Strain-associated virulence factors of Streptococcus iniae in hybrid-striped bass

Streptococcus iniae is a major fish pathogen producing invasive infections that result in economic losses in aquaculture. Development of in vitro models of S. iniae virulence may provide insight to the pathogenesis of infection in vivo. Three S. iniae strains (K288, 94-426, and 29178) were tested for virulence in a hybrid-striped bass (HSB) model using intraperitoneal injection. S. iniae strains K288 and 94-426 caused high levels of mortality in HSB (lethal dose 2x10(5)CFU) while strain 29178 was avirulent even upon IP challenge with 1000-fold higher inocula. In vitro assays were developed to test for the presence of characteristics previously associated with virulence in other species of pathogenic Streptococcus in animals and humans. In vitro differences relevant to virulence were not detected for beta-hemolysin activity, sensitivity to antimicrobial peptides, or adherence and invasion of epithelial cell layers. However, in whole-blood killing assays, the pathogenic strains were resistant to blood clearance, while 29178 was cleared (P<0.001) and more sensitive to complement (P<0.001). The avirulent strain 29178 was most efficiently phagocytosed and was most susceptible to intracellular killing (P<0.01) by the carp leukocyte cell line (CLC). When exposed to reactive oxygen species, strain 29178 was most susceptible. When the oxidative burst of CLC cells was inhibited, intracellular survival of 29178 was rescued fivefold, while no significant enhancement in survival of K288 or 94-426 was detected. Our results indicate that resistance to phagocytosis, oxidative killing, and associated phagocytic clearance is a significant factor in S. iniae virulence.     

Effectiveness of controlled internal drug release device treatment to alleviate reproductive seasonality in anestrus lactating or dry Barki and Rahmani ewes during non‐breeding season

This study aimed to evaluate the effectiveness of hormonal treatments on ovarian activity and reproductive performance in Barki and Rahmani ewes during non‐breeding season. Forty‐eight multiparous ewes, 24 Barki and 24 Rahmani ewes were divided into two groups, 12 lactating and 12 dry ewes for each breed. Controlled internal drug release (CIDR) device was inserted in all ewes for 14 days in conjunction with intramuscular 500 IU equine chronic gonadotrophin (eCG) at day of CIDR removal. Data were analysed using PROC MIXED of SAS for repeated measures. Breed, physiological status and days were used as fixed effects and individual ewes as random effects. Barki ewes recorded higher (p < .05) total number of follicles, number of large follicles, serum estradiol concentration and estradiol: progesterone (E₂:P₄) ratio compared to Rahmani ewes. Lactating ewes recorded higher (p < .05) number of small follicles and lower concentration of total antioxidant capacity (TAC) compared to dry ewes. Number and diameter of large follicles recorded the highest (p < .05) values accompanied with disappearance of corpora lutea at day of mating. Serum progesterone concentration recorded lower (p < .05) value at day of mating and the highest (p < .05) value at day 35 after mating. CIDR‐eCG protocol induced 100% oestrous behaviour in both breeds, but Rahmani ewes recorded longer (p < .05) oestrous duration compared to Barki. Conception failure was higher (p < .05) in Barki compared to Rahmani ewes. In conclusion, CIDR‐eCG protocol was more potent in improving ovarian activity in Barki compared to Rahmani ewes, but this protocol seems to induce hormonal imbalance in Barki ewes that resulted in increasing conception failure compared to Rahmani ewes.     

Can the Genetic Origin Affect Rabbit Seminal Plasma Protein Profile along the Year?

The study was designed to evaluate the influence of genetic origin on rabbit seminal plasma protein profile variation along the year. Seminal plasma of rabbits from line A (maternal line) and R (paternal line) collected during a natural year was subjected to polyacrylamide gel electrophoresis (SDS‐PAGE). The electrophoretic profile of rabbit seminal plasma resulted in multiple protein bands of different intensity ranging from 9 to 240 kDa. Results showed that seven protein bands were significantly different between genetic lines, and among these, three protein bands were significantly different between seasons. The differentially expressed proteins were identified by MALDI‐TOF/TOF or LC‐MS/MS analysis and were the following ones: FAM115E‐like (220, 113 and 59 kDa), ectonucleoside triphosphate diphosphohydrolase 3 isoform X2 (72 kDa), annexin A5 (32 kDa), lipocalin allergen Ory c 4 precursor (19 kDa), and haemoglobin subunit zetalike (13 kDa) between genetic lines and FAM115E‐like (113 kDa), haemoglobin subunit zetalike (13 kDa) and β‐nerve growth factor (12 kDa) between seasons. These results indicate that proteins from rabbit seminal plasma are under both seasonal control and genetic control. Furthermore, the differential presence of these proteins could be one of the causes explaining the differences observed in fertility and seminal parameters between these two lines in earlier studies.     

Cryopreservation of Immature Bovine Oocytes to Reconstruct Artificial Gametes by Germinal Vesicle Transplantation

Joining immature gamete cryopreservation and germinal vesicle transplantation (GVT) technique could greatly improve assisted reproductive technologies in animal breeding and human medicine. The present work was aimed to assess the most suitable cryopreservation protocol between slow freezing and vitrification for immature denuded bovine oocytes, able to preserve both nuclear and cytoplasmic competence after thawing. In addition, the outcome of germinal vesicle transfer procedure and gamete reconstruction was tested on the most effective cryopreservation system. Oocytes, isolated from slaughterhouse ovaries, were stored after cumulus cells removal either by slow freezing or by vitrification in open pulled straws. After thawing, oocytes were matured for 24 h in co-culture with an equal number of just isolated intact cumulus enclosed oocytes, and fixed in order to evaluate the stage of meiotic progression and cytoskeleton organization. Our results showed that after warming, vitrified oocytes reached metaphase II (MII) in a percentage significantly higher than oocytes cryopreserved by slow freezing (76.2% and 36.5% respectively, p < 0.05). Moreover, vitrification process preserved the organization of cytoskeleton elements in a higher proportion of oocytes than slow freezing procedure. Therefore vitrification has been identified as the elective method for denuded immature oocytes banking and it has been applied in the second part of the study. Our results showed that 38.3% of oocytes reconstructed from vitrified gametes reached the MII of meiotic division, with efficiency not different from oocytes reconstructed with fresh gametes. We conclude that vitrification represents a suitable method of GV stage denuded oocyte banking since both nuclear and cytoplasmic components derived from cryopreserved immature oocytes can be utilized for GVT.     

Sulfur-associated polioencephalomalacia in cattle grazing plants in the Family Brassicaceae

Polioencephalomalacia was diagnosed histologically in cattle from two herds on the Darling Downs, Queensland, during July–August 2007. In the first incident, 8 of 20 18-month-old Aberdeen Angus steers died while grazing pastures comprising 60% Sisymbrium irio (London rocket) and 40% Capsella bursapastoris (shepherd's purse). In the second incident, 2 of 150 mixed-breed adult cattle died, and another was successfully treated with thiamine, while grazing a pasture comprising almost 100% Raphanus raphanistrum (wild radish). Affected cattle were either found dead or comatose or were seen apparently blind and head-pressing in some cases. For both incidents, plant and water assays were used to calculate the total dietary sulfur content in dry matter as 0.62% and 1.01% respectively, both exceeding the recommended 0.5% for cattle eating more than 40% forage. Blood and tissue assays for lead were negative in both cases. No access to thiaminase, concentrated sodium ion or extrinsic hydrogen sulfide sources were identified in either incident. Below-median late summer and autumn rainfall followed by above-median unseasonal winter rainfall promoted weed growth at the expense of wholesome pasture species before these incidents.     

Activated coagulation times in normal cats and dogs using MAX-ACTTM tubes

Objective  To establish reference values for activated coagulation time (ACT) in normal cats and dogs, by visual assessment of clot formation using the MAX-ACT^TM tube.
Subjects  We recruited 43 cats and 50 dogs for the study; 11 cats and 4 dogs were excluded from the statistical analysis because of abnormalities on clinical examination or laboratory testing including anaemia, prolonged prothrombin time (PT) or activated partial thromboplastin time (APTT), or insufficient plasma volume for comprehensive laboratory coagulation testing.
Procedure  Blood samples were collected via direct venipuncture for MAX-ACT, packed cell volume/total solids, manual platelet estimation and PT/APTT measurement. Blood (0.5 mL) was mixed gently in the MAX-ACT tube at 37°C for 30 s, then assessed for clot formation every 5 to 10 s by tipping the tube gently on its side and monitoring for magnet movement. The endpoint was defined as the magnet lodging in the clot. The technique was tested with 10 dogs by collecting two blood samples from the same needle insertion and running a MAX-ACT on each simultaneously.
Results  In normal cats the mean MAX-ACT was 66 s (range 55–85 s). In normal dogs the mean was 71 s (range 55–80 s). There was no statistical difference between the first and second samples collected from the same needle insertion.
Conclusions and Clinical Relevance  In both cats and dogs, a MAX-ACT result >85 s should be considered abnormal and further coagulation testing should be performed. Additionally, failure to discard the first few drops of the sample does not appear to significantly affect results.     

欧拉羊与山谷型藏羊杂交F_1代羔羊补饲兼放牧育肥试验效果观察

用欧拉羊杂交改良山谷型藏羊,对杂交F1代羔羊补饲兼放牧育肥试验,补饲阶段试验结果表明：经过90 d补饲,试验组增重10.88kg,比对照组高4.32 kg（P〈0.01）,平均日增重0.121 kg,比对照组高0.048 kg;放牧育肥阶段试验结果表明：经过3个月放牧育肥,试验组增重9.09 kg,比对照组高2.67 kg（P〈0.05）,平均日增重0.101 kg,比对照组高0.03 kg。     

Postbloom fruit drop of citrus and key lime anthracnose are caused by distinct phylogenetic lineages of Colletotrichum acutatum

Colletotrichum acutatum causes two diseases of citrus, postbloom fruit drop (PFD) and Key lime anthracnose (KLA). PFD is a disease restricted to flowers of sweet orange and most other citrus, and symptoms include petal necrosis, abscission of developing fruit, and the formation of persistent calyces. KLA is a disease of foliage, flowers, and fruits of Key lime only, and symptoms include necrotic lesions on leaves, fruits, twigs, flowers, and blight of entire shoots. The internal transcribed spacers 1 and 2 and the gene encoding the 5.8S ribosomal RNA subunit within the nuclear ribosomal cluster (ITS) and intron 2 of the glyceraldehyde-3-phosphate dehydrogenase gene (G3PD) were sequenced for isolates from PFD-affected sweet orange and KLA-affected Key limes collected in the United States (Florida), Brazil (S?o Paulo), Mexico, Belize, Costa Rica, and the Dominican Republic to determine if there are consistent genetic differences between PFD and KLA isolates over the geographic area where these diseases occur. Based on the sequence data, isolates clustered into two well-supported clades with little or no sequence variation among isolates within clades. One clade (PFD clade) contained PFD isolates from all countries sampled plus a few isolates from flowers of Key lime in Brazil. The other clade (KLA clade) contained KLA isolates from Key lime foliage from all countries sampled and one isolate from flowers of sweet orange in Mexico. In greenhouse inoculations with PFD and KLA isolates from Florida, isolates from both clades produced PFD symptoms on Orlando tangelo flowers, but KLA-clade isolates produced significantly less severe symptoms. PFD-clade isolates were not pathogenic to Key lime foliage, confirming previous studies. The differentiation of PFD and KLA isolates into two well-supported clades and the pathogenicity data indicate that PFD and KLA are caused by distinct phylogenetic lineages of C. acutatum that are also biologically distinct. PFD is a recently described disease (first reported in 1979) relative to KLA (first reported in 1912) and it had been proposed that strains causing PFD evolved from strains causing KLA eventually losing pathogenicity to Key lime foliage. We reject the hypothesis that PFD strains have diverged from KLA strains recently based on estimated divergence times of haplotypes and it appears that PFD and KLA strains have been dispersed throughout the Americas independently in association with each host.