An inexpensive and rapid diagnostic method of Koi Herpesvirus (KHV) infection by loop-mediated isothermal amplification

BACKGROUND: Koi Herpesvirus (KHV) affects both juvenile and adult common carp and koi, and is especially lethal to fry. The high mortalities caused by the disease have had a negative impact on the international koi trade. Different diagnostic techniques have been used to detect KHV, including: isolation of the virus in cell culture, electron microscopy, several PCR tests, ELISA and in situ hybridisation. All of these methods are time consuming, laborious and require specialised equipment. RESULTS: A rapid field diagnosis of KHV in common and koi carp was developed using loop-mediated isothermal amplification (LAMP). The LAMP reaction rapidly amplified nucleic acid with high specificity and efficiency under isothermal conditions using a simple water bath. Two methods of extracting DNA from host tissue were compared: extraction by boiling and by using a commercial extraction kit. A set of six primers – two inner primers, two outer primers and two loop primers – was designed from a KHV amplicon. The reaction conditions were optimised for detection of KHV in 60 min at 65°C using Bst (Bacillus stearothermophilus) DNA polymerase. When visualised by gel electrophoresis, the products of the KHV LAMP assay appeared as a ladder pattern, with many bands of different sizes from 50 base-pairs (bp) up to the loading well. The KHV LAMP product could also be simply detected visually by adding SYBR Green I to the reaction tube and observing a colour change from orange to green. All samples positive for KHV by visual detection were confirmed positive by gel electrophoresis. The KHV LAMP had the same sensitivity as a standard PCR assay for the detection of KHV. CONCLUSION: This paper describes an accelerated LAMP assay for diagnosis of KHV. The entire procedure took only 90 minutes to produce a result: 15 minutes for DNA extraction; 60 min for the LAMP reaction; 2 min for visual detection using SYBR Green I. The test can be used under field conditions because the only equipment it requires is a water bath

Loop-mediated isothermal amplification as an emerging technology for detection of Yersinia ruckeri the causative agent of enteric red mouth disease in fish

<p>Abstract</p> <p>Background</p> <p>Enteric Redmouth (ERM) disease also known as Yersiniosis is a contagious disease affecting salmonids, mainly rainbow trout. The causative agent is the gram-negative bacterium <it>Yersinia ruckeri</it>. The disease can be diagnosed by isolation and identification of the causative agent, or detection of the <it>Pathogen </it>using fluorescent antibody tests, ELISA and PCR assays. These diagnostic methods are laborious, time consuming and need well trained personnel.</p> <p>Results</p> <p>A loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for detection of <it>Y. ruckeri </it>the etiological agent of enteric red mouth (ERM) disease in salmonids. The assay was optimised to amplify the <it>yruI/yruR </it>gene, which encodes <it>Y. ruckeri </it>quorum sensing system, in the presence of a specific primer set and <it>Bst </it>DNA polymerase at an isothermal temperature of 63°C for one hour. Amplification products were detected by visual inspection, agarose gel electrophoresis and by real-time monitoring of turbidity resulted by formation of LAMP amplicons. Digestion with <it>Hph</it>I restriction enzyme demonstrated that the amplified product was unique. The specificity of the assay was verified by the absence of amplification products when tested against related bacteria. The assay had 10-fold higher sensitivity compared with conventional PCR and successfully detected <it>Y. ruckeri </it>not only in pure bacterial culture but also in tissue homogenates of infected fish.</p> <p>Conclusion</p> <p>The ERM-LAMP assay represents a practical alternative to the microbiological approach for rapid, sensitive and specific detection of <it>Y. ruckeri </it>in fish farms. The assay is carried out in one hour and needs only a heating block or water bath as laboratory furniture. The advantages of the ERM-LAMP assay make it a promising tool for molecular detection of enteric red mouth disease in fish farms.</p

Construction and Screening of an Expression cDNA Library from the Triactinomyxon Spores of Myxobolus cerebralis, the causative agent of Salmonid Whirling Diseases

The ZAP Express cDNA library was constructed using mRNA extracted from the triactinomyxon spores. First-strand cDNA was synthesized using Moloney Murine leukaemia virus reverse transcriptase. Following second-strand cDNA synthesis, the double-stranded cDNA was digested with Xho I restriction enzyme, cDNA fragments less than 400bp were removed and the remaining cDNA was ligated with the lambda ZAP Express vector. The recombinants were packaged in vitro using Gigapack III gold packaging extract. The primary cDNA library titre contained 0.5 X 106 clones, with 97% recombinant and only 3% non-recombinant clones. The cDNA library was then screened using the anti-triactinomyxon antibodies. Positive clones were selected and re-screened twice more to give a final selection of 526 clones. One clone (46-5) was selected randomly and subjected to in vivo excision of the pBK-CMV phagemid from the ZAP express vector. The sequence of the entire clone was obtained using rapid amplification of the cDNA ends. A search of the clone sequence against GenBank revealed that it related to ribosomal protein L23 and it had a high percentage similarity to this protein from different species. A conserved domain for ribosomal protein L23 was also identified in the clone sequenc

Impact of Betaine Supplementation as Anti Stress on Some Hematological Parameters and Thermoregulatory Responses of Aberdeen Angus Cows in Arid Subtropical Regions

This study aimed to investigate the effects of betaine supplementation on some hematological and physiological parameters of Aberdeen Angus cows. Twelve cows were divided randomly into two equal groups (6 cows each) and each group was treated for 150 successive days. All experimental animals were fed 60% of their requirements as a concentrate mixture and the rest of other requirements was covered from wheat strew ad-libitum. In addition to basal diet, animals in experimental group were supplemented with 30 g betaine hydrochloride per day. Air temperature and relative humidity were recorded during the experimental days to calculate the current temperature–humidity index (THI). Blood samples were collected during experimental period from jugular vein. Rectal temperature (RT), pulse rate (PR), respiration rate (RR), skin temperature (ST) and hair temperature (HT) were recorded during the experimental days. The obtained results showed that the average values of THI were between 71.6 to 74.78 at 08:00 am and 77.09 to 83.01 at 02:00 pm during the experimental period which indicates exposure of animals to heat stress. There were no significant differences among groups in WBC, HCT, MCV, MCH, MCHC and PLT. Moreover, RBC and HGB was higher (P < 0.05) in betaine group than control. adding betaine had no significant decrease in RR, ST, HT and ET. While, significant decrease in RT and PR was observed at 02:00 pm of experimental animal. It is concluded that betaine may be improved some hematological parameters and thermoregulatory responses of Aberdeen Angus cows under New Valley arid areas

Identitätsstiftende Praktiken in Gangsta-Rap-, Türsteher:in-, Kraft- und Kampfsport- sowie Shisha-Bar-Szene: Methodische und theoretische Annäherung an die Szenenfelder

In öffentlichen Debatten taucht immer häufiger der Begriff „Clan“ auf. Dabei wird der Begriff gegenwärtig meist mit „Clankriminalität“ in unmittelbare Verbindung gesetzt. Hierdurch wird ein Zusammenhang zur (organisierten) Kriminalität vermittelt. Häufig entsteht auch eine Verknüpfung zur sogenannten Paralleljustiz. Mangelnde Integrationsbereitschaft sticht in diesem Zusammenhang ebenfalls als Schlagwort hervor. In der medialen Berichterstattung und behördlichen Berichten werden dabei die Begriffe „Clan“ und „Clankriminalität“ weitgehend undifferenziert verwendet. Was einen „Clan“ und dessen Kriminalität ausmacht, bleibt in den gesellschaftspolitischen Debatten vage. Sozialwissenschaftliche Erkenntnisse zur Struktur und Funktionsweise der im Fokus stehenden Gemeinschaften bzw. Solidarverbände, häufig nahöstlicher Herkunft, stellen ein Desiderat dar. Eine sozialwissenschaftliche Betrachtungsweise ist jedoch unerlässlich. Insbesondere auch eine, die die bisherigen, spärlichen Kenntnisse in den Blick nimmt. Dieser wird in erster Linie durch die Linse der ermittlungsbehördlichen Wahrnehmung geprägt. „Clans“ werden demnach weitgehend als eine aus Familienangehörigen bestehende kriminelle Vereinigung verstanden. Qua Familienzugehörigkeit erfolgt also eine Zuschreibung, der ein kriminelles oder deviantes Handeln innewohne. Ausschlaggebendes Kriterium sei dabei der Familienname. Das vorliegende Working Paper leistet einen sozialwissenschaftlichen Beitrag zur Versachlichung der Debatte. Es zeigt die Grundstruktur eines vom Bundesministerium für Bildung und Forschung geförderte Forschungsvorhabens auf, welches gegenwärtig von den Autor:innen gemeinsam durchgeführt wird. Ziel ist es, wissenschaftlich fundierte Einblicke in den Alltag von Angehörigen der Familien zu erhalten, denen die Zuschreibung zuteilwird. Auf Empirie gestützter Basis sollen wissenschaftlich fundierte Erkenntnisse über Lebenswelten einschlägiger großfamiliärer Strukturen und ihrer Umwelt, insbesondere der Gesamtgesellschaft, gewonnen werden. Hierbei wird die Methodologie der Grounded Theory (GT) angewendet. Im Sinne des nachfolgend näher beschriebenen GT-Ansatzes wird der Fokusgruppe möglichst offen begegnet. Dabei wird vor dem Feldeinstieg auf die Formulierung eng zugeschnittener Forschungsfragen verzichtet. Was den Alltag der Fokusgruppen in welcher Weise prägt, wird nicht vorab durch eine feste Fragestellung antizipiert, sondern aus der qualitativen Forschung im Feld herausgearbeitet. Eine praxeologische Sichtweise soll demgemäß bei der Generierung von Einblicken in die Lebenswelt(en) der Akteur:innen helfen. Zu identifizierende Praktiken der Fokusgruppe dienen dabei als Analyseeinheit. Ziel ist es, den Blick auf Alltägliches zu schärfen und mit dem der öffentlichen Wahrnehmung abzugleichen. In der durch Medien geprägten Öffentlichkeit werden mit dem Begriff der „Clankriminalität“ – und somit mit den der „Clankriminalität“ zugeordneten Familienangehörigen – häufig auch bestimmte Szenenfelder genannt. So beispielsweise die Gangsta-Rap-, Shisha-Bar-, Kampfsport- und Türsteher:innenszene. Auffällig ist hierbei, dass die Lebensrealitäten derjenigen, die sich in eben jenen Feldern aufhalten, kaum Beachtung erfährt und, wie bereits erwähnt, sozialwissenschaftlich kaum erforscht ist. Aus diesem Grund fokussiert die hier dargestellte Forschung diese vier Szenen, um gängige Narrative konzeptionell zu durchdringen und die Lebenswelten derjenigen, die sich in den genannten vier Szenenfelder aufhalten, möglichst präzise abzubilden. Zu betonen ist hierbei auch, dass das Forschungsprojekt eine Analyse der Szenen vornehmen und somit die Frage beantworten möchte, ob und wo sich das Konstrukt „Clan“ überhaupt als relevant zeigt. Das vorliegende Working Paper ist entlang der Logik der GT strukturiert. Ausgangspunkt ist eine kurze Einführung in den Themenkomplex „Clankriminalität“. Danach erfolgt eine kritische Auseinandersetzung mit Begriffsbestimmungen der „Clan-“ und organisierte Kriminalität. Darauf aufbauend stellen wir unser Erkenntnisinteresse dar, artikulieren unsere Forschungsfragen mit Blick auf die ausgewählten Szenenfelder und deren Akteur:innen. Da unser Vorhaben sich an der GTM orientiert, messen wir insbesondere den Methoden rund um die GT und ihrer Methodologie ein nicht unerhebliches Gewicht bei. Unsere Daten generieren wir im Feld, wobei im sprichwörtlichen Vorfeld möglichst wenig konzeptionelle Vorannahmen für das Feld getroffen werden sollen. Aus diesem Grund verstehen wir unser sozialwissenschaftliches Vorwissen als sensibilisierende Konzepte, die erst am Ende dieses Working Papers ihren Raum finden. Abschließend versuchen wir uns an einem Ausblick. In diesem Ausblick werfen wir Fragen auf, derer Beantwortung wir im Rahmen des Projektes anstreben. Wie für einen „Ausblick“ typisch dürften dabei auch Gesichtspunkte aufkommen, die es über die Projektlaufzeit (2020–2023) hinaus noch weiter aufzuarbeiten lohnt

#SocialScienceOnSlides. Wissenstransfer via Instagram

Vorstellung der Wissenschaftskommunikation via Instagram eines Milieu- und Szenefeldforschungsteam

Vertical transmission of Tetracapsuloides bryosalmonae (Myxozoa), the causative agent of salmonid proliferative kidney disease

license: Copyright © Cambridge University Press 2013 0000-0001-7279-715Xlicense: Copyright © Cambridge University Press 2013. The attached document is the authors' final accepted version of the journal article. You are advised to consult the publisher's version if you wish to cite from it

Effect of Antioxidant supplementation on Some Hematological Parameters and Thermoregulatory Responses of Aberdeen Angus Cows During Hot Season in Arid Subtropical Regions

This work aims to investigate the impact of some antioxidants, i.e., zinc sulfate (ZnS) administration, vitamin E and selenium (E-Sel) injection on hematological parameters and thermoregulatory responses of Aberdeen Angus cows. Sixteen cows were randomly divided into 4 equal groups (4 cows each), First group served as a control (G1); second group was received E-Sel injection at rate of 15 ml/ head / 15day) (G2); third group was fed on ZnS with an average rate of 200 mg/head/ daily (G3) and the fourth one was fed on ZnS in combination with E-Sel injection (G4). All experimental cows were fed 60% of their requirements as CFM and the rest of other requirements was covered from wheat strew ad libitum. In addition, the AT and RH% were recorded during the experimental days to calculate the current THI. Blood samples were collected during experimental period from jugular vein. Physiological parameter measurements were recorded during the experimental days. The obtained results showed that the average values of THI were between 69.04 and 85.46 during the experimental period. Treated cows with E-Sel recorded the highest values of hematological parameters in terms of WBC, RBC, HB, HCT and PLT compared with other groups. Using antioxidant agents had significant decrease in RR, PR, and HT. While, slightly decrease in RT, ST and ET was recorded of experimental animal. It is concluded that some antioxidants could improve hematological parameters and thermoregulatory responses of Aberdeen Angus cows under hot climatic conditions

Ketamine plus Bupivacaine versus Nalbuphine plus Bupivacaine Caudal Anaesthesia in Pediatric Subumbilical Surgeries

Background: Caudal anaesthesia is used as pain control for many sub umbilical surgical procedures. Objectives: This study intended to Estimate length of analgesia (1ry outcome) Track haemodynamics and detect side effects Patients and methods: This prospective randomized double blind observational study was conducted on 60 healthy paediatric patients recruited from Qena university hospital, South Valley University, Qena, Egypt, undergoing elective lower abdominal and pelvic surgeries were allocated to two groups according to mode of blocking agents and caudal region during the study duration from May 2019 to August 2020 Results: efficacy of ketamine bupivacaine versus nalbuphine bupivacaine to provide intraoperative and postoperative pain relief. The time to first analgesic administration was longer in the ketamine bupivacaine group about (8 hrs) than nalbuphine bupivacaine about (5.8 hrs)., Conclusion: Caudal nalbuphine and caudal ketamine are safe in pediatric surgeries in the lower half of the body and effectively reducing postoperative pain with longer duration of analgesia in ketamine group than nalbuphine group with no obvious side effects in both groups