally separated sources of sheep scrapie from Great Britain
Molecular basis of phenotypic variability in sporadic Creutzfeldt-Jakob disease
Experimental transmission of a Kuru-like syndrome to chimpanzees
Jr Transmission of two subacute spongiform encephalopathies of man (Kuru and Creutzfeldt-Jakob disease) to new world monkeys
Experimental transmission of BSE and scrapie to the common marmoset
Transmission and characterization of the agents of spongiform virus encephalopathies: kuru
Prion protein gene variation among primates
Infectious amyloid precursor gene sequences in primates used for experimental transmission of human spongiform encephalopathy
Transgenic mice expressing porcine prion protein resistant to classical scrapie but susceptible to sheep bovine spongiform encephalopathy and atypical scrapie
Temporary and permanent modifications to a single strain of mouse scrapie on transmission to rats and hamsters
Prion strain mutation determined by prion protein conformational compatibility and primary structure
Continuum of prion protein structures enciphers a multitude of prion isolate-specified phenotypes
Atypical scrapie prions from sheep and lack of disease in transgenic mice overexpressing human prion protein
Chronic wasting disease and atypical forms of bovine spongiform encephalopathy and scrapie are not transmissible to mice expressing wild-type levels of human prion protein
Defining sporadic Creutzfeldt-Jakob disease strains and their transmission properties
Atypical scrapie/Nor98 in a sheep from New Zealand
Kuru in the 21st century--an acquired human prion disease with very long incubation periods
Atypical/Nor98 scrapie infectivity in sheep peripheral tissues
Which PrP haplotypes in a French sheep population are the most susceptible to atypical scrapie
Early detection of PrPres in BSE-infected bovine PrP transgenic mice
The use of transgenic mice in the investigation of transmissible spongiform encephalopathies
Screening of 145 anti-PrP monoclonal antibodies for their capacity to inhibit PrPSc replication in infected cells
Dynamics and genetics of PrPSc placental accumulation in sheep
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We are greatly in debt with Dr Marion Simmons and Dr Raymond Bujdoso for their critical reading of this article
This work was funded by the EU FP7 project ‘Priority CT2009-222887’
The EU FEDER/INTERREG EFA205/11 ‘CONCOSTA’ and the Spanish Plan Nacional de I+D+I RTA2012-00004 and AGL2012-37988-C04 projects
Hervé Cassard and Juan-Maria Torres: These authors contributed equally to this work
Juan-Maria Torres &amp; Juan-Carlos Espinosa
UR892 Virologie et Immunologie Moléculaires Centre de Recherche de Jouy-en-Josas
The authors declare no competing financial interests
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The Senate overwhelmingly passed the mammoth fiscal 2023 spending package in a burst of activity on the floor Thursday after finally nailing down an amendments deal it took all day Wednesday and into the morning to hammer out
The vote was 68-29 in support of the 4,155-page legislation
It includes&nbsp;the dozen annual spending bills for every federal agency
supplemental aid for the war in Ukraine and natural disaster victims
and a series of unrelated policies ranging from retirement savings incentives to driftnet fishing regulations
&#8220;This is one of the most significant appropriations packages we have done in a very long time,&#8221; Senate Majority Leader <a class="memberLink" href="https://www.rollcall.com/members/371?utm_source=memberLinks&amp;utm_medium=memberlinks&amp;personid=371" title="Click to view member info in a new window" target="_blank" rel="noopener">Charles E. Schumer</a> said before final passage
&#8220;The range of people it helps is large and deep.&#8221;
The package includes $858 billion in defense spending
a nearly 10 percent increase over the previous fiscal year
close to an 8 percent increase.&nbsp;It also would provide roughly&nbsp;$85 billion in supplemental funding for Ukraine and disaster relief.&nbsp;
Senate passage paves the way for the House to clear the legislation ahead of government spending running out at midnight on Friday.&nbsp;
The House planned to take up the measure Friday as early as 9 a.m., Majority Leader <a class="memberLink" href="https://www.rollcall.com/members/230?utm_source=memberLinks&#038;utm_medium=memberlinks&#038;personid=230" title="Click to view member info in a new window" target="_blank" rel="noopener">Steny H. Hoyer</a> announced hours after the Senate vote
House leaders initially had hoped to clear the bill Thursday night but then determined it would not be ready for floor action in their chamber before midnight
Most of the amendment attempts were unsuccessful
It wasn&#8217;t immediately clear how long it would take to package up all the amended paperwork for transmittal to the House
it will take time for the measure to be enrolled for Pelosi&#8217;s signature
the last step before it&#8217;s ready to go to the White House
a stopgap measure that would extend current funding through Dec
The latest enacted continuing resolution is set to expire at midnight Friday
House Appropriations Chair <a class="memberLink" title="Click to view member info in a new window" href="https://www.rollcall.com/members/92?utm_source=memberLinks&amp;utm_medium=memberlinks&amp;personid=92" target="_blank" rel="noopener">Rosa DeLauro</a>
said she didn&#8217;t think that would be necessary
And she expressed confidence the omnibus wouldn&#8217;t face any hurdles in the House
It&#8217;s going to happen,&#8221; DeLauro said
Thursday’s passage followed drama Wednesday night when a dispute over an amendment related to Trump-era border restrictions during the public health emergency delayed passage
Republicans wanted a simple majority vote on Lee&#8217;s amendment to bar the Biden administration from ending the Title 42 pandemic-era asylum restriction policy
claiming it was germane to the underlying spending bill
in addition to blocking funds for ending Title 42
would appropriate $8.7 billion for border security and migrant care.&nbsp;
Both amendments were defeated in a bit of procedural and political theater
enabling the spending bill to advance without being weighed down by controversy when it arrives in the House
enabling border state and other Democrats to comfortably back that proposal instead of Lee&#8217;s
Lee <a href="https://twitter.com/SenMikeLee/status/1605948247012306944">wrote on Twitter</a> after the vote that the Sinema-Tester amendment is a &#8220;wolf in sheep&#8217;s clothing to&nbsp;mislead the American people to believe Dems are doing something to secure our borders.&#8221; He added that it &#8220;merely provides them cover to vote against my extension of Title 42 protections.&#8221;
The Senate’s passage of the legislation came the day after Ukrainian President Volodymyr Zelenskyy <a href="https://www.rollcall.com/2022/12/21/ukraines-zelenskyy-pleads-with-congress-for-more-military-aid/">addressed a joint meeting of Congress</a>
thanking lawmakers for their support and pleading for more as Ukraine continues to fend off Russia’s invasion
The bill includes nearly $47 billion in new military
economic and humanitarian aid for Ukraine.&nbsp;
Republicans praised the measure&#8217;s higher level of defense spending and smaller increase on the nondefense side as a win in negotiations
and highlighted the retention of the Hyde amendment
that blocks federal funding for abortion in most cases,&nbsp;and flat-funding the IRS.&nbsp;
Senate Minority Leader <a class="memberLink" href="https://www.rollcall.com/members/202?utm_source=memberLinks&amp;utm_medium=memberlinks&amp;personid=202" title="Click to view member info in a new window" target="_blank" rel="noopener">Mitch McConnell</a> said earlier this week the package “equips our armed forces with the resources they need while cutting nondefense
nonveteran spending in real dollars.” &nbsp;&nbsp;
Democrats also highlighted priorities in the package
including the first funding increase for the National Labor Relations Board in over a decade
increased clean energy funding in the Energy-Water bill and more funding for affordable housing.&nbsp;
Pelosi said Thursday the package featured “the highest nondefense
domestic number ever” though noted “we always want more.”&nbsp;
The legislation also included a bevy of provisions unrelated to the annual appropriations process
including legislation banning TIkTok on government phones and horseracing industry rules
High-profile legislation clarifying the role of the vice president in counting electoral votes and raising the number of members needed to object to electoral votes was also included in the package.&nbsp;
where Democrats will not need Republican support if all of their members vote in favor of the legislation.&nbsp;
Minority Leader <a class="memberLink" href="https://www.rollcall.com/members/25162?utm_source=memberLinks&amp;utm_medium=memberlinks&amp;personid=25162" title="Click to view member info in a new window" target="_blank" rel="noopener">Kevin McCarthy</a>
who is struggling to secure the votes needed to become House speaker in January
Just nine Republicans voted for the one-week CR needed last week to pave way for omnibus passage
Lindsey McPherson contributed to this report
At the beginning of the episode, Emma Dutton (Marley Shelton) attends to the grave of her <a href="https://www.businessinsider.com/1923-star-confused-by-yellowstone-dutton-family-tree-as-fans-2023-1" rel="" target="_self" data-track-click="{&quot;element_name&quot;:&quot;body_link&quot;,&quot;event&quot;:&quot;tout_click&quot;,&quot;index&quot;:&quot;bi_value_unassigned&quot;,&quot;product_field&quot;:&quot;bi_value_unassigned&quot;}">husband, John (James Badge Dale)</a>
and we learn that the date of the fateful ambush by Banner Creighton (Jerome Flynn) and his men was August 28
when Spencer (Brandon Sklenar) and Alexandra (Julia Schlaepfer) try to book passage from Kenya to the United Kingdom (as there are no direct voyages from Africa to the United States)
they are told that the ship won't leave for another three weeks and the sail date is November 11
With the concrete dates given in this episode
it turns out no more than two months have passed
Hank explains to Teonna that the area they're in isn't part of the reservation
nor is it a part of Montana that the government counts as their own land
Like the so-called "train station" in "Yellowstone," the land covers a zone where people
it would make sense for the Broken Rock tribe to bring the flock there to ensure that they can keep hold of them
As Hank explains: "The sheep belong to the people
but if Jacob Dutton (Harrison Ford) hadn't given the sheep to the Broken Rock tribesmen then Hank would have no reason to be in the deserted area and would not have come upon Teonna and rescued her
Teonna — whose surname is Rainwater — is a direct ancestor of&nbsp; Chief Thomas Rainwater (Gil Birmingham in "Yellowstone")
the Rainwater line might have ended with Teonna being eaten alive by the wolves
When Hank (Michael Greyeyes) finds Teonna Rainwater (Aminah Nieves) hiding out among his sheep
he tells her that she can't go home to her family as it is the first place they'll look for her
"Maybe we should go to Canada," she suggests
"No Canada," he responds with a steely look."Canada's worse."&nbsp;
Like the American residential school depicted in the show
Canada's residential school system was run by the Catholic Church and saw Indigenous children torn away from their families and experience physical and sexual abuse in the name of "civilizing" them
prompting the country and its leaders to reckon with its history for the first time
This line takes on even more significance when you consider that Greyeyes is an Indigenous Canadian actor himself whose parents were both forced to attend residential schools before they were closed down
Shortly after the remains of 215 children were found at an Indigenous residential school site in British Columbia in May 2021, the actor <a href="https://twitter.com/michaelgreyeyes/status/1398976120863789056?lang=en" target="_blank" rel="noopener nofollow" data-track-click="{&quot;click_type&quot;:&quot;other&quot;,&quot;element_name&quot;:&quot;body_link&quot;,&quot;event&quot;:&quot;outbound_click&quot;}">tweeted</a>: "Both my parents survived Canada's Indian residential school system
My sister and I were the first generation in our family not to be taken by the CDN government."
the ship that Spencer and Alexandra are first offered a place on
didn't actually operate between Mombasa and London
realizing that they will have to make their own way across the Atlantic Ocean
they send a wire via another liner docked in Mombasa: the RMS Mauretania
However, this ship couldn't have been there as, crucially, in 1923, it was <a href="https://collectionsprojects.org.uk/mauretania/story-final.html" target="_blank" rel="noopener nofollow" data-track-click="{&quot;click_type&quot;:&quot;other&quot;,&quot;element_name&quot;:&quot;body_link&quot;,&quot;event&quot;:&quot;outbound_click&quot;}">undergoing a major overhaul</a>&nbsp;in Southampton
It did not return to Atlantic service until the following year
meaning Spencer couldn't have sent a wire from it to tell the Duttons of his imminent departure
During his first meeting with Spencer in the bar
Lucca (Peter Stormare) is shown with a nasty
he is seen carrying a bloodied handkerchief
when he takes over the watch during the night
he steered the tugboat right into the path of the ghost ship that he remarked on earlier
It's worth remembering that viewers saw Lucca getting pretty handsy with the beans and bread he served the couple earlier on in the day
dipping his fingers in the beans and then licking them to check they were hot enough
if it is some sort of infectious disease such as tuberculosis that has killed him
Spencer and Alexandra may find themselves getting sick and perish before they can reach Montana
As the screen fades to black after a final shot of the overturned tugboat and the ghost ship nowhere to be seen
an in-memoriam message appears on the screen before the credits start rolling
His cause of death hasn't been shared but in a <a href="https://www.gofundme.com/f/derek-chavez-memorial-fund" target="_blank" rel="noopener nofollow" data-track-click="{&quot;click_type&quot;:&quot;other&quot;,&quot;element_name&quot;:&quot;body_link&quot;,&quot;event&quot;:&quot;outbound_click&quot;}">GoFundMe</a> organized by the show's cast and crew
it states that his passing was sudden and left those close to him — including his wife Kristen and daughters Ava and Emmy— "shocked and saddened."
The inclusion of the tribute in the closing credits shows just how much of an integral role Chavez played in bringing the show to life
the Transhumance Festival cancels this year's edition: there will be no thousands of sheep crossing the capital
for which there is already a date of celebration in 2024
&#8220;the role of transhumance and extensive livestock farming as a tool for biodiversity conservation and combating climate change&#8220;
 You can consult the map and chronogram of the passage of the flock in <a href="https://diario.madrid.es/cieacasadecampo/wp-content/uploads/sites/61/2024/10/trashuman-243.jpg" rel="nofollow noopener noreferrer" target="_blank" class="shortcode-outbound-link" data-has-ga="true" data-ga-type="click" data-label="https://diario.madrid.es/cieacasadecampo/wp-content/uploads/sites/61/2024/10/trashuman-243.jpg" data-action="text_cta_2" data-category="click_non_sales">this link</a>
Continued land improvement works and reseeding has allowed a progressive sheep farmer to increase ewe numbers on 150ac of both owned and rented ground in the past number of years
This now sees the farm operating a mixed enterprise of 20 continental sucklers plus followers alongside a 200-ewe lowland flock of Suffolk crosses and Texel-crosses
The shed itself is a four-bay double-slatted sheep shed with an internal feed passage
The shed was sourced from Brennan Engineering in Swinford
with shed erection and all concrete work completed by Merdoc Construction Ltd
This side of the shed was raised to allow for a roofed handling unit to be created  between the two sheds in the future
Slatted pens either side of this passageway are 5.9m wide
Due to the layout of the walkthrough troughs
the pens at both ends of the shed are smaller
The two middle pens on both sides of the shed are larger and measure 4.8m x 5.9m
There are three walkthrough troughs on each side of the shed measuring 5m in length
which greatly increases feeding space available
Feed space for the smaller pens equates to 8.1m
With the larger pens having access to two walkthrough feeders
larger pens can accommodate 25-30 ewes and smaller pens can accommodate 14-16 ewes
the farmer had 28-30 twin-bearing ewes in these larger pens and felt they had adequate feeding and lying space
Additional rails are fitted on both the barriers and walk-through troughs
“This was done to accommodate working with ewes and lambs inside during the summer to prevent lambs escaping through the barriers
so I’m hopeful this will prevent that re-occurring,” the farmer outlined
An adjustable double railing was specced to prevent lambs escaping during summer work
At the end of each walkthrough feeder is a hinged 3ft gate allowing ewes to be moved between pens without having to be brought into the feed passageway
The farmer noted that this small feature was of great benefit when scanning and batching ewes earlier in the spring
The generous 4.7m feed passageway allows for easy access and feeding
It is intended that the double row of 18-20 lambing pens in the centre of the passage will be set up at lambing time
with enough room available at either end to drop a block or bale of silage
access to good-quality straw at an affordable price is becoming an increasingly difficult for this farmer
in his existing four-bay solid-floor sheep shed
he felt that he was spending more time bedding ewes than he was feeding
it was decided to install MIK plastic sheep slats
The sloping nature of the site means much of the tank wall is exposed on one side
They were installed over a 1.8m shuttered concrete tank
with outside agitation points at either end of the tank
was not required as the existing cattle shed has a suspended passage ensuring more than adequate slurry storage on the farm
The sloping nature of the site added some extra costs and workload to the project
One side of the tank is mainly above ground level
The girders for the shed frame sit on the tank wall
which is 300mm in width to accommodate this load
Although the shed could have been located elsewhere in the yard
the farmer felt the chosen location allowed the new build to sit well within the existing farmyard
The location of the shed also allows the farmer to roof in between the existing cattle shed and new sheep shed at some stage in the future if desired
Plastic slats and walkthrough feeders were used to create ample feed space and reduce labour
This side of the sheep shed was purposely left higher to allow for girders to be grafted on
Door spaces were left at either end of the new shed
with plans to knock doors from the cattle shed into this space as well
A cattle crush is currently being installed
with plans to have two dry calving pens and two dry pens as an overflow for ewes and lambs post-lambing in this space
Raising the new shed height will allow for a cost-effective roofed handling facility as well as extra calving/lambing facilities
A total of 24 reinforced skylights are fitted in the shed
leaving the area extremely bright and comfortable to work in
vented sheeting with a gap between the roof and side sheeting for greater air intake was chosen
A central canopy over the feed passage acts as an outflow for stale air
the shed was bright and airy with no drafts
All work was carried out to TAMS specifications
with the farmer qualifying for a 40% grant on this build
Extra concrete around the shed resulted in €1,000 extra
as well as a €1,000 cost for modifications to the shed frame to raise the height on one side to allow for future developments – both of these costs are accounted for above
Stainless steel runners for under the plastic slats were chosen for longevity
Money was saved on the total project by the farmer carrying out some work themselves
including digging and backfilling the tank
installing barriers and completing all necessary plumbing work
The farmer hopes to receive his full €32,000 back from his TAMS grant
leaving a net cost to him of roughly €91,341
This leaves the cost per ewe at €456.70 across 200 ewes
materials used will ensure that the shed will have a long lifespan and it is a long-term investment on the farm to allow for future expansion
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Mother’s Day is not something everyone celebrates
Some people have a tortured relationship with their mom
their mom may have died and this day is a reminder
And some may desperately want to be mothers
Leave space for people to feel whatever emotions they have on this day
there is an ancient tradition within Christianity of seeing Jesus as our Mother
the Roman soldier pierced him with a sword
Many have seen this as Jesus giving birth to something new
Have there been other men in your life who have been a mother figure to you
Main Conversation: John 10:22-30 – Good Shepherd Sunday
John 10 describes Jesus as the Good Shepherd
Ezekiel said the Shepherd was supposed to care for the flock
the ancient kings of Israel were bad shepherds
They amassed wealth at the expense of the poor
Jesus is walking through Solomon’s portico
He was one of the greatest kings in Israel’s history
Israel was freed from Egypt because the Egyptians forced them into slavery to build their temples
Solomon built the Temple in Jerusalem through forced labor
Solomon also made a lot of money through buying and selling weapons – namely horses and chariots from Egypt
Jesus’ opponents ask him to be upfront and tell him if he is the Messiah
Jesus tells them that he has already shown him that he is the Messiah
Lots of ink has been spilled trying to figure out what that means
“The Bible and God are one.” Many people treat the Bible as if it were one with God
God is like the one who has nothing to do with sacrificing or killing people
and loving people with acts of nonviolence
Jesus is the Good Shepherd in contrast to the bad shepherds who sacrifice their sheep
Jesus leads his sheep away from sacrifice and into eternal life
This is not an eternal life in the future after you die
We receive eternal life by becoming Jesus’ sheep – by listening to his voice and following him
But Jesus seems to make a promise he can’t keep
He says that no one can take his sheep away from him
11 of your 12 original disciples were killed
and the 12 was exiled to a horrible island called Patmos
I think your sheep were take away from you and sacrificed…right
maybe Jesus meant no one can take his love away
Jesus’ disciples knew that they were loved by God
They know that in a world committed to violence
the disciples knew that death doesn’t have the last word
for the Good Shepherd leads us into eternal life
<a href="https://commons.wikimedia.org/wiki/File:StJohnsAshfield_StainedGlass_GoodShepherd_Portrait.jpg" class=" decorated-link" target="_blank" rel="nofollow">Image: Wikimedia Commons.</a>