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

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

Bone seeking matrix metalloproteinase-2 inhibitors prevent bone metastatic breast cancer growth

Bone metastasis is common during breast cancer progression. Matrix metalloproteinase-2 (MMP-2) is significantly associated with aggressive breast cancer and poorer overall survival. In bone, tumor or host derived MMP-2 contributes to breast cancer growth and does so by processing substrates including type I collagen and transforming growth factorβ (TGFβ) latency proteins. These data provide strong rationale for the application of MMP-2 inhibitors to treat the disease. However, in vivo, MMP-2 is systemically expressed. Therefore, to overcome potential toxicities noted with previous broad-spectrum MMP inhibitors (MMPIs), we used highly selective bisphosphonic based MMP-2 inhibitors (BMMPIs) that allowed for specific bone targeting. In vitro, BMMPIs impacted the viability of breast cancer cell lines and osteoclast precursors but not osteoblasts. In vivo, we demonstrated using two bone metastatic models (PyMT-R221A and 4T1) that BMMPI treatment significantly reduced tumor growth and tumor associated bone destruction. Additionally, BMMPIs are superior in promoting tumor apoptosis compared to the standard of care bisphosphonate, zoledronate. We demonstrated MMP-2 selective inhibition in the bone microenvironment using specific and broad spectrum MMP probes. Further, compared to zoledronate, BMMPI treated mice had significantly lower levels of TGFβ signaling and MMP generated type I collagen carboxy-terminal (ICTP) fragments. Taken together, our data show the feasibility of selective inhibition of MMPs in the bone metastatic breast cancer microenvironment. We posit that BMMPIs could be easily translated to the clinical setting for the treatment of bone metastases given the well-tolerated nature of bisphosphonates

Groin recurrence in patients with early vulvar cancer following superficial inguinal node dissection

AbstractObjectiveTo investigate the causes of groin recurrence in patients with vulval cancer who previously had negative nodes following superficial inguinal node dissection (SIND).Material and methodsForty-one patients with squamous cell carcinoma of the vulva (stage I or II) were operated upon. The primary treatment was wide local excision with 2cm safety margin and superficial inguinal lymphadenectomy. Six patients had ipsilateral and one patient had bilateral groin recurrence. Those patients were subjected to deep inguinal node dissection (one patient required bilateral node dissection).ResultsThe mean age at time of diagnosis was 59years (range 51–68). The median follow-up period for all patients was 63months (range 24–71) and that of the recurrent cases was 20months (range 12–38). The mean depth of invasion of the recurrent cases was 5.5mm (range 5–5.9mm) and the mean diameter of the primary tumor in recurrent cases was 3.8cm (range 3–4.5cm). All recurrent cases had a high grade of the primary tumor. The median interval to recurrence was 21months (range 12–57). The groin recurrence rate after negative SIND was 17% (7/41 patients).The mean number of nodes resected per groin was eight (range 1–17). The nodes ranged in size from 0.2 to 4.0cm.ConclusionCarcinoma of the vulva with the following criteria (size of tumor is greater than 3cm, depth of invasion greater than 5mm, and high grade tumors) is at high risk of recurrence

Survival rate in acute kidney injury superimposed COVID-19 patients: a systematic review and meta-analysis

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