Holstein haplotype (HH) 1, 3 and 4 are lethal mutations, responsible for early embryonic losses in Holstein Friesian (HF) cattle, worldwide. Three PCR based assays – tetra Amplification Refractory ...Mutation System PCR, PCR primer induced restriction analysis and PCR-restriction fragment length polymorphism techniques for screening of HH1, 3 and 4, respectively were developed and validated. During screening, six among 60 HF bulls were found as carrier for either of three mutations. These PCR assays are highly accurate and reproducible and can be used for screening of the haplotypes in HF cattle.
•PCR assays were developed for detection of Holstein haplotypes-1, 3, 4 associated with embryonic death in cattle.•These assays are economical, easier to handle and highly reliable.•Screening of these haplotypes resulted in presence of carriers in Indian Holstein cattle with 10 percent of frequencies.•The assays can also be used for cross-validation of the results obtained from DNA Chip based screening.
Global cultured shrimp production has sustained considerable losses due to acute hepatopancreatic necrosis disease (AHPND) caused by some strains of Vibrio that secrete PirAB binary toxin. Previous ...experiments have revealed changes in mortality due to AHPND under variable environmental conditions. In order to determine whether and how environmental factors affect the pathogenicity of a strain of
Vibrio parahaemolyticus
that causes AHPND (Vp
AHPND
), a Vp
AHPND
strain was grown at different temperatures (28, 30, and 33 °C) and proportions of NaCl (2.0, 3.0, and 3.5%). PirA gene expression was assessed in these bacterial cultures, and bioassays by immersion challenge were performed at 20, 30, and 35 practical salinity units (PSU). The results showed that salinities and temperatures tested do not inhibit Vp
AHPND
growth. However, maximum PirA gene expression at 3.5, 3.0, and 2.0% NaCl reached 21.1, 13.0, and 0.03 millions of copies per ng of RNA, respectively, and at 28, 30, and 33 °C reached 48.5, 21.1, and 11.3 millions of copies per ng of RNA, respectively. Furthermore, the mortality rate in the bioassay challenges corresponded proportionally to the expression of the PirA gene, reaching 83.3, 46.6, and 0 mortality at 35, 30, and 20 PSU, respectively; and 90.0, 63.3, and 43.3 mortality at 28, 30, and 33 °C, respectively. These results indicate that NaCl concentration and temperature modify the expression of the PirA gene and regulate Vp
AHPND
virulence. Since the virulence of Vp
AHPND
is sensitive to environmental factors, it may be a potent target for therapeutic intervention in seasonal bacterial disease.
Early mortality syndrome (EMS) or acute hepatopancreatic necrosis disease (AHPND) is currently the most significant disease of shrimp in farms of Vietnam, Thailand, Malaysia, China and Mexico, and ...there is a great risk that it may spread to other shrimp farming countries. Although, an array of sophisticated detection tools for AHPND available, there is a need for a sensitive, simple and rapid detection method. In this study, a simple, sensitive, rapid and polyclonal antibody‐based farmer‐friendly flow‐through assay (FTA) test has been developed for the detection of AHPND pathogen. The recombinant Photorhabdus insect‐related (Pir) A toxin‐like protein of AHPND pathogen was used to immunize rabbits at 21‐day interval observed for highest antibody titre after third booster by ELISA. The raised rabbit antiserum was purified by affinity chromatography and characterized by Western blot. The antiserum showed no cross‐reactivity with AHPND‐free Vibrio parahaemolyticus, V. anguillarum, White Spot Virus (WSV), Aeromonashydrophila and Aphanomycesinvadans. This polyclonal rabbit antiserum was used to develop a farmer‐friendly FTA test for the detection of AHPND pathogen. This simple FTA testis is more sensitive and could detect PirAVP toxin up to 0.121 µg/ml, compared with 0.242 µg/ml by immunodot assay. Furthermore, FTA test requires only 8–10 min for completion, compared with 3 hr by immunodot thus found to be more sensitive, specific and cost‐effective. Collectively, sensitive FTA test would help shrimp farmers to take real‐time management decisions, especially emergency harvest and finally be a better hope for the prevention of AHPND.
Acute hepatopancreatic necrosis disease (AHPND) is a lethal shrimp disease. The pathogenic agent of this disease is a special Vibrio parahaemolyticus strain that contains a pVA1 plasmid. The protein ...products of two toxin genes in pVA1, pirAvp and pirBvp, targeted the shrimp’s hepatopancreatic cells and were identified as the major virulence factors. However, in addition to pirAvp and pirBvp, pVA1 also contains about ~90 other open-reading frames (ORFs), which may encode functional proteins. NCBI BLASTp annotations of the functional roles of 40 pVA1 genes reveal transposases, conjugation factors, and antirestriction proteins that are involved in horizontal gene transfer, plasmid transmission, and maintenance, as well as components of type II and III secretion systems that may facilitate the toxic effects of pVA1-containing Vibrio spp. There is also evidence of a post-segregational killing (PSK) system that would ensure that only pVA1 plasmid-containing bacteria could survive after segregation. Here, in this review, we assess the functional importance of these pVA1 genes and consider those which might be worthy of further study.
Dans les poèmes de jeunesse et les premiers recueils de Salvatore Quasimodo, le fanciullo est décrit comme un enfant de Dieu, un messager des étoiles systématiquement associé à des éléments ...témoignant de son rapport privilégié à l’invisible. On ne peut l’envisager sans prendre en considération l’influence fondamentale du très fervent Giorgio La Pira, ami et confident de Quasimodo, à qui ce dernier dédie le poème qui constitue leur manifeste d’un « être fanciullo », « Il fanciullo canuto ». Bien plus qu’un simple motif littéraire, l’enfant se révèle être, dans les poèmes de l’un et les lettres de l’autre, la métaphore d’une condition existentielle, l’expression d’un idéal de vie et une « révélation de l’Indicible ». Avatar du faible, de l’humble, du vagabond ou du mendiant, le fanciullo est l’être dont le « linguaggio canuto » donne accès au « dono sublime di saper parlare al cuore dell’uomo ».
Puccinia kuehnii and P. melanocephala cause orange and brown rust of sugarcane, respectively. Puccinia kuehnii has been confirmed in Asia, Australia and recently, the Caribbean basin, whereas P. ...melanocephala is distributed among the majority of sugarcane growing regions. Differentiating these two economically significant pathogens visually is problematic and limited to material exhibiting mature disease symptoms or spores. Partial ITS1, ITS2 and complete 5·8S sequences were generated from P. kuehnii and P. melanocephala isolates from around the world. PCR primers and dual labelled hydrolysis probes were designed for each pathogen for use in real-time PCR and optimized using locked nucleic acids (LNA). The primers amplified DNA from their target pathogens and not from other species of Puccinia or fungal species isolated from sugarcane leaves. Optimized real-time PCR conditions allowed the detection of 0·19 pg of P. kuehnii or P. melanocephala genomic DNA and differentiated the pathogens on sugarcane leaves prior to observing typical symptoms in the field. Primer-introduced restriction analysis-PCR (PIRA-PCR) was used to detect a single nucleotide polymorphism (Pk ITS1 183A>G) in ITS1 of P. kuehnii. Allele 183A was observed in all samples, whereas 183G was detected in 52% of samples from Asia and Australia yet absent from all Caribbean basin samples. Long distance spore dispersal, dispersal through an intermediate location or improper movement of contaminated material could explain the introduction of P. kuehnii to the Western hemisphere. However, the current proliferation of the pathogen in the Americas is limited to isolates which contain only the 183A allele.
The global trend towards a colder and more arid climate at the Eocene-Oligocene boundary triggered significant changes in the European flora. Here we study the latest Eocene (Priabonian) flora from ...lacustrine deposits at Sarral in the Ebro Basin, Catalonia, Spain, to evaluate vegetation changes near this boundary. Three main taphofacies are recognised: (1) assemblages composed of small, size-sorted leaves represented by Zizyphus, Fabaceae leaflets and Pinus needles, inferred to be wind transported from open woodlands located distal from the lake; (2) assemblages composed of whole unsorted leaves belonging to Salicaceae, Myricaceae (Myrica arensi and Comptonia schrankii), Lauraceae (Daphnogene and Laurophyllum) and helophytic plant stems, interpreted as remains of a riparian community; (3) assemblages comprising disarticulated organs of Tetraclinis, Cupressaceae and a few torn leaves of Myrica arensi and Salicaceae, interpreted as derived from riparian or open woodland vegetation upstream from the lake. The Priabonian Sarral flora is similar to the nearby early Oligocene flora of Cervera, making it difficult to distinguish the effects of the Eocene-Oligocene climatic crisis upon the vegetation. Sedimentological analysis indicates that the Sarral leaf bed represents deposition during a drier period of small-order climate cycles, whereas the Cervera flora represents vegetation during a wetter period of a similar cycle but under the cooler and more arid environmental conditions of the early Oligocene, thereby disguising large-scale Eocene-Oligocene climatic change. Understanding such short-term sedimentary and climatic cycles is crucial in order to correctly interpret the regional climatic signal based on leaf physiognomy.
•The Priabonian flora from Sarral is dominated by small, size-biased xeric leaves.•It was deposited during the drier terms of short, climatically driven sedimentary cycles.•Riverine plants include Salicaceae, Myrica arensi, Comptonia schrankii and Lauraceae.•Open woodlands were dominated by Fabaceae, Zizyphus, Pinus and Tetraclinis.•The Eocene-Oligocene boundary floral change is masked by small-order climatic cycles.
In April 2019, a so-called 'dissident' republican New IRA gun-man killed journalist Lyra McKee, whilst firing at police during a riot in Derry in the North of Ireland. The New and Continuity IRAs ...remain wedded to an armed campaign for Irish sovereignty, drawing legitimacy from partition and the ongoing British 'presence' in Northern Ireland - and rejecting the significance of altered conditions within the state. Conversely, independent 'dissident' republicans, formerly in the Provisional IRA, criticise the ongoing campaign by the groups as futile. This article examines key areas of debate within the 'dissident'/radical republican base, on armed actions at present - drawing on unpublished qualitative interviews with independents, the RSF Movement, and Saoradh,- the organisation believed to be the political wing of the New IRA. This article assesses the nature of the campaign waged by the Continuity and New IRAs and examines whether it represents a continuation of the Provisional IRA campaign, or a new departure.
Complex vertebral malformation (CVM) has considerable economic impact on dairy cattle breeding due to extensive use of artificial insemination (AI). Identifying the carrier is an important factor to ...reduce the incidence of the genetic disorder. The study was conducted to identify the carriers of CVM in Frieswal cattle by polymerase chain reaction-primer-introduced restriction analysis (PCR-PIRA) method, which was further confirmed by sequencing. Carrier prevalence of 1% was observed in the Frieswal cattle. The results of the study clearly demonstrated the existence of carriers of CVM among Frieswal bull calves. Due to the widespread use of AI it is recommended to screen young bulls at early stages for this defective allele in order to avoid its rapid spread within the population.
Acute hepatopancreatic necrosis disease (AHPND) is a contagious disease for the shrimp cultivation, thus early detection of disease is an unmet need. This present study documented for the first time ...a simple lateral flow immunoassay (LFIA) strip using polyclonal antibodies was created for the rapid detection both of PirA
vp
and PirB
vp
protein simultaneously. LFIA method based on the principle of sandwich format. The label is the colloidal gold. The polyclonal antibody was conjugated with the colloidal gold acting as biorecognition element and coated onto the conjugate pad. The rabbit anti-Pir
vp
, anti-PirB
vp
antibodies, and goat anti-rabbit IgG antibody were separately sprayed onto a nitrocellulose membrane to form two test lines and one control line, respectively. The appearance of red bands at the control line and the test line indicated a positive result. A single coloured band at control area indicated a negative result. The limit of detection of LFIA was found to be 125 ng, which could be visually detected by naked eye within 15 min. There was no cross-reactivity observed with VP
non-AHPND
. Furthermore, the sensitivity and specificity of LFIA were 94.0% and 98.0%, respectively. The developed test strip could be a game changer for early and in situ diagnosis of AHPND.
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