Yazarlar vektör kaynaklı hastalıkların Hayvan Kaynakları Şubesi Bölümü teşekkür etmek istiyorum, özellikle Andrea Peterson, Lisa Massoudi, Elçin O&#39;Brien ve fare ve tavşan bakımı ve bakım için John Liddell. Biz de bu yazının doğru katkıları için Amy Ullmann, Theresa Russell, ve Barbara J. Johnson teşekkür etmek istiyorum. Son olarak, bu yazının filme ile ilgili tüm legalities yönlendirmek için grafik çizimler ve Judy Lavelle üretmek için CDC, İletişim için Associate Direktörü Office Alissa Eckert kabul etmek istiyorum.

1. Slayt hazırlanması için hemolimf toplama (Film 1) <ol><li> Yavaşça yüzeyi sterilize ila 10 dakika süreyle% 70 etanol içinde daha sonra 5 dakika boyunca% 3 topikal hidrojen peroksit ve bir hayvan yerden aktif besleme kenelerin kaldırmak ve. </li><li> Pap kalem silan kaplı mikroskop lamı üzerine bir daire çizin ve pap kalem daire içinde kene yerleştirin ile. <ol><li> Silan kaplı slaytlar mikroskop lamı için hemolimf en iyi yapışması için kullanılır. </li></ol></li><li> Bir mikroskop (1X objektif, 10X mercek, 3.5x büyütme) altında kene görüntüle. </li><li> Yavaşça kene bacaklarının yayılmak ve kene immobilize etmek için forseps ile kenenin sırtı aşağı bastırın. </li></ol> Not: Bu ortabağırsak veya ponksiyon kene bozabilir ve hemolimf kontamine olabilir, çünkü kene immobilizing çok sert basmayın zaman. <ol start="5"><li> Th kenenin bacak veya bacakları organınıtek neşter ince bir nokta ile e distal eklem. Enfektivite belirlemek için hemolimf üzerinden sadece 1 bacağı ampute edilmesi gerekir. Slaytta hemolimf toplanması için birkaç bacakları ampute olabilir. </li></ol> Not: Bu hemolimf bir ortabağırsak kontaminasyona neden olabilir, çünkü vücudun çok yakın bacak kesmeyin. <ol start="6"><li> Bacak veya bacakları kesildikten sonra yavaşça slayt üzerine bacaklar dışarı salgılaması için hemolimf için kenenin sırtından basınç uygulamaya devam edeceklerdir. Yavaşça hemolimf yaymak için slayt etrafında kene taşıyın. </li></ol> 2. Tükürük bezinin çıkarılması (Movies 2 &amp; 3) <ol><li> Fosfat Nokta birkaç 25 ul havuzları bir mikroskop lamı üzerine (PBS) tamponlanmış ve PBS havuzlarından birine tick koyun. </li><li> Bir mikroskop (1X objektif, 10X mercek, 3.5x büyütme) altında kene görüntüle. </li><li> Ince uçlu forsepsle ile kene stabilizetemelinde capitulum (ağızdan parçalar) veya kene arka tutarak s. </li><li> Kene arka içine ince uçlu pens takın ve kene organları açığa sırtı var kadar dilim. İsterseniz ortabağırsak şu anda kaldırılmış ve bir mikroskop lamı üzerine PBS taze bir havuz veya PBS içeren bir mikrofuge&#39;de tüpüne aktarılabilir. </li><li> Bilateral kene bacakları yanında bulunan tükürük bezlerinde (üzüm salkımı benzeri) çifti bulun. Tükrük bezleri kene enkaz tükürük bezlerinin bozulması ve kaybını azaltmak için PBS taze bir havuz, hala tükürük bezleri içeren, ana kene kısmı hareket arasında görünmüyorsa. </li></ol> Not: Daha önce bir beslenme içinde, tükrük bezleri daha sonra beslenme üzerine karşılaştırıldığında onlar gibi gelişmiş olmadığından bulmak zordur. <ol start="6"><li> Ince uçlu pens ve PBS taze bir havuz yer olan kene ile tükürük bezleri çıkarın. </li><li> Wiince inci forseps herhangi bir dış mikroorganizmalar ve kene enkaz kaldırmak için bu yıkama adımı 3-4 defa daha tekrarlayın, başka bir temiz 25 ul PBS havuzuna tükürük bezlerinde transfer uçlu. </li></ol> Not: Bireysel tükürük bezlerinin kaybı ve kesintileri azaltmak için hafifçe tükürük bezi kümeleri yıkayın. <ol start="8"><li> Bir silan kaplı slaytta PBS temiz bir havuza veya PBS içeren bir mikrofuge&#39;de tüp bezleri yerleştirin. </li></ol> 3. Tükürük toplama (Movie 4) <ol><li> Yavaşça tavşan veya tam tıkalı onlar düşüyorlar hemen önce ince uçlu pens, yaklaşık 5-7 gün post-eki kullanarak başka bilgisayardan yetişkin dişi kene kaldırın. </li><li> Seloteyip ile bir mikroskop lamı bir ucuna yaklaşık tıkanık kene uyun. Bant yaklaşık konulmalıdır ¾ baş doğru kenenin sırtı kadar yolu, kenenin temelinde capitulum (ağız parçaları) maruz bırakarak.</li><li> Bandı% 5 pilokarpin çözeltisi kenelerin dorsal yüzeyini pipet 5 ul (metanol içinde) ön kenarının birleştiği yerde. Bant pilokarpin kenenin temelinde capitulum ile temas etmesine izin vermeden, kenenin sırtı pilokarpin fitil izin verin. </li><li> Mikroskop lamı üzerine toksik olmayan modelleme kil parçasının kenenin ağız yaklaşık bir inç monte edin. </li><li> Ince uçlu pens istenilen çapa çekti kılcal tüp 28 ucunu kırın kullanılması. </li><li> Bir mikroskop altında kenenin temelini capitulum görüntüle. </li><li> Yavaşça maksiller palps kılcal boru dışına ikamet için izin çekti kapiler tüpe kenenin hypostome uyacak. </li><li> Kılcal tüp yerinde tutmak için kil modelleme içine kılcal tüpün ucunu basın. </li><li> Yüksek nem (örneğin, ıslak pap ile kaplı bir strafor kapaklı kutusu ile karanlık bir odası içinde monte salyalar kene yerleştirin<html> er havlu). Yerçekimi tükürük toplama yardımcı sağlayan hypostome noktaları böylece, kabın dibine kayar eğim. </li><li> Oda sıcaklığında bir kap yerleştirin. </li><li> Yakından ilk saat için salyalar keneler izlemek ve bir Pasteur pipeti ampul kılcal tüpler dışarı atarak oluşturulan, tükürükteki toplamak. İlk saat sonra, en az 4 saat süreyle birikimi her saat kontrol edin. Oluşturulduğunda gibi tükürük toplamak için devam edin. </li></ol> Not: Yeterince tükürük yapılmaktadır Araştırma için toplanan bir kez Tükürük toplama durdurulabilir. <ol start="12"><li> Keneler salyalar değildir veya daha fazla tükürük gerekiyorsa, salivasyon bazen hypostome masaj kılcal tüpü kullanılarak oluşturulan olabilirsem. </li><li> Ihtiyaç kadar -80 ° C de tükürük ve mağaza proteaz inhibitörü kokteyl 0.1 hacimleri ekleyin. </li></ol> 4. Temsilcisi Sonuçlar e_content &quot;&gt; Film 1 kısmen beslenen I. scapularis perisi tutun ve bir mikroskop lamı üzerine hemolimf toplamak için bacaklarını kesmek zorunda gösterilmiştir. bacak veya bacakları açık bir sıvı (Şekil 1A ve 1B) salgılanır ampute edildikten sonra. Eğer ortabağırsak hemolimf olduğu gibi bulanık görünüyorsa ampute bacak (lar) (Şekil 1C ve 1D) çıkan delinmiş veya yırtılmış. Perisi 48 veya 72 saat beslenme sonra tükürük bezlerinin çıkarılması filmleri 2 ve 3&#39;te gösterilmiştir. Kene enkaz bir sürü genellikle yoktur delinirse sonra (trakea, Malpighi tüpleri tübüller, kan oluşan bağ dokusu vb) tükürük bezlerinin kaybı veya bozulmasını önlemek için, PBS taze bir havuz kene taşıyın. Perisi kesmişler sonra tükürük bezleri bulunur Şekil 2A ve 2B gösterisi ve şekil 2C PBS bir havuzda tükürük bezi başlıkların çıkarılması gösterir. Tükürük toplama I. den kurmak scapularis yetişkin kadın i<html> s film 4 ve Şekil 3&#39;te gösterilmiştir. Bir tüp içine salyalar bir kene film 5 gözlenmektedir. Salivasyon teşvik etmek ve yetişkin dişi kene başına tükürük 20 ul fazla verim alınabilir pilokarpin kullanılan tükürük toplama yöntemi. <img alt="Şekil 1.." src="/files/ftp_upload/3894/3894fig1.jpg" /> Şekil 1. Bir I. scapularis nympha Etiketli yapılar. Film 1. Ixodes scapularis hemolimf koleksiyon. <a href="http://www.jove.com/files/ftp_upload/3894/3894movie1.avi">film izlemek için tıklayınız</a> . <img alt="Şekil 2." src="/files/ftp_upload/3894/3894fig2.jpg" /> Şekil 2. Kirlenmemiş (A &amp; B) ve perisi bacağını gelen yayan (C &amp; D) hemolimf kontamine. 48 saatlik bir beslenen I. Film 2. Tükürük bezi çıkarma scapularis perisi.pload/3894/3894movie2.avi &quot;&gt; film izlemek için tıklayınız. 72 saat beslenen I. Film 3. Tükürük bezi çıkarma scapularis perisi. <a href="http://www.jove.com/files/ftp_upload/3894/3894movie3.avi">film izlemek için tıklayınız</a> . <img alt="Şekil 3." src="/files/ftp_upload/3894/3894fig3.jpg" /> Şekil 3. Ixodes scapularis perisi tükrük bezleri. (A &amp; B) önce çıkarılması için bir 72 saat beslenmiş perisi olarak tükürük bezlerinin örnekleri. (C) tükürük bezi küme kaldırıldı. Film 4. Tükürük toplama bir yetişkin I. den kurmak scapularis dişi kene. <a href="http://www.jove.com/files/ftp_upload/3894/3894movie4.avi">film izlemek için tıklayınız</a> . <img alt="Şekil 4." src="/files/ftp_upload/3894/3894fig4.jpg" /> Yetişkin I. gelen Şekil 4. Tükürük toplama scapularis kadın ticks. (A &amp; B) kil modelleme tarafından tutulan kapiler tüpün unpulled sonu ile kılcal boru çekti sonunda onun hypostome bir slayt monte işaretleyin. (C &amp; D) salyalar yetişkin I. içeren Nemlendirilmiş odası scapularis dişi keneler. Film 5. Bir tüp içine salyalar scapularis dişi kene Ixodes. <a href="http://www.jove.com/files/ftp_upload/3894/3894movie5.avi">film izlemek için tıklayınız</a> .

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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Borreliacidal+activity+of+saliva+of+the+tick+Amblyomma+americanum.">Borreliacidal activity of saliva of the tick Amblyomma americanum.</a> Med. Vet. Entomol. 19, 90-95 (2005).</li><li>Ribeiro, J. M., Zeidner, N. S., Ledin, K., Dolan, M. C., Mather, T. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+much+pilocarpine+contaminates+pilocarpine-induced+tick+saliva?.">How much pilocarpine contaminates pilocarpine-induced tick saliva?.</a> Med. Vet. Entomol. 18, 20-24 (2004).</li><li>Barker, R. W., Burris, E., Sauer, J. R., Hair, J. 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>

Biz ifşa hiçbir şey yok.

Kene hemolimf, tükrük bezleri ve tükürük toplama kene kaynaklı patojen bulaşma çalışmada önemlidir, yaygınlık, yaygınlaştırma, proliferasyon ve kene ve ana 6,11-13,20,23,29 hem de kalıcılık. Bir kene 30,31 incelemek için çeşitli yollar vardır. Ancak, tükrük bezleri alırken düzgün böylece tükürük bezlerinin yırtılması veya kenenin kalıntıların kayıp değildir kene incelemek için önemlidir. Tükürük bezleri onlar ortabağırsak kontaminasyonu gidermek için ...

<table border="1"><tbody><tr><td> Reaktif </td><td> Şirket </td><td> Katalog Numarası </td></tr><tr><td> Hidrojen peroksit </td><td> Balıkçı </td><td> H312-500 </td></tr><tr><td> Etanol </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 Güzel forseps (3C) </td><td> Balıkçı </td><td> NC9906085 </td></tr><tr><td> Silan tedavi mikroskop </td><td> Bioworld </td><td> 42763007-1 </td></tr><tr><td> Pap kalem </td><td> Bioworld </td><td> 21750008-1 </td></tr><tr><td> Süper don artı mikroskop </td><td> Balıkçı </td><td> 12-550-18 </td></tr><tr><td> Pilokarpin </td><td> Sigma </td><td> P6503-5G </td></tr><tr><td> Proteaz inhibitörü kokteyl </td><td> Sigma </td><td> P2714 </td></tr><tr><td> # 11 bistüri atılabilir </td><td> Tüy </td><td> 2975 # 11 </td></tr><tr><td> Zehirli kil modelleme </td><td&gt; Fisher </td><td> S17307 </td></tr><tr><td> Kapiler tüpler </td><td> Chase bilimsel Cam, inc </td><td> 40A502 </td></tr></tbody></table>

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

Keneler dünya çapında bulunur ve birçok kene kaynaklı hastalıklar insanlarda eziyet edilir. Keneler, Lyme hastalığı ve kene kaynaklı tekrarlayan ateş (Borrelia spp.), Rocky Mountain Benekli Ateşi (Rickettsia rickettsii), Ehrlichiosis (Ehrlichia chaffeensis ve E. equi), anaplazmoz (Anaplasma phagocytophilum), ansefalit (kene neden patojenler için vektörleri kaynaklı ensefalit virüsü), babesiosis (Babesia spp.), Kolorado kene ateşi (Coltivirus) ve tularemi (Francisella tularensis) 1-8. Düzgün konak içine aktarılacak bu enfeksiyon ajanları farklı şekilde, gen ekspresyonu düzenleyen kene proteinleri ile etkileşim ve kene 3,9-13 yoluyla göç ederler. Örneğin, Lyme hastalığı ajanı, Borrelia burgdorferi, kenenin enzootik döngüsü 14,15 şölen ve kıtlık aşamalarına farklı gen ifade aracılığıyla uyarlar. Dahası, bir Ixodes kene gibi bir tüketirbloodmeal Borrelia çoğaltmak ve ortabağırsak gelen onlar tükürük bezlerinin seyahat ve sınırdışı tükürük 9,16-19 ile ev sahibi içine iletilir hemocoel, göç. Kene beslenirken ev sahibi genellikle güçlü bir hemostatik ve immün yanıtı 11,13,20-22 ile yanıt verir. Bu konak yanıtları rağmen, I. kene tükürük 3,11,20,21,23 besleyen kene yardım immünomodülatör olan proteinlerin parçalayıcı ajanlar, antikoagülanlar ve fibrinolysins içerdiğinden scapularis birkaç gün süreyle besleyebilir. Kene tükürük veya tükürük bezi ekstraktı (SGE) tarafından sahip immünomodülatör faaliyetleri çok sayıda kene kaynaklı patojenlerin 3,20,24-27 iletimi, çoğalması ve yayılması kolaylaştırmak. Daha da kenelerin beslenme aktif incelemek ve kene tükürük toplamak esastır enfeksiyöz ajanlar hastalığı nasıl neden kene kaynaklı anlamak için. Bu video protokol için diseksiyon teknikleri gösteriyorhemolimf toplanması ve aktif I. beslenmesini tükürük bezlerinin çıkarılması scapularis periler 48 sonra ve 72 saat sonrası fare yerleşimi. Ayrıca yetişkin bir dişi I. tükürük toplama göstermek scapularis kene.

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

Keneler hemolimf, tükrük bezleri ve tükürük toplama patojenler hastalıklara nasıl neden kene kaynaklı incelemek önemlidir. Bu protokolü biz beslenmesini hemolimf ve tükrük bezleri toplamak nasıl gösterilmektedir<em&gt; Scapularis Ixodes</em&gt; Periler. Ayrıca kadın tükürük toplama göstermek<em&gt; I. scapularis</em&gt; Yetişkinler.

Ixodes scapularis Keneler gelen tükürük, Tükürük Bezi ve hemolimf Koleksiyonu

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

29.4K Görüntüleme.  Centers for Disease Control and Prevention. Keneler hemolimf, tükrük bezleri ve tükürük toplama patojenler hastalıklara nasıl neden kene kaynaklı incelemek önemlidir. Bu protokolü biz beslenmesini hemolimf ve tükrük bezleri toplamak nasıl gösterilmektedir Scapularis Ixodes Periler. Ayrıca kadın tükürük toplama göstermek I. scapularis Yetişkinler.

Video: 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.

Keneler de RNA Girişim

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

İmmünoloji ve Enfeksiyon

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.

Melezleme<em&gt; In situ</em&gt; Bezler, Yumurtalık ve Vektör Sivrisinek ve Embriyoların

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.

Lyme Hastalığı Araştırma İletim ve Xenodiagnosis için Hayvanlar Keneler besleme

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

Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and infections using cost effective, high-throughput approaches would be indispensable. This manuscript describes the development and analysis of a bead-based multiplex immunoassay capable of measuring the presence of antibodies in human saliva to multiple pathogens simultaneously. Saliva is particularly attractive in this application because it is noninvasive, cheaper and easier to collect than serum. Antigens from environmental pathogens were coupled to carboxylated microspheres (beads) and used to measure antibodies in very small volumes of human saliva samples using a bead-based, solution-phase assay. Beads were coupled with antigens from Campylobacter jejuni, Helicobacter pylori, Toxoplasma gondii, noroviruses (G I.1 and G II.4) and hepatitis A virus. To ensure that the antigens were sufficiently coupled to the beads, coupling was confirmed using species-specific, animal-derived primary capture antibodies, followed by incubation with biotinylated anti-species secondary detection antibodies and streptavidin-R-phycoerythrin reporter (SAPE). As a control to measure non-specific binding, one bead set was treated identically to the others except it was not coupled to any antigen. The antigen-coupled and control beads were then incubated with prospectively-collected human saliva samples, measured on a high throughput analyzer based on the principles of flow cytometry, and the presence of antibodies to each antigen was measured in Median Fluorescence Intensity units (MFI). This multiplex immunoassay has a number of advantages, including more data with less sample; reduced costs and labor; and the ability to customize the assay to many targets of interest. Results indicate that the salivary multiplex immunoassay may be capable of identifying previous exposures and infections, which can be especially useful in surveillance studies involving large human populations.

In the current climate of scarce resources, new technologies are emerging that allow researchers to conduct studies cheaper, faster and with more precision. Here we describe the development of a bead-based salivary antibody multiplex immunoassay to measure human exposure to multiple environmental pathogens simultaneously.

Bir Tükürük Antikor Multiplex Immuoassay Gelişmekte Çevre Patojenler İnsan Pozlama&#39;yı Tedbir

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and ...

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.

yetiştirme<em&gt; Geyik kenesi,</em&gt; Siyah Ayaklı Tik Tak: Fare üzerinde Olgunlaşmamış Evreleri Besleme

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.

Tükürük bezi işlevi farelerde ölçülmesi için bir yöntem

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.

Dik Intravital mikroskopi için fare Submandibular Tükürük bezi hazırlanması

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.

Salispheres veya Monolayers olarak In vitro büyüme için ınsan tükürük bezlerinden tükürük epitelyal hücrelerin yalıtım

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.

Yüksek Verim Ekstrasellüler Vezikül Preparatlarının Virüsten Uzaklaştırılması

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.

Hemolymph toplama ve Metarhizyum blastosporların omurgasız patoloji çalışmalarına doğru Rhipicephalus Microplus ticks içine inoculation

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

Ixodes scapularis Keneler gelen tükürük, Tükürük Bezi ve hemolimf Koleksiyonu

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