Detection of Paranannizziopsis australasiensis in tuatara (Sphenodon punctatus) using fungal culture and a generic fungal PCR

AIMS: To describe the methods used at the Animal Health Laboratory (AHL, Ministry for Primary Industries) to identify Paranannizziopsis australasiensis.

METHODS: Skin biopsy samples from two adult male tuatara were submitted to the AHL in March 2014. Approximately half of each sample was processed for fungal culture and incubated on mycobiotic agar containing cycloheximide at 30°C. Following morphological examination of the culture products, DNA was extracted from suspect colonies. PCR was used to amplify the internal transcribed spacer (ITS) region of fungal rRNA using primers ITS1 and ITS4. Positive amplicons were subjected to DNA sequencing and the results were compared to published sequences. In addition, DNA was extracted from the remaining skin samples and the same PCR was carried out to compare the results.

RESULTS: After 7 days of incubation, colonies morphologically resembling P. australasiensis were observed. DNA extracted from these isolates tested positive for P. australasiensis by PCR and DNA sequencing. Samples of DNA extracted directly from the infected skin samples tested negative for P. australasiensis using the generic fungal PCR.

CONCLUSIONS AND CLINICAL RELEVANCE: Isolation and identification of P. australasiensis was carried out using a combination of fungal culture and molecular testing available at AHL. Results were available in significantly less time than in the past, when isolates had to be sent overseas. PCR and sequencing of fungal isolates is a valuable tool for identification of species that have few, if any, unique macroscopic or microscopic features to aid identification. Further sampling from captive and wild New Zealand reptiles will provide important information on the epidemiology of P. australasiensis, and the conservation and management implications for tuatara and other native reptile species.     

Shortening the Postpartum Anoestrous Interval in Suckled Crossbred Dual Purpose Cows Using Progestagen Intravaginal Sponges plus eCG and PGF2α

One hundred and twenty‐six suckled crossbred cows (Bos taurus × Bos indicus), with body condition score ≥3 (1–5 point scale), were employed in the present study to evaluate the effectiveness of intravaginal progestin‐releasing sponges (IVS) for shortening anoestrous interval. Fifty‐four cows were assigned to control group. Seventy‐two cows were treated with IVS impregnated with 250 mg of medroxy‐acetate‐progesterone (MAP) as follows: day 0, IVS plus 5 mg of 17β‐E and 50 mg of MAP i.m.; day 6, 500 IU of equine chorionic gonadotropin and 25 mg prostaglandin F_2α i.m.; day 8, IVS withdrawal and day 9, 1 mg 17β‐E i.m. Cows were also grouped according to postpartum days (dpp) at treatment: MAP <70 days (n = 25); control <70 days (n = 22); MAP >70 days (n = 47); control >70 days (n = 32). From IVS removal, cows were detected in oestrus and inseminated. Cows not detected in oestrus were timed artificial insemination 72 h after sponge removal. Treatment effect on oestrous rate (ER), conception rate (CR), pregnancy rate (PR) and treatment to conception intervals (TCI) and calving to conception intervals (CCI) were evaluated. The ER, CR and PR were analysed using proc logistic , while TCI and CCI with proc glm of SAS. The groups MAP <70 days and MAP >70 days showed higher (p < 0.01) ER than control <70 days and control >70 days (84.0% and 76.6% vs 31.8% and 31.3% respectively). The PR was higher (p < 0.01) in MAP <70 days vs control <70 days (64.0% vs 22.7%) and also higher (p < 0.05) in MAP >70 days vs control <70 days (40.4% vs 18.8%). The TCI and CCI were shorter (p < 0.01) in MAP <70 days vs control <70 days (36.0 and 95.8 days; 95.3 and 158.6 days respectively). In conclusion, only cows treated with IVS before 70 dpp had a CCI shorter than 100 days, consequently this treatment shortened postpartum anoestrous interval in crossbred dual purpose cattle.     

Diagnosis of Endometritis in the Bitch: A New Approach

There are a few investigations into endometritis in the bitch and its relationship with failure to conceive remains unclear. This may be because of the difficulty in collecting uterine samples for further investigations. Recently, transcervical catheterization by vaginal endoscopy has been introduced allowing the evaluation of the endometrium. In this study, uterine cytology and bacteriology were evaluated in 26 infertile bitches. Endometritis was bacterial in origin in most cases (70% of affected bitches), but these results may be underestimated, as some other pathogens (anaerobic bacteria, mycoplasms and fungi) were not investigated. Endometritis, in our opinion, should be investigated in each case of unexplained infertility in bitches. The method used here seems reliable although defining more accurate classification criteria will improve the efficiency of this non-invasive technique.     

Anthocyanin-based natural colorants: a new source of antiradical activity for foodstuff

The antiradical capacity (radical scavenger capacity, RSC) of anthocyanin-based fruit extracts prepared in the laboratory (black chokeberry, black-thorn, and strawberry) was studied by using the 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH(*)). To determine their RSC, the second-order rate constant (k(2)) for the oxidation of these extracts by DPPH(*) was calculated. The value of k(2) was compared to that used in the food industry as natural (alpha-tocopherol) or synthetic (butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA)) antioxidants, as well as for a commercial elderberry concentrate and a synthetic colorant (Ponceau 4R). The k(2) values ((mg/mL)(-)(1) s(-)(1)), in methanol at 25 degrees C, were 1.87, 0.7, 0.42, 0.2, 0.05, 0.03, and 0.008 for alpha-tocopherol, black chokeberry, BHA, black-thorn, BHT, strawberry, and elderberry, respectively. Ponceau 4R lacked RSC. Therefore, these natural colorants proved to be a combined source of color and RSC for food material.     

液体潲食对猪生产性能和健康状况的影响

用液体稻食作为猪口粮,可降低10％～17％的成本有数据表明：液体路食可提高猪生产性能,改善其健康状况并降低死亡率70％液体潲食富合碳水化合物如：液体小麦淀粉,乳酪及熟马铃薯度等液体潲食有一些利于贮藏发酵的特点和优势,发酵可能是由于液体潲食所含合成食品和水份所引起．研究人员采用试验研究5种不同液体饲料贮藏6d后的理化特征变化并统计所有可测变化值,如PN值下降到35－3B;乳酸水平上升为15～30g／kg．本文数据综合自几个试验     

Synthesis of the antioxidant hydroxytyrosol using tyrosinase as biocatalyst

Hydroxytyrosol (HTyr), a natural ortho-diphenolic antioxidant with health-beneficial properties that mainly occurs in virgin olive oil and olive oil mill waste waters (also known as vegetative waters), has been enzymatically synthesized using mushroom tyrosinase. This o-diphenol (not commercially available) was obtained from its monophenolic precursor tyrosol (commercially available) in the presence of both tyrosinase and ascorbic acid. The reaction synthesis is continuous, easy to perform, and adaptable to a bioreactor for industrial purposes. The HTyr concentration is time-predicted, and the yield of reaction can be 100%. The synthesis method reported here is an alternative approach to obtain this compound in an environmentally friendly way.     

Characterization of the total free radical scavenger capacity of vegetable oils and oil fractions using 2,2-diphenyl-1-picrylhydrazyl radical

The total free radical scavenger capacity (RSC) of 57 edible oils from different sources was studied: olive (24 brands of oils), sunflower (6), safflower (2), rapeseed (3), soybean (3), linseed (2), corn (3), hazelnut (2), walnut (2), sesame (2), almond (2), mixture of oils for salad (2), "dietetic" oil (2), and peanut (2). Olive oils were also studied according to their geographical origins (France, Greece, Italy, Morocco, Spain, and Turkey). RSC was determined spectrophotometrically by measuring the disappearance of the radical 2,2-diphenyl-1-picrylhydrazyl radical (DPPH(*)) at 515 nm. The disappearance of the radical followed a double-exponential equation in the presence of oils and oil fractions, which suggested the presence of two (fast and slow) groups of antioxidants. RSC was studied for the methanol-soluble phase ("methanolic fraction", MF) of the oil, the fraction nonsoluble in methanol ("lipidic fraction", LF), and the nonfractionated oil ("total oil"; TF = MF + LF). Only olive, linseed, rapeseed, safflower, sesame, and walnut oils showed significant RSC in the MF due to the presence of phenolic compounds. No significant differences were found in the RSC of olive oils from different geographical origins. Upon heating at 180 degrees C the apparent constant for the disappearance of RSC (k(T)) and the half-life (t1/2) of RSC for MF, LF, and TF were calculated. The second-order rate constants (k2) for the antiradical activity of some phenolic compounds present in oils are also reported.     

Caseins and casein hydrolysates. 1. Lipoxygenase inhibitory properties

Whole casein from bovine origin, the different casein subtypes alpha, beta, and kappa, and the related dephosphorylated proteins were assayed as modulators of soybean lipoxygenase 1 activity and were found to inhibit it. To define the lipoxygenase inhibitory domain, whole casein and beta-casein were digested by proteases (trypsin, clostripain, and subtilisin). The beta-casein tryptic digest and the tryptic and subtilisin digests of whole casein retained their inhibitory properties. The tryptic beta-casein digest was the most potent inhibitor of lipoxygenase activity and was further fractionated by FPLC or HPLC. The collected peptides inhibited the lipoxygenase-catalyzed reaction to different extents. The active fractions were analyzed by ESI-MS, and the sequences of several lipoxygenase inhibitory peptides, corresponding mainly to the C-terminal moiety of beta-casein, were identified.     

Impact of Maillard reaction on immunoreactivity and allergenicity of the hazelnut allergen Cor a 11

Few studies exist on the influence of processing methods on structural changes and allergenic potential of hazelnut proteins. This study focused on the effect of glycation (Maillard reaction) on the immunoreactivity and degranulation capacity of the purified hazelnut 7S globulin, Cor a 11. After heating, the extent of the Maillard reaction, sensitivity to proteolysis, binding of human IgE or rabbit IgG, and degranulation capacity were analyzed. Changes in electrophoretic mobility, amount of free amino groups, and contents of bound sugar and fructosamine indicated that glycation of Cor a 11 occurred at all conditions. Glycation at 37 °C did not influence the specific IgG or IgE binding and was decreased after heating at 60 and 145 °C. Heating, with or without glucose, at 145 °C increased basophil degranulation capacity. The results suggest that glycation of Cor a 11 at 60 and 145 °C may decrease the IgE/IgG binding properties but not the degranulation capacity of basophils. This is possibly related to aggregation of the proteins as a result of the Maillard reaction.