Die Autoren möchten sich bei der Aufteilung des Vector-Borne Diseases Animal Resources Abteilung bedanken, speziell Andrea Peterson, Lisa Massoudi, Verna O&#39;Brien, John Liddell und für ihre Pflege und Wartung der Mäuse und Kaninchen. Wir möchten auch an Amy Ullmann, Theresa Russell, und Barbara J. Johnson für ihre Beiträge zu diesem Manuskript danken. Schließlich möchten wir Alissa Eckert in das Amt des Associate Director für Kommunikation an der CDC anerkennen für die Herstellung der grafischen Darstellungen und Judy Lavelle zum Lenken alle Gesetzmäßigkeiten mit den Dreharbeiten zu dieser Handschrift verbunden.

1. Hämolymphe Sammlung der Präparation der Objektträger (Movie 1) <ol><li> Vorsichtig entfernt aktiv Zuführen Zecken aus einem Tier und in 3% Wasserstoffperoxid topische für 5 Minuten und dann in 70% Ethanol für 10 Minuten an die Oberfläche zu sterilisieren. </li><li> Mit einem Pap-Stift zeichnen Sie einen Kreis auf einem Silan beschichtete Objektträger und steckt die Zecke innerhalb der PAP PEN Kreis. <ol><li> Silan beschichtete Objektträger für die besten Einhaltung der Hämolymphe auf den Objektträger verwendet. </li></ol></li><li> Sehen Sie das Häkchen unter einem Binokular (1X Ziel, 10x Okular, 3,5 facher Vergrösserung). </li><li> Drücken Sie vorsichtig auf der Zecke mit einer Pinzette zu Dorsum der Zecke die Beine spreizen und immobilisieren die Zecke. </li></ol> Hinweis: Bei der Immobilisierung die Zecke nicht zu fest drücken, da dies die Punktion oder Mitteldarm der Zecke kann stören und verunreinigen das Hämolymphe. <ol start="5"><li> Amputieren der Zecke am Bein oder die Beine an the distale Gelenk mit einer feinen Spitze Einweg-Skalpell. Um die Infektiosität zu ermitteln via Hämolymphe nur ein Bein muss amputiert werden. Für Sammlung von Hämolymphe auf einer Folie mehrere Beine amputiert werden kann. </li></ol> Hinweis: Schneiden Sie nicht das Bein zu nahe an den Körper, weil diese Mitteldarm Kontamination der Hämolymphe verursachen können. <ol start="6"><li> Nachdem das Bein oder die Beine abgeschnitten sind weiterhin üben Sie sanften Druck an der Zecke Dorsum für die Hämolymphe zur Sekretion aus den Beinen auf die Folie. Bewegen Sie das Häkchen um auf der Folie, um die Hämolymphe zu verbreiten. </li></ol> 2. Speicheldrüse Entfernung (Filme 2 &amp; 3) <ol><li> Spot 25 ul mehrere Pools von Phosphat-gepufferter Salzlösung (PBS) auf einen Objektträger und legen einen Haken in eine der PBS-Pools. </li><li> Sehen Sie das Häkchen unter einem Binokular (1X Ziel, 10x Okular, 3,5 facher Vergrösserung). </li><li> Stabilisieren Sie die Zecke mit feinen Spitzen forceps, indem Sie die Basis Capitulum (Mundwerkzeuge) oder den hinteren Teil der Zecke. </li><li> Legen Sie die feinen Pinzetten in die hintere der Zecke und in Scheiben schneiden bis die Zecke ist Dorsum, um die Organe freizulegen. Falls gewünscht kann der Mitteldarm zu diesem Zeitpunkt entfernt werden und in ein frisches Pool von PBS auf einem Objektträger oder einem Mikrozentrifugenröhrchen mit PBS. </li><li> Finden Sie das Paar Speicheldrüsen (Traube-wie Cluster) bilateral neben den Beinen der Zecke befinden. Wenn die Speicheldrüsen sind nicht unter die Zecke Trümmer bewegen Sie den wichtigsten Teil Tick, immer noch mit den Speicheldrüsen, in ein frisches Pool von PBS, um die Störung und Verlust der Speicheldrüsen reduzieren sichtbar. </li></ol> Hinweis: Frühere in einer Fütterung sind die Speicheldrüsen schwieriger zu lokalisieren, weil sie nicht so weit entwickelt sind als im Vergleich zu einem späteren Zeitpunkt in der Fütterung. <ol start="6"><li> Entfernen Sie die Speicheldrüsen der Zecke aus mit feinen Pinzetten und in ein frisches Pool von PBS. </li><li> With feinen Pinzetten übertragen Sie die Speicheldrüsen zu einem anderen sauberen 25 ul PBS-Pool, wiederholen Dieser Waschschritt 3-4 weitere Male, um alle externen Mikroorganismen und Zecken zu entfernen. </li></ol> Hinweis: Waschen Sie die Speicheldrüse Cluster sanft, um den Verlust und die Störung der einzelnen Speicheldrüsen zu reduzieren. <ol start="8"><li> Platzieren Sie die Drüsen in einem sauberen Pool von PBS auf einem mit Silan beschichtete Folie oder in einem Mikrozentrifugenröhrchen mit PBS. </li></ol> 3. Speichelsammlung (Movie 4) <ol><li> Entfernen Sie vorsichtig erwachsene weibliche Zecken aus dem Kaninchen oder anderen Host mit feinen Pinzetten, kurz bevor sie abfallen sich prall an, etwa 5-7 Tage nach der Befestigung. </li><li> Beachten Sie die fast engorged Tick auf das eine Ende eines Objektträgers mit Tesafilm. Das Band sollte etwa platziert werden ¾ der Weg nach oben der Zecke Rücken in Richtung des Kopfes, so dass der Zecke Grundlage Capitulum (Mundwerkzeuge) ausgesetzt.</li><li> Wo die Band den vorderen Rand der Zecken Dorsalfläche Pipette 5 ul 5% Pilocarpin-Lösung (in Methanol) erfüllt. Lassen Sie das Band, um das Pilocarpin über die Zecke Dorsum Docht, ohne dass das Pilocarpin in Kontakt mit der Zecke Grundlage Capitulum kommen. </li><li> Bringen Sie ein Stück ungiftig Modelliermasse auf den Objektträger etwa einen Zoll von der Zecke Mundwerkzeuge. </li><li> Mit feinen Pinzetten brechen Sie die Spitze eines beigezogenen Kapillarrohr 28 auf den gewünschten Durchmesser. </li><li> Ansicht der Zecke Grundlage Capitulum unter einem Binokular. </li><li> Sanften Sitz der Zecke Hypostom in die Kapillare gezogen so dass die oberen Palpen auf der Außenseite des Kapillarrohrs befinden. </li><li> Drücken Sie die entgegengesetzten Ende des Kapillarrohrs in der Modelliermasse um das Kapillarrohr in Position zu halten. </li><li> Stellen Sie das montierte salivating Zecke in einer dunklen Kammer mit hoher Luftfeuchtigkeit (z. B. ein Deckel Styroporbox mit nassen Brei gefüttert<html> ER Handtücher). Drücken Sie den Träger so den Hypostom Punkten auf den Boden des Behälters, so dass die Schwerkraft beim Speichelprobe zu unterstützen. </li><li> Stellen Sie den Behälter bei Raumtemperatur. </li><li> Genau beobachten salivating Zecken für die erste Stunde, und sammeln Speichel, wie es durch Austreiben es aus der Kapillarröhrchen mit einer Pasteur-Pipette Glühbirne erzeugt wird. Nach der ersten Stunde, überprüfen Anhäufung jede Stunde für mindestens 4 Stunden. Weiter, um den Speichel, wie es erzeugt wird, zu sammeln. </li></ol> Hinweis: Der Speichel Erwerb kann gestoppt werden, sobald genügend Speichel für die Prüfung durchgeführt wird, gesammelt werden. <ol start="12"><li> Wenn die Zecken sind nicht Speichelfluss oder wenn mehr Speichel benötigt wird, Speichelfluss kann gelegentlich mit der Kapillare eine Hypostom massieren induziert werden. </li><li> Geben Sie 0,1 Volumina von Protease Inhibitor Cocktail auf die Speichel-und Speicher bei -80 ° C bis sie benötigt werden. </li></ol> 4. Repräsentative Ergebnisse e_content &quot;&gt; Movie 1 veranschaulicht, wie eine nur teilweise eingezogenen I. scapularis Nymphe halten und die Beine amputieren, um Hämolymphe auf einen Objektträger zu sammeln. Sobald das Bein oder Beine amputiert werden eine klare Flüssigkeit abgesondert wird (Abbildung 1A und 1B). Wenn der Mitteldarm gebrochen wird die Hämolymphe erscheint trübe, wie es ist kommt aus dem amputierten Bein (e) (Abbildung 1C und 1D). Die Gewinnung von Speicheldrüsen nach der Nymphe hat sich für 48 oder 72 Stunden gefüttert wird in Filmen 2 und 3 gezeigt. Nachdem die Zecke punktiert wird es im Allgemeinen eine Menge Schutt (bestehend aus Luftröhre, Malpighischen Gefäße, Blut, Bindegewebe etc.) zu bewegen, um den Verlust oder die Störung der Speicheldrüsen der Zecke zu verhindern, um einen frischen Pool von PBS. 2A und 2B zeigen, wo die Speicheldrüsen befinden, nachdem die Nymphe aufgeschnittenen sind und 2C zeigt entfernt Speicheldrüse Cluster in einem Pool von PBS. Speichelsammlung aus I festgelegt scapularis erwachsenen Weibchen i<html> s in Film 4 und Abbildung 3 dargestellt. Ein Häkchen salivating in ein Kapillarrohr ist in Film 5 beobachtet. Diese Methode der Speichel-Auflistung verwendet, um den Speichelfluss anzuregen Pilocarpin und kann über 20 ul Speichel pro erwachsene weibliche Zecke liefern. <img alt="Abbildung 1." src="/files/ftp_upload/3894/3894fig1.jpg" /> Abbildung 1. Markierten Strukturen einer I. scapularis Nymphe. Movie 1. Ixodes scapularis Hämolymphe Sammlung. <a href="http://www.jove.com/files/ftp_upload/3894/3894movie1.avi">Hier klicken, um Film zu sehen</a> . <img alt="Abbildung 2." src="/files/ftp_upload/3894/3894fig2.jpg" /> Abbildung 2. Nicht kontaminierte (A &amp; B) und verunreinigt (C &amp; D) Hämolymphe exsudierenden von der Nymphe Bein. Movie 2. Speicheldrüse Extraktion aus einem 48 Stunden gefüttert I. scapularis Nymphe.pload/3894/3894movie2.avi &quot;&gt; Klicken Sie hier, um Film zu sehen. Movie 3. Speicheldrüse Extraktion aus einem 72 Stunden gefüttert I. scapularis Nymphe. <a href="http://www.jove.com/files/ftp_upload/3894/3894movie3.avi">Klicken Sie hier, um Film zu sehen</a> . <img alt="Abbildung 3." src="/files/ftp_upload/3894/3894fig3.jpg" /> Abbildung 3. Ixodes scapularis Nymphe Speicheldrüsen. (A &amp; B) Beispiele für Speicheldrüsen in 72 Stunden gefüttert Nymphe, vor der Extraktion. (C) entfernt Speicheldrüse Cluster. Movie 4. Speichelsammlung up von einem erwachsenen I festgelegt scapularis weibliche Zecke. <a href="http://www.jove.com/files/ftp_upload/3894/3894movie4.avi">Klicken Sie hier, um Film zu sehen</a> . <img alt="Abbildung 4." src="/files/ftp_upload/3894/3894fig4.jpg" /> Abbildung 4. Speichelsammlung aus adulten I. scapularis weibliche tIcks. (A &amp; B) Markieren auf einem Objektträger mit Hypostom in der ausgezogenen Ende des Kapillarröhrchens mit dem unpulled Ende des Kapillarrohrs durch Modelliermasse gehalten. (C &amp; D) befeuchteten Kammer mit salivating erwachsenen I. scapularis weiblichen Zecken. Movie 5. Ixodes scapularis weibliche Zecke salivating in ein Kapillarrohr. <a href="http://www.jove.com/files/ftp_upload/3894/3894movie5.avi">Klicken Sie hier, um Film zu sehen</a> .

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composition+of+tick+oral+secretions+obtained+by+three+different+collection+methods.">Composition of tick oral secretions obtained by three different collection methods.</a> J. Med. Entomol. 10, 198-201 (1973).</li><li>Burgdorfer, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemolymph+test.+A+technique+for+detection+of+rickettsiae+in+ticks.">Hemolymph test. A technique for detection of rickettsiae in ticks.</a> Am. J. Trop. Med. Hyg. 19, 1010-1014 (1970).</li></ol>

Wir haben nichts zu offenbaren.

Die Sammlung von Zecken Hämolymphe, Speicheldrüsen, und Speichel ist in der Studie von Zecken übertragene Erreger Übertragung wichtig, Prävalenz, Verbreitung, Vermehrung und Persistenz sowohl in der Zecke und dem Host 6,11-13,20,23,29. Es gibt mehrere Möglichkeiten, um ein Häkchen 30,31 sezieren. Allerdings, wenn das Sammeln Speicheldrüsen ist es wichtig, die Zecke richtig, so dass die Speicheldrüsen nicht gebrochen oder verloren in der Zecke die Überreste zu sezieren. Sobald die Speichel...

<table border="1"><tbody><tr><td> Reagens </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> Wasserstoffperoxid </td><td> Fischer </td><td> H312-500 </td></tr><tr><td> Ethanol </td><td> Acros </td><td> 61509-5000 </td></tr><tr><td> PBS </td><td> Boston Bioproducts </td><td> BM-2205 </td></tr><tr><td> Dumont feinen Pinzette (3C) </td><td> Fischer </td><td> NC9906085 </td></tr><tr><td> Silan behandelten Objektträger </td><td> Bioworld </td><td> 42763007-1 </td></tr><tr><td> Pap-pen </td><td> Bioworld </td><td> 21750008-1 </td></tr><tr><td> Super-Frost Plus Objektträger </td><td> Fischer </td><td> 12-550-18 </td></tr><tr><td> Pilocarpin </td><td> Sigma </td><td> P6503-5G </td></tr><tr><td> Protease-Inhibitor-Cocktail </td><td> Sigma </td><td> P2714 </td></tr><tr><td> Nr. 11 Einweg-Skalpell </td><td> Feder </td><td> 2975 # 11 </td></tr><tr><td> Ungiftige Knetmasse </td><td&gt; Fisher </td><td> S17307 </td></tr><tr><td> Kapillarrohre </td><td> Chase Scientific Glass, inc. </td><td> 40A502 </td></tr></tbody></table>

saliva, salivary gland, and hemolymph collection from ixodes scapularis ticks

Zecken sind weltweit und plagen die Menschen mit vielen Zecken-übertragene Erkrankungen. Zecken sind Vektoren für Krankheitserreger, die Lyme-Borreliose und Frühsommer-Rückfallfieber (Borrelia spp.), Rocky Mountain Spotted Fever (Rickettsia rickettsii), Ehrlichiose (Ehrlichia chaffeensis und E. equi), Anaplasmose (Anaplasma phagocytophilum), Enzephalitis (tick-verursachen Meningo-Enzephalitis-Virus), Babesiose (Babesia spp.), Colorado Zeckenbissen (Coltivirus) und Tularämie (Francisella tularensis) 1-8. Um richtig in den Wirt übertragen werden diese Erreger unterschiedlich die Genexpression regulieren, interagieren mit Zecken-Proteine ​​und wandern durch die Zecke 3,9-13. Zum Beispiel, passt sich die Lyme-Borreliose-Agent, Borrelia burgdorferi, durch differentiellen Genexpression zu dem Fest und Hungersnot Stadien der Zecke enzootischen Zyklus 14,15. Darüber hinaus verbraucht als ein Ixodes Zecken einBlutmehl Borrelia replizieren und wandern aus dem Mitteldarm in die Haemocoel, wohin sie wandern in die Speicheldrüsen und werden in den Wirt mit dem Speichel vertrieben 9,16-19 übertragen. Wie eine Zecke führt der Host antwortet in der Regel mit einer starken hämostatischen und angeborene Immunantwort 11,13,20-22. Trotz dieser Wirtsreaktionen, I. scapularis kann für mehrere Tage zu ernähren, da Zecken Speichel enthält Proteine, die immunmodulatorische sind, lytische Mittel, Antikoagulanzien und fibrinolysins, die Zecke 3,11,20,21,23 Fütterung zu helfen. Die immunmodulatorischen Aktivitäten von Zeckenspeichel oder Speicheldrüsen-Extrakt (SGE) besessen zu erleichtern Übertragung, Vermehrung und Verbreitung von zahlreichen Zecken übertragenen Krankheitserregern 3,20,24-27. Um weiter zu verstehen, wie durch Zecken übertragene Erreger eine Krankheit verursachen es wichtig, aktiv zu sezieren Fütterung Zecken und sammeln Zeckenspeichel ist. Dieses Video demonstriert Protokoll Dissektion Techniken fürdie Sammlung von Hämolymphe und die Entfernung der Speicheldrüsen von der aktiven Zuführen I. scapularis Nymphen nach 48 und 72 Stunden nach der Platzierung der Maus. Wir zeigen auch, Speichel Kollektion von einer erwachsenen Frau I. scapularis tick.

Watch this Scientific Journal Video about Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks at JoVE.com

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Die Sammlung von infizierten Zecke Hämolymphe, Speicheldrüsen, und Speichel ist wichtig, zu untersuchen, wie durch Zecken übertragene Erreger eine Krankheit verursachen. In diesem Protokoll haben wir demonstrieren, wie man Hämolymphe und Speicheldrüsen von der Fütterung sammeln<em&gt; Ixodes scapularis</em&gt; Nymphen. Wir zeigen auch, Speichel Sammlung von weiblichen<em&gt; I. scapularis</em&gt; Erwachsene.

Speichel, Speicheldrüse, und Hämolymphe Abholung Ixodes scapularis Zecken

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

saliva-salivary-gland-hemolymph-collection-from-ixodes-scapularis

Research

JoVE Journal

Immunology and Infection

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

29.2K Aufrufe.  Centers for Disease Control and Prevention. Die Sammlung von infizierten Zecke Hämolymphe, Speicheldrüsen, und Speichel ist wichtig, zu untersuchen, wie durch Zecken übertragene Erreger eine Krankheit verursachen. In diesem Protokoll haben wir demonstrieren, wie man Hämolymphe und Speicheldrüsen von der Fütterung sammeln Ixodes scapularis Nymphen. Wir zeigen auch, Speichel Sammlung von weiblichen I. scapularis Erwachsene.

Serie: Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

RNA Interference in Ticks

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors of human diseases1 and the most important vectors affecting cattle industry worldwide2. Ticks are classified in the subclass Acari, order Parasitiformes, suborder Ixodida and are distributed worldwide from Arctic to tropical regions3. Despite efforts to control tick infestations, these ectoparasites remain a serious problem for human and animal health4,5.
RNA interference (RNAi)6 is a nucleic acid-based reverse genetic approach that involves disruption of gene expression in order to determine gene function or its effect on a metabolic pathway. Small interfering RNAs (siRNAs) are the effector molecules of the RNAi pathway that is initiated by double-stranded RNA (dsRNA) and results in a potent sequence-specific degradation of cytoplasmic mRNAs containing the same sequence as the dsRNA trigger7-9. Post-transcriptional gene silencing mechanisms initiated by dsRNA have been discovered in all eukaryotes studied thus far, and RNAi has been rapidly developed in a variety of organisms as a tool for functional genomics studies and other applications10.
RNAi has become the most widely used gene-silencing technique in ticks and other organisms where alternative approaches for genetic manipulation are not available or are unreliable5,11. The genetic characterization of ticks has been limited until the recent application of RNAi12,13. In the short time that RNAi has been available, it has proved to be a valuable tool for studying tick gene function, the characterization of the tick-pathogen interface and the screening and characterization of tick protective antigens14. Herein, a method for RNAi through injection of dsRNA into unfed ticks is described. It is likely that the knowledge gained from this experimental approach will contribute markedly to the understanding of basic biological systems and the development of vaccines to control tick infestations and prevent transmission of tick-borne pathogens15-19.

A method for RNA interference (RNAi) by injection of dsRNA into unfed ticks is described. RNAi is the most widely used gene-silencing technique in ticks where the use of other methods of genetic manipulation has been limited.

RNA-Interferenz in Ticks

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors ...

Forschung

Immunologie und Infektion

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes. Investigation of transcripts and gene regulators that are expressed in tissues in which the mosquito host and pathogen interact, and in organs involved in reproduction are of great interest for strategies to reduce mosquito-borne disease transmission and disrupt egg development. A number of tools have been employed to study and validate the temporal and tissue-specific regulation of gene expression. Here, we describe protocols that have been developed to obtain spatial information, which enhances our understanding of where specific genes are expressed and their products accumulate. The protocol described has been used to validate expression and determine accumulation patterns of transcripts in tissues related to mosquito-borne pathogen transmission, such as female salivary glands, as well as subcellular compartments of ovaries and embryos, which relate to mosquito reproduction and development. 
 The following procedures represent an optimized methodology that improves the efficiency of various steps in the protocol without loss of target-specific hybridization signals. Guidelines for RNA probe preparation, dissection of soft tissues and the general procedure for fixation and hybridization are described in Part A, while steps specific for the collection, fixation, pre-hybridization and hybridization of mosquito embryos are detailed in Part B.

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Temporal and spatial gene expression analyses have a crucial role in functional genomics. Whole-mount hybridization in situ is useful for determining the localization of transcripts within tissues and subcellular compartments. Here we outline a hybridization in situ protocol with modifications for specific target tissues in mosquitoes.

Hybridisierung<em&gt; In-situ-</em&gt; Der Speicheldrüsen, Ovarien und Embryonen von Vektor-Mücken

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes.  Investigation of transcripts and gene ...

Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the attachment and blood feeding of Ixodes species ticks on mammalian hosts. In nature, this zoonotic bacterial pathogen may use a variety of reservoir hosts, but the white-footed mouse (Peromyscus leucopus) is the primary reservoir for larval and nymphal ticks in North America. Humans are incidental hosts most frequently infected with B. burgdorferi by the bite of ticks in the nymphal stage. B. burgdorferi adapts to its hosts throughout the enzootic cycle, so the ability to explore the functions of these spirochetes and their effects on mammalian hosts requires the use of tick feeding. In addition, the technique of xenodiagnosis (using the natural vector for detection and recovery of an infectious agent) has been useful in studies of cryptic infection. In order to obtain nymphal ticks that harbor B. burgdorferi, ticks are fed live spirochetes in culture through capillary tubes. Two animal models, mice and nonhuman primates, are most commonly used for Lyme disease studies involving tick feeding. We demonstrate the methods by which these ticks can be fed upon, and recovered from animals for either infection or xenodiagnosis.

Lyme disease is the most commonly-reported vector-borne disease in North America. The causative agent, Borrelia burgdorferi is a spirochete bacterium transmitted by Ixodid ticks. Transmission and detection of infection in animal models is optimized by the use of tick feeding, which we describe here.

Fütterung von Zecken auf Tiere, die zur Übertragung und Xenodiagnosis in Lyme Disease Research

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the  attachment and blood feeding of Ixodes species ticks on ...

Rearing Ixodes scapularis, the Black-legged Tick: Feeding Immature Stages on Mice

Ixodes scapularis, the vector of Lyme disease, is one of the most important disease vectors in the eastern and Midwestern United States. This species is a three host tick that requires a blood meal from a vertebrate host for each development stage, and the adult females require a blood meal for reproduction. Larval ticks attach to their host for 3 - 5 days for feeding and drop off the host when fully engorged. This dependency on several different hosts and the lengthy attachment time for engorgement complicates tick rearing in the laboratory setting. However, to understand tick biology and tick-pathogen interactions, the production of healthy, laboratory-reared ticks is essential. Here, we demonstrate a simple, cost-effective protocol for immature tick feeding on mice. We modified the existing protocols for decreased stress on mice and increased tick feeding success and survival by using disposable cages without mesh bottoms to avoid contact of ticks with water contaminated with mice urine and feces.

Rearing Ixodes scapularis, the Black-legged Tick: Feeding Immature Stages on Mice

The production of healthy laboratory-reared ticks is essential to studies on tick biology, and tick-pathogen interactions. Here we demonstrate a simple protocol for immature tick feeding that is cost-effective and less stressful to mice.

Aufzucht<em&gt; Ixodes scapularis,</em&gt; Die schwarzbeinigen Tick: Fütterung unreife Stadien auf Mäusen

Ixodes scapularis, the vector of Lyme disease, is one of the most important disease vectors in the eastern and Midwestern United States. This species is a ...

A Method for the Measurement of Salivary Gland Function in Mice

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Salivary gland hypofunction is a frequent consequence of autoimmune disease and radiation therapy. Reproducible evaluation of salivary gland function in mouse models of these diseases is a technical challenge. Here, a simple method for accurate and reproducible measurement of saliva production in mice is described.

Eine Methode zur Messung der Speicheldrüse Funktion bei Mäusen

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...

Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, including cell biology, oncology, dentistry, and immunology. The SMG is an exocrine gland comprised of secretory epithelial cells, myofibroblasts, endothelial cells, nerves, and extracellular matrix. Dynamic cellular processes in the rat and mouse SMG have previously been imaged, mostly using inverted multi-photon microscope systems. Here, we describe a straightforward protocol for the surgical preparation and stabilization of the murine SMG in anesthetized mice for in vivo imaging with upright multi-photon microscope systems. We present representative intravital image sets of endogenous and adoptively transferred fluorescent cells, including the labeling of blood vessels or salivary ducts and second harmonic generation to visualize fibrillar collagen. In sum, our protocol allows for surgical preparation of mouse salivary glands in upright microscopy systems, which are commonly used for intravital imaging in the field of immunology.

We describe a protocol to surgically expose and stabilize the murine submandibular salivary gland for intravital imaging using upright intravital microscopy. This protocol is easily adaptable to other exocrine glands of the head and neck region of mice and other small rodents.

Vorbereitung der murinen mandibulären Speicheldrüse aufrecht Intravitalen Mikroskopie

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, ...

Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to restore function of glands damaged by radiation therapies or due to pathologies associated with Sj&#246;gren's syndrome. A second goal of researchers is to define the mechanisms by which viruses replicate within glandular tissue where they can then gain access to salivary fluids important for horizontal transmission. These goals highlight the need for a robust and accessible in vitro salivary gland model that can be utilized by researchers interested in the above mentioned as well as related research areas. Here we discuss a simple protocol to isolate epithelial cells from human salivary glands and propagate them in vitro. Our protocol can be further optimized to meet the needs of individual studies. Briefly, salivary tissue is mechanically and enzymatically separated to isolate single cells or small clusters of cells. Selection for epithelial cells occurs by plating onto a basement membrane matrix in the presence of media optimized to promote epithelial cell growth. These resulting cultures can be maintained as three-dimensional clusters, termed "salispheres", or grown as a monolayer on treated plastic tissue culture dishes. This protocol results in the outgrowth of a heterogenous population of mainly epithelial cells that can be propagated for 5-8 passages (15-20 population doublings) before undergoing cellular senescence.

We present a method for isolating and cultivating primary human salivary gland-derived epithelial cells. These cells exhibit gene expression patterns consistent with them being of salivary epithelial origin and can be grown as salispheres on basement membrane matrices derived from Engelbreth-Holm-Swarm tumor cells or as monolayers on treated culture dishes.

Isolierung von Speicheldrüsenepithelzellen aus menschlichen Speicheldrüsen für In-Vitro-Wachstum als Speicheldrüsen oder Monolayer

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to ...

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

Reinigung von hochertragreichen extrazellulären Vesikelpräparaten weg vom Virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight loss, depreciation of the animals&#39; leather and also can act as a vector of several pathogens. Due to the exorbitant costs to control these parasites, damage to the environment caused by the inappropriate use of chemical acaricides, and the increased resistance against traditional parasiticides, alternative control of ticks, by the use of entomopathogenic fungi, for example, has been considered an interesting approach. Nevertheless, few studies have demonstrated how the tick&#39;s immune system acts to fight these entomopathogens. Therefore, this protocol demonstrates two methods used for entomopathogen inoculation into engorged females and two techniques used for hemolymph collection and hemocytes harvesting. Inoculation of pathogens at the leg insertion in the tick female&#39;s body allows evaluation of females biologic parameters unlike the inoculation between the scutum and capitulum, which frequently damages Gen&#233;&#39;s organ. Dorsal hemolymph collection yielded a higher volume recovery than collection through the legs. Some limitations of tick hemolymph collection and processing include i) high rates of hemocytes&#39; disruption, ii) hemolymph contamination with disrupted midgut, and iii) low hemolymph volume recovery. When hemolymph is collected through the leg cutting, the hemolymph takes time to accumulate at the leg opening, favoring the clotting process. In addition, fewer hemocytes are obtained in the collection through the leg compared to the dorsal collection, even though the first method is considered easier to be performed. Understanding the immune response in ticks mediated by entomopathogenic agents helps to unveil their pathogenesis and develop new targets for tick control. The inoculation processes described here require very low technological resources and can be used not only to expose ticks to pathogenic microorganisms. Similarly, the collection of tick hemolymph may represent the first step for many physiological studies.

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

Analysis of tick hemolymph represents an important source of information on how some pathogens cause disease and how ticks immunologically respond to this infection. The present study demonstrates how to inoculate fungal propagules and collect hemolymph from Rhipicephalus microplus engorged females.

Die Sammlung der Häkolymphe und die Inokulation von Metarhizium-Blastospores in Rhipicephalus Microplus Ticks Towards Pathology Studies

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight ...

Echtzeit-Live-Bildgebung der Regeneration der submandibulären Drüse der Maus

Interrogating Cell-Cell Interactions in the Salivary Gland via Ex Vivo Live Cell Imaging

Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal role played by macrophages in driving the regenerative response. However, our understanding of this critical role has primarily relied on static views obtained from fixed tissue biopsies. To overcome this limitation and gain insights into these interactions in real time, this study outlines a comprehensive protocol for culturing salivary gland tissue ex vivo and capturing live images of cell migration.
The protocol involves several key steps: First, mouse submandibular salivary gland tissue is carefully sliced using a vibratome and then cultured at an air-liquid interface. These slices can be intentionally injured, for instance, through exposure to radiation, to induce cellular damage and trigger the regenerative response. To track specific cells of interest, they can be endogenously labeled, such as by utilizing salivary gland tissue collected from genetically modified mice where a particular protein is marked with green fluorescent protein (GFP). Alternatively, fluorescently-conjugated antibodies can be employed to stain cells expressing specific cell surface markers of interest. Once prepared, the salivary gland slices are subjected to live imaging using a high-content confocal imaging system over a duration of 12 h, with images captured at 15 min intervals. The resulting images are then compiled to create a movie, which can subsequently be analyzed to extract valuable cell behavior parameters. This innovative method provides researchers with a powerful tool to investigate and better understand macrophage interactions within the salivary gland following injury, thereby advancing our knowledge of the regenerative processes at play in this dynamic biological context.

Autor im Rampenlicht: Untersuchung von Makrophagen-Epithelzell-Interaktionen bei der Regeneration von Speicheldrüsen nach Verletzungen 

Immunofluorescent imaging is constrained by the ability to observe complex, time-dependent biological processes in just a single snapshot in time. This study outlines a live-imaging approach conducted on precision-cut mouse submandibular gland slices. This approach allows for the real-time observation of cell-cell interactions during homeostasis and the processes of regeneration and repair.

Untersuchung von Zell-Zell-Interaktionen in der Speicheldrüse mittels ex vivo Lebendzell-Bildgebung

Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal ...