Exophiala angulospora infection in hatchery‐reared lumpfish (Cyclopterus lumpus) broodstock

Samples from moribund lumpfish were collected in a marine hatchery in Scotland in 2015. Black nodules were noted on the skin, and gills and fungal hyphae were extensively distributed in musculature and internal organs. Multifocal chronic inflammatory lesions displaced structures in all affected organs. Mortalities commenced on completion of spawning in May and were evenly distributed over the second year in the temperature range 11–15°C. The main systemic infection causing agent was initially identified based on morphological characteristics as an Exophiala species. Ribosomal DNA (rDNA) ITS regions of the isolates were subsequently sequenced confirming the isolates belonged to Exophiala genus. All isolates fell in a single phylogenetic cluster, which is represented by Exophiala angulospora. Fish were treated with either formalin or Bronopol or a combination of both, but there was no effect on the pattern or numbers of mortalities. Isolates were also tested against three different concentrations of Latrunculin A, Amphotericin B and Itraconazole with no success. It is of utmost importance to increase the knowledge on pathogen–host interactions to successfully develop sustainable control methods.     

Isolation and selection of Hemileia Vastatrix antagonists

Organic coffee growing is rapidly increasing in Brazil, and many diseases, especially coffee leaf rust (Hemileia vastatrix), are threatening its production. This study is a first step towards a biocontrol program for coffee rust on organically grown plants. In three organic coffee farms in the state of Minas Gerais, 393 microbial strains including 154 bacterial and 239 fungal strains were isolated from leaves, leaf debris, and soil samples, and in 6 month-old coffee plants, 17 of these isolates reduced both the infection frequency (IF) and the number of H. vastatrix urediniospores produced per leaf (UPL) by more than 70 %. The isolates were identified as eight bacteria isolates, seven Bacillus spp. and one Pseudomonas sp., and nine fungal isolates, four Fusarium spp., two Penicillium spp., one Aspergillus sp., one Acremonium sp. and one Cladosporium sp. Each fungal and bacterial isolate was applied 0, 4, 8, 12 or 16 days before and 0, 4, 8, 12 or 16 days after H. vastatrix inoculation, and the efficiency in reducing both IF and UPL was evaluated. The efficiency was higher and lasted longer when the bacterial isolates were applied before H. vastatrix inoculation. Six Bacillus (B10, B25, B143, B157, B171, B175), two Fusarium (F205, F281), and one Pseudomonas (B286) isolates are potentially efficient as biocontrol agents of H. vastatrix and will be tested using field experiments.     

Molecular detection of Leishmania sp. in cats (Felis catus) from Andradina Municipality, São Paulo State, Brazil

The aim of this work was to molecularly detect Leishmania species in 52 cats from Andradina Municipality, S?o Paulo State, Brazil. The direct parasitological test was performed by using imprints of poplited lymph node, bone marrow and spleen to verify amastigote forms of Leishmania spp. The samples that were positive parasitological tests were subjected to molecular analysis (PCR) and sequencing. Infection was detected for 5.76% (3/52) of the examined cats and two had presence of amastigote forms of Leishmania spp. in lymph nodes. Polymerase chain reaction (PCR) of kinetoplast minicircle DNA, indicated positive amplification for samples of spleen and lymph nodes and the sequencing resulted in 97% similarity with Leishmania (L.) chagasi. This study proved the occurrence of infection with Leishmania (L.) chagasi in felines from Andradina municipality, S?o Paulo State.     

Changes in c‐erbB‐2 Immunoexpression in Feline Endometrial Adenocarcinomas

Human epidermal growth factor receptor type 2 (c‐erbB‐2), an oncoprotein with potential prognostic marker and therapeutic use, is overexpressed in several human and animal tumours. But information regarding this molecule in feline tumours is scarce. This study aimed to assess the changes in the immunohistochemical expression of c‐erbB‐2 in feline endometrial adenocarcinomas (FEA) compared to normal endometrium. An immunohistochemistry assay using a specific antibody against c‐erbB‐2 was performed in FEA samples (n = 34) and in normal endometrium in the follicular (FS; n = 12) and luteal (LS; n = 11) stages. In FEA, the c‐erbB‐2 immunoexpression was assessed in neoplastic epithelial cells whilst in normal endometria it was individually evaluated in the surface and the superficial and deep glandular epithelia (SE, SGE and DGE, respectively). In FS and in LS, all the epithelia were positive for c‐erbB‐2; positivity was higher in the SE and the SGE than in DGE. Twenty of the 34 FEA samples (58.8%) were positive for c‐erbB‐2 immunolabelling. Nevertheless, its expression was higher in all the epithelia in the FS compared to FEA (p ≤ 0.0001) or the LS (p = 0.016). The results presented herein suggest that c‐erbB‐2 molecule is differently expressed in the feline endometrium through the oestrous cycle and though it may also be involved in feline endometrial carcinogenesis, a question remains unanswered on the importance of additional pathways of epithelial proliferation in the neoplastic changes in feline endometrium.     

Interaction between ascorbic acid and follicle-stimulating hormone maintains follicular viability after long-term in vitro culture of caprine preantral follicles

This study evaluates the effects of ascorbic acid and its interaction with follicle-stimulating hormone (FSH) on the morphology, activation, and in vitro growth of caprine preantral follicles. Ovarian fragments were cultured for 1, 7, or 14 d in minimum essential medium (MEM) containing ascorbic acid (50 or 100 μg/mL), FSH (50 ng/mL), or both of these substances. Ovarian tissue that was either fresh (control) or cultured for 1, 7, or 14 d was processed for histological and ultrastructural evaluation. The results showed that after 14 d of culture, medium supplemented with 50 μg/mL of ascorbic acid alone or combined with FSH showed higher rates of follicular survival compared with MEM. After 7 d of culture, FSH, ascorbic acid at 50 μg/mL with or without FSH, and ascorbic acid at 100 μg/mL increased the percentage of follicular activation compared to fresh control. In addition, FSH alone significantly increased the percentage of growing follicles after 14 d. The combination of 50 μg/mL of ascorbic acid and FSH promoted a significant increase in oocyte and follicular diameter after 7 d of culture. Ultrastructural and fluorescent analysis confirmed the integrity of follicles cultured with 50 μg/mL of ascorbic acid and FSH after 14 d. In conclusion, the combination of 50 μg/mL of ascorbic acid and FSH maintained follicular integrity and promoted follicular activation and growth after long-term in vitro culture of caprine preantral follicles.     

Ion,Protein, Phospholipid and Energy Substrate Content of Oviduct Fluid During the Oestrous Cycle of Buffalo (Bubalus bubalis)

The aim of this research was to analyse the composition of oviduct fluid (ODF) in buffalo cows at different oestrous cycle phases to fulfil the requirements of buffalo embryos in vitro. ODF was collected by chronic cannulation from three cows that were synchronized by administering a synthetic prostaglandin. Based on hormonal profiles, the pre‐ovulatory, ovulatory, post‐ovulatory and luteal phases of the oestrous cycle were defined. The volume of ODF produced (ml/24 h) was influenced by the oestrous cycle, with values (mean ± SE) around ovulation (1.0 ± 0.2) greater (p < 0.05) than in both the luteal (0.4 ± 0.1) and the post‐ovulatory phases (0.5 ± 0.1), but not different from the intermediate values in the pre‐ovulatory phase (0.8 ± 0.2). Among cycle phases, no differences were found in sodium, potassium, calcium and magnesium concentrations (130.0 ± 1.1, 5.1 ± 0.3, 2.8 ± 0.1 and 0.59 ± 0.04 mmol/l respectively). Interestingly, the chloride secretion (μm /24 h) was higher (p < 0.05) at ovulation (150.2 ± 16.5) than during both the luteal (73.7 ± 22.0) and the post‐ovulatory phases (63.7 ± 11.2), with intermediate values in the pre‐ovulatory phase (113.4 ± 23.5). Glucose concentration (mmol/l) was higher (p = 0.056) in the pre‐ovulatory phase (0.06 ± 0.02) than in the luteal (0.02 ± 0.01) and post‐ovulatory (0.02 ± 0.01) phases but not different from values in the ovulatory phase (0.04 ± 0.02). Concentrations of pyruvate and lactate among oestrous cycle phases were similar (0.08 ± 0.01 and 1.0 ± 0.1 mmol/l respectively). The total quantity of phospholipids (μmol/24 h) was greater (p < 0.05) at ovulation (0.21 ± 0.02) compared with the luteal, pre‐ovulatory and post‐ovulatory phases of the cycle (0.09 ± 0.02, 0.13 ± 0.02 and 0.09 ± 0.01 respectively). No differences were found in either the protein concentration (1.8 ± 0.3 mg/ml) or the quantity of proteins secreted in 24 h (1.8 ± 0.4 mg) among oestrous cycle phases. In conclusion, this study provides the first characterization of buffalo ODF during the oestrous cycle, showing species‐specific differences that may be useful for developing suitable media for buffalo in vitro embryo production.     

Corpus Luteum Development and Function after Supplementation of Long‐Acting Progesterone During the Early Luteal Phase in Beef Cattle

Strategic supplementation of P4 may be used to increase conception rates in cattle, but timing of supplementation in relation to ovulation, mass of supplementary P4 and formulation of the P4‐containing supplement has not been determined for beef cattle. Effects of supplementation of long‐acting progesterone (P4) on Days 2 or 3 post‐ovulation on development, function and regression of corpus luteum (CL) were studied in beef cattle. Cows were synchronized with an oestradiol/P4‐based protocol and treated with 150 or 300 mg of long‐acting P4 on Day 2 or 3 post‐ovulation (6–7 cows/group). Colour‐doppler ultrasound scanning and blood sample collection were performed from Day 2–21.5. Plasma P4 concentrations were greater (p < 0.05) from Day 2.5–5.5 in the Day 2‐treated groups and from Day 3.5–5.5 in the Day 3‐treated cows than in the control group. CL area and blood flow during Day 2–8.5 did not differ (p > 0.05) among groups, suggesting no effect of P4 treatment on luteal development. The frequency of cows that began luteolysis before Day 15 was greater (p < 0.04) in cows treated with 300 mg than in the controls, but there were no differences between non‐treated and 150 mg‐treated cows. The interval from pre‐treatment ovulation to functional and structural luteolysis was shorter (p < 0.01) in the combined P4‐treated groups than in the control cows. In conclusion, was showed for the first time that long‐acting P4 supplementation on Day 2 or 3 post‐ovulation increases P4 concentrations for ≥3 day, has no effect on luteal development, but anticipates the beginning of luteolysis in beef cattle.