The development of an ELISA-assay for semi-quantitative detection of dihydrogriesenin, a sesquiterpene lactone from Geigeria

Certain species of Geigeria contain sesquiterpene lactones which cause vomiting disease in sheep. Dihydrogriesenin (DHG), a sesquiterpene lactone from G. asperta, contains an alpha-methylene function which can spontaneously react with thiol groups on proteins to form a covalent adduct. A specific antiserum against a DHG-protein adduct can be used to determine the fate of DHG in poisoned animals. The preparation of such an antiserum is reported in this paper. DHG was reacted with cysteine and subsequently coupled to serum albumin using the carbodiimide reaction. When rabbits were immunized with one such conjugate (DHG-bovine serum albumin), it was found that the carrier determinants were immunodominant. A DHG-specific anti-serum of sufficient (ELISA) titre could, however, be obtained by alternating serum albumin carriers for DHG in booster immunizations. The ELISA antigen-antibody reaction could be inhibited by prior reaction of the antisera with cysteinyl-DHG in solution.     

Compatibility between livestock databases used for quantitative biosecurity response in New Zealand

AIM: To characterise New Zealand's livestock biosecurity databases, and investigate their compatibility and capacity to provide a single integrated data source for quantitative outbreak analysis.

METHODS: Contemporary snapshots of the data in three national livestock biosecurity databases, AgriBase, FarmsOnLine (FOL) and the National Animal Identification and Tracing Scheme (NAIT), were obtained on 16 September, 1 September and 30 April 2014, respectively, and loaded into a relational database. A frequency table of animal numbers per farm was calculated for the AgriBase and FOL datasets. A two dimensional kernel density estimate was calculated for farms reporting the presence of cattle, pigs, deer, and small ruminants in each database and the ratio of farm densities for AgriBase versus FOL calculated. The extent to which records in the three databases could be matched and linked was quantified, and the level of agreement amongst them for the presence of different species on properties assessed using Cohen's kappa statistic.

RESULTS: AgriBase contained fewer records than FOL, but recorded animal numbers present on each farm, whereas FOL contained more records, but captured only presence/absence of animals. The ratio of farm densities in AgriBase relative to FOL for pigs and deer was reasonably homogeneous across New Zealand, with AgriBase having a farm density approximately 80% of FOL. For cattle and small ruminants, there was considerable heterogeneity, with AgriBase showing a density of cattle farms in the Central Otago region that was 20% of FOL, and a density of small ruminant farms in the central West Coast area that was twice that of FOL. Only 37% of records in FOL could be linked to AgriBase, but the level of agreement for the presence of different species between these databases was substantial (kappa >0.6). Both NAIT and FOL shared common farm identifiers which could be used to georeference animal movements, and there was a fair to substantial agreement (kappa 0.32–0.69) between these databases for the presence of cattle and deer on properties.

CONCLUSIONS: The three databases broadly agreed with each other, but important differences existed in both species composition and spatial coverage which raises concern over their accuracy. Importantly, they cannot be reliably linked together to provide a single picture of New Zealand's livestock industry, limiting the ability to use advanced quantitative techniques to provide effective decision support during disease outbreaks. We recommend that a single integrated database be developed, with alignment of resources and legislation for its upkeep.     

Vesicles Cytoplasmic Injection: An Efficient Technique to Produce Porcine Transgene‐Expressing Embryos

The use of vesicles co‐incubated with plasmids showed to improve the efficiency of cytoplasmic injection of transgenes in cattle. Here, this technique was tested as a simplified alternative for transgenes delivery in porcine zygotes. To this aim, cytoplasmic injection of the plasmid alone was compared to the injection with plasmids co‐incubated with vesicles both in diploid parthenogenic and IVF zygotes. The plasmid pcx‐egfp was injected circular (CP) at 3, 30 and 300 ng/μl and linear (LP) at 30 ng/μl. The experimental groups using parthenogenetic zygotes were as follows: CP naked at 3 ng/μl (N = 105), 30 ng/μl (N = 95) and 300 ng/μl (N = 65); Sham (N = 105); control not injected (N = 223); LP naked at 30 ng/μl (N = 78); LP vesicles (N = 115) and Sham vesicles (N = 59). For IVF zygotes: LP naked (N = 44) LP vesicles (N = 94), Sham (N = 59) and control (N = 79). Cleavage, blastocyst and GFP+ rates were analysed by Fisher's test (p < 0.05). The parthenogenic CP naked group showed lower cleavage respect to control (p < 0.05). The highest concentration of plasmids to allow development to blastocyst stage was 30 ng/μl. There were no differences in DNA fragmentation between groups. The parthenogenic LP naked group resulted in high GFP rates (46%) and also allowed the production of GFP blastocysts (33%). The cytoplasmic injection with LP vesicles into parthenogenic zygotes allowed 100% GFP blastocysts. Injected IVF showed higher cleavage rates than control (p < 0.05). In IVF zygotes, only the use of vesicles produced GFP blastocysts. The use of vesicles co‐incubated with plasmids improves the transgene expression efficiency for cytoplasmic injection in porcine zygotes and constitutes a simple technique for easy delivery of plasmids.     

Ultrasonographic and Endocrine Characterization of Follicular Waves in Llamas with a Special Reference to the Overlapping Phenomenon During Successive Waves

Ovarian follicular growth in non‐mated llamas occurs in successive waves that generally superimpose their origin on the regression of the preceding wave (overlapping), originating prolonged sexual receptivity in the species. The aim of this study was to perform an ultrasonographic and endocrine characterization of individual and successive waves in non‐mated llamas with a special interest on the overlapping phenomenon. Twelve llamas were examined daily by transrectal ultrasonography for at least two consecutive waves. In six females, blood samples were collected daily at the end of each examination. The development of the largest follicle (F) showed a wavelike pattern with a mean duration of 25 days. All waves evaluated were partially overlapped on the preceding wave and emerged at a mean interval of 15.8 ± 0.5 days. This interwave interval determines a mean overlapping degree of 32% of the wave length. Similarly, mean plasma oestradiol‐17β (E₂) concentrations followed a wavelike pattern. However, E₂ concentrations started to decline before the structural regression of the F was observed. Mean basal E₂ concentrations remain higher than 10.9 ± 0.6 pmol/l. In conclusion, follicular activity in non‐mated llamas is characterized by continuous emergence of successive waves that always overlap the preceding wave with variable degrees. E₂ production during the follicular wave is shorter in duration than the morphological development of the F. Finally, the overlapping phenomenon maintains increased plasma E₂ concentrations persistently and this could explain the prolonged periods of sexual receptivity registered in llamas.     

Proteomic analysis of plasma from Holstein cows testing positive for mycobacterium avium subsp. Paratuberculosis (MAP)

Johne's disease (JD) is a widespread and economically important chronic inflammatory disease of the small intestine of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Although there are several techniques available for diagnosis of JD, their sensitivity is questionable. New proteome profiling methods, such as serum/plasma protein fingerprinting by 2-Dimensional Fluorescence Difference Gel Electrophoresis (2D-DIGE), may therefore be useful for identifying novel protein biomarkers of MAP infection. In this study, plasma samples were collected from 380 Holstein cows and screened for the presence of MAP infection using the M.pt. Johne's antibody Kit (IDEXX). Five negative (MAP-), and 5 strongly positive (MAP+) cows were selected for proteomic analysis. Highly abundant proteins were depleted from the plasma samples using the ProteoMiner technology (Bio-Rad) to enhance the resolution of low abundance proteins. Plasma samples from MAP-, MAP+, and a pooled internal control were labelled with different fluorescent dyes and separated based on their isoelectrical point (IP) and then their molecular weight. Gel images of the fluorescent plasma protein maps were acquired using a Typhoon scanner and analyzed using the DeCyder software. Proteins that were differentially expressed were excised from the gels, trypsin digested, and subjected to MS/MS analysis for identification. Six proteins were identified as being up-regulated at least 2-fold in MAP+ cows including: transferrin, gelsolin isoforms α & β (actin binding protein - ABP), complement subcomponent C1r, complement component C3, amine oxidase - copper containing 3 (AOC3), and coagulation factor II (thrombin) (p<0.05). Two proteins that were down-regulated approximately 2-fold in the MAP+ cows included coagulation factor XIII -B polypeptide (COAFXIII), and fibrinogen γ chain (FGG) and its precursor.     

First insights into insecticidal activity against Aedes aegypti and partial biochemical characterization of a novel low molecular mass chymotrypsin‐trypsin inhibitor purified from Lonchocarpus sericeus seeds

BACKGROUND

Arboviroses such as dengue, Zika and chikungunya represent a serious public health issue as a consequence of the absence of approved vaccines or specific antiviral drugs against the arboviruses that cause them. One way to prevent these diseases is by combating the vector mosquito, Aedes aegypti (Diptera), which has serine proteases in the midgut. Protease inhibitors are molecules that can block enzyme activity, impairing digestion and nutrition, which can lead to death. Thus, we purified and characterized a novel chymotrypsin‐trypsin inhibitor (LsCTI) from Lonchocarpus sericeus seeds and investigated its effect upon Ae. aegypti egg hatching, larval development and digestive proteases.

RESULTS

LsCTI showed a single protein band in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE), and the molecular mass determined by matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) was 8870.45 Da. Kinetics analyses revealed a noncompetitive type of inhibition and low inhibition constant (K_i) for chymotrypsin (8.24 x 10^‐8 m ). The thermal resistance was remarkable, even at 100 °C for 180 min. The inhibitor concentration required for 50‐percent enzyme inhibition (IC₅₀) of LsCTI was 4.7 x 10^‐7 m for Ae. aegypti midgut larval enzymes. LsCTI did not affect egg hatchability at 0.3 mg mL^‐1, but caused a high larval mortality rate (77%) and delayed development (37%).

CONCLUSIONS

LsCTI is a novel protease inhibitor with remarkable biochemical characteristics and is a potential tool to control Ae. aegypti development. © 2017 Society of Chemical Industry

     

中国六个白灵菇(Pleurotus nebrodensis) 商业菌株的DNA指纹图谱分析

Pleurotus nebrodensis(Inzengae)Qulet wasfirst described by Inzengae in1863asAgaricusnebrodensis,and subsequently renamedPleurotusnebrodensisin1886(VENTURELLA,2000).InChina,the species is found only in the Xinjiangregion(MAO,2000).Xinjiang researcherscommenced domesticationofthis mushroomin1987,andthe first commercial strain was obtainedin1997(HUANG,1997).Since then,the popularity ofP.nebrodensisin Asia has increased due to itsdesirable organoleptic qualities and market value,and man…     

Oestrogen and Progesterone Receptors and COX‐2 Expression in Endometrial Biopsy Samples During Maternal Recognition of Pregnancy in Llamas (Lama glama)

Endometrial expression of oestrogen receptor‐α (ERα), progesterone receptor (PR) and cyclooxigenase‐2 (COX‐2) was evaluated in non‐pregnant and pregnant llamas during the period when luteolysis/maternal recognition of pregnancy is expected to occur. Females (n = 28) were divided into two groups: non‐pregnant llamas were induced to ovulate with a Buserelin injection, and endometrial biopsies were obtained on day 8 (n = 5) or 12 (n = 5) post‐induction of ovulation. Animals of the pregnant group (n = 18) were mated with a fertile male. Pregnancy was confirmed by the visualization of the embryo collected by transcervical flushing in 5 of 9 animals on day 8 post‐mating and by progesterone profile on day 12 post‐mating in 4 of 9 animals, when endometrial biopsies were obtained. An immunohistochemical technique was used to evaluate receptors population and COX‐2 expression. Pregnant llamas showed a higher percentage of positive cells and stronger intensity for ERα than for non‐pregnant llamas in stroma on day 8 and in the luminal epithelium on day 12 post‐induction of ovulation, while a deep decrease in endometrial PR population was reported in pregnant llamas on that day in luminal and glandular epithelia and stroma. In the luminal epithelium, COX‐2 expression was lower in pregnant than in non‐pregnant animals. Briefly, the increase of ERα in pregnant llamas gives further support to the hypothesis that oestrogens are involved in the mechanism of maternal recognition of pregnancy. Endometrial PR decrease in pregnant llamas might be a necessary event to allow the expression of proteins involved in conceptus attachment, a mechanism widely accepted in other species. Moreover, embryo seems to attenuate maternal PGF_2α secretion during early pregnancy by decreasing the endometrial expression of COX‐2 in the luminal epithelium of pregnant llamas.