Effect of Dimethyl Sulfoxide on Cell Cycle Synchronization of Goldfish Caudal Fin Derived Fibroblasts Cells

Several studies have previously been conducted regarding cell cycle synchronization in mammalian somatic cells. However, limited work has been performed on the control of cell cycle stages in the somatic cells of fish. The aim of this study was to determine the cell cycle arresting effects of several dimethyl sulfoxide (DMSO) concentrations for different times on different cell cycle stages of goldfish caudal fin‐derived fibroblasts. Results demonstrated that the cycling cells or control group (68.29%) yields significantly higher (p < 0.05) arrest in G0/G1 phase compared with the group treated for 24 h with different concentrations (0.5%, 1.0% or 1.5%) of DMSO (64.88%, 65.70%, 64.22% respectively). The cell cycle synchronization in the treatment of cells with 1.0% DMSO at 48 h (81.14%) was significantly higher than that in the groups treated for 24 h (76.82%) and the control group (77.90%). Observations showed that treatment of DMSO resulted in an increase in the proportion of cells at G0/G1 phase for 48 h of culture. However, high levels of apoptotic cells can be detected after 48 h of culture treated with 1% concentration of DMSO.     

Adenomatous hyperplasia of the thyroid gland in beluga whales (Delphinapterus leucas) from the St. Lawrence Estuary and Hudson Bay, Quebec, Canada

We evaluated thyroid gland lesions in beluga whales (Delphinapterus leucas) from the St. Lawrence Estuary (n = 16) and Hudson Bay (n = 14). Follicular cysts and nodules of adenomatous hyperplasia of the thyroid gland were found in eight and nine adults from the St. Lawrence Estuary (n = 10), respectively, and in four and six adults from Hudson Bay (n = 14), respectively. The total volume of the lesions of thyroid adenomatous hyperplasia was positively correlated with age in both populations. Comparison between populations could not be performed because of differences in age structures of sample groups. Beluga whales from both populations have unique thyroid lesions among marine mammals.     

Triazole fungicides and the selection of resistance to medical triazoles in the opportunistic mould Aspergillus fumigatus

Azole resistance is an emerging problem in the opportunistic mould Aspergillus fumigatus. The triazoles are the most important agents for the management of Aspergillus diseases in humans. Selection for acquired resistance may occur in the hospital setting through exposure to high doses of azoles during azole therapy, but evidence is accumulating that A. fumigatus may become resistant to medical triazoles through environmental exposure to fungicides. The recovery of A. fumigatus isolates resistant to the medical triazoles from azole‐naive patients as well as from the environment strongly indicates an environmental route of resistance selection. Molecule alignment studies have identified five fungicides that share a very similar molecule structure with the medical triazoles, and thus may have selected for mechanisms that confer resistance to both groups of compounds. It is important to explore further the presumed fungicide‐driven route of resistance selection in order to implement effective preventive measures as the prevalence of azole resistance in A. fumigatus continues to increase and causes major challenges in the management of azole‐resistant Aspergillus diseases. Copyright © 2012 Society of Chemical Industry     

Characterization of Transgenic Pigs That Express Human Decay Accelerating Factor and Cell Membrane‐tethered Human Tissue Factor Pathway Inhibitor

The expression of human complement regulatory proteins (hCRP; hDAF, hCD59, and hMCP) in pig tissues has been suggested as one of strategies to overcome the hyperacute rejection (HAR) in pig‐to‐human transplantation. Expression of human tissue factor pathway inhibitor (hTFPI) in porcine endothelial cells has been suggested as a remedy to overcome microvascular thrombosis. To investigate the effects of these combined transgenes, we established transformed pig cells expressing human decay accelerating factor (hDAF) under the control of enhancer promoter (5′LTR‐PCMVIE), and the fusion protein (hTFPI/hCD4) consisting of the functional domains (K1 and K2) of hTFPI and membrane‐tethering domains (D3 and D4) of hCD4 under the control of PCMVIE. Transgenic pigs were generated with the transformed porcine cells through somatic cell nuclear transfer (SCNT) technology. Analysis of quantitative PCR and real‐time quantitative RT‐PCR showed that four copies of hDAF were integrated and 391 copies of hDAF mRNA expressed in the cells of the transgenic pig. The enhancing activity of 5′LTR was approximately 2 fold compared to CMVIE promoter only. The cell viability test showed that more than 80% of ear cells were viable in the presence of 50% human serum. The chromogenic substrate assay and immunocytochemical staining with tail cells showed that the TFPI activity of fusion protein was observed on the cell membrane. The membrane localization of hDAF and hTFPI proteins was observed by immunocytochemical staining, and the expression of transgenes in heart and liver tissues was also confirmed by immunohistochemistry.     

Captive Breeding of Cheetahs in South Africa – 30 Years of Data from the de Wildt Cheetah and Wildlife Centre

The de Wildt Cheetah and Wildlife Centre was established in 1971 and the first cheetah cubs were born in 1975. During the period 1975–2005, 242 litters were born with a total of 785 cubs. Mean cub survival from 1 to 12 months and greater than 12 months of age was 71.3 and 66.2%, respectively. The majority of losses (84.9%) occurred during the first month postpartum whereas only 15.1% deaths took place between 1 and 12 months of age. Females were first bred at an age of approximately 3 years, reached maximum reproductive age at 6–8 years, where after fertility declined. Males reached peak reproduction at 6 and maintained this for up to 12 years of age. Male fertility was best correlated with sperm morphology. During recent years, for practical purposes, males were allocated to 'good' (≥70% normal), 'fair' (40–70% normal) and 'poor' (<40% normal) categories according to sperm morphology count. The breeding males were selected from the good (preferably) and fair categories but poor category males were also used at times. Average litter sizes for 'good', 'fair' and 'poor' males were 3.44 (n = 21), 3.14 (n = 18) and 2.28 (n = 18), respectively. In females the heritability for litter size was high at 0.5848 (532 progeny, 1975–2007) and the maternal heritability for cub mortality was estimated to be 0.596. The data from the de Wildt Cheetah and Wildlife Centre and two other centres in the world (Kapama and Wassenaar) demonstrate that cheetah can be bred successfully in captivity.     

Artificial Insemination of Swine: Fecundity of Boar Semen Stored in Beltsville TS (BTS), Modified Modena (MM), or MR-A and Inseminated on One,Three and Four Days After Collection12

Contents: A field trial was conducted on 1,463 farms in The Netherlands to compare the fertilizing capacity of boar spermatozoa extended in Beltsville TS (BTS), Modified (MM), or MR-A, and inseminated in 2896 sows and gilts on the first, third or fourth day following collection. Semen was collected, extended, and stored at 18°C at six different AI centers and inseminated by inseminators on their regular routes. Sows inseminated with BTS and MR-A extended semen had higher farrowing rates than MM (79.3, 77.6 us 50.4, P <.0001), higher total pigs per litter (11.4, 11.1 vs 10.0, P <.0001) and higher total pigs born alive (10.7, 10.5 vs 9.4, P <. 0001). The farrowing rate of gilts inseminated with BTS extended semen was superior to MM (73.5 vs. 50.2%, P <.004), while MR-A gave farrowing rates greater than MM (64.1 vs 50.2%, P =.06). There was no difference in litter data for gilts. Farrowing rates for 1 and 4 day semen were superior to 3 day semen (73.2, 73.8 vs 60.3, P <.0001). The semen inseminated on day 4 contained 6 billion sperm per dose rather than the 3 billion per dose for 1 and 3 day semen. Based on the results of this study, BTS and MR-A are effective diluents for extension and storage of boar semen for use within the same week of collection. In addition, semen extended in either BTS of MR-A and stored and inseminated on the fourth day after collection can give fertility equal to first day insemination if the sperm per insemination dose is doubled. Inhalt: Künstliche Besamung beim Schwein: Fruchtbarkeitsergebnisse von Ebersamen nach Flussigkonservierung mit BTS, MR-A oder modifiziertem Modena-Verdünner (MM) und Besamung am ersten, dritten und vierten Tag nach Samengewinnung In einem in den Niederlanden durchgeführten Feldversuch wurden 3 verschiedene Verdünner-Medien für Ebersamen in vivo verglichen. Die Medien waren Beltsville TS (BTS), modifizierter Modena-Verdünner (MM) und das spanische MR-A-Medium. Insgesamt wurden 2896 Jung- und Altsauen in 1463 Herden mit 1, 3 oder 4 Tage altem Sperma besamt. Der Versuch wurde als “split-sample”-Versuch an 6 Eberstationen durchgeführt, der verdunnte Samen bei + 18°Cgelagert und von den Besamungsbeauftragten im Rahmen ihrer regulären Fahrten eingesetzt. BTS- und MR-A-konserviertes Sperma gab bei Altsauen gegenüber MM höhere Abferkelraten (79,3, 77,6 vs 50,4%, P < 0.0001), eine höhere totale Wurfzahl (11,4, 11,1 vs 10,0, P < 0.0001) und eine höhere Anzahl lebendgeborener Ferkel)10,7, 10,5 vs 9,4, P < 0.0001). Die mit BTS-uerdünntem Samen inseminierten Jungsauen wiesen gegenüber MM höhere Abferkelraten auf(73.5 vs 50,2%, P < 0.004). MR-A-Sperma lag ebenfalls hüher als MM-Samen (641 vs 50,2%, P=0.06). Die Wurfgröβen der Jungsauen reigten keine Unterschiede. Die Abferkelergebnisse nach Besamung mit 1 und 4 Tage altem Samen lagen höher als die des 3 rage alten Spermas (73,2, 73,8 vs 60,3%; P < 0.0001). Die am Tag 4 verwendeten Samenportionen enthielten 6 Milliarden Spermien gegenüber 3 Milliarden der nach 1 bzw. 3 Tagen inseminierten Portionen. Die Ergebnkse dieses Versuches zeigen, daβ BTS und MR-A effektive Verdünnermedien sind und die Lagerung von Eberfrischsperma zum Einsatz innerhalb der Entnahmewoche ermoglichen. Darüber hinaus können mit BTS oder MR-A verdünntes und am Tag 4 nach Gewinnung eingesetrtes Sperma Fruchtbarkeitsergebnisse erzielt werden, die ebenso gut sind wie nach Verwendung des 1-Tage-Spermas, wenn die Spermienzahl pro Besamungsdosis verdoppelt wird.     

Nitrogen losses from outdoor pig farming systems

Abstract. Nitrogen losses via nitrate leaching, ammonia volatilization and nitrous oxide emissions were measured from contrasting outdoor pig farming systems in a two year field study. Four 1‐ha paddocks representing three outdoor pig management systems and an arable control were established on a sandy loam soil in Berkshire, UK. The pig management systems represented: (i) current commercial practice (CCP) ‐ 25 dry sows ha^?1 on arable stubble; (ii) ‘improved’ management practice (IMP) ‐ 18 dry sows ha^?1 on stubble undersown with grass, and (iii) ‘best’ management practice (BMP) 12 dry sows ha^?1 on established grass. Nitrogen (N) inputs in the feed were measured and N offtakes in the pig meat estimated to calculate a nitrogen balance for each system. In the first winter, mean nitrate‐N concentrations in drainage water from the CCP, IMP, BMP and arable paddocks were 28, 25, 8 and 10 mg NO₃ l^?1, respectively. On the BMP system, leaching losses were limited by the grass cover, but this was destroyed by the pigs before the start of the second drainage season. In the second winter, mean concentrations increased to 111, 106 and 105 mg NO₃‐N l^?1 from the CCP, IMP and BMP systems, respectively, compared to only 32 mg NO₃‐N l^?1 on the arable paddock. Ammonia (NH₃) volatilization measurements indicated that losses from outdoor dry sows were in the region of 11 g NH₃‐N sow^?1 day^?1. Urine patches were identified as the major source of nitrous oxide (N₂O) emissions, with N₂O‐N losses estimated at less than 1% of the total N excreted. The nitrogen balance calculations indicated that N inputs to all the outdoor pig systems greatly exceeded N offtakes plus N losses, with estimated N surpluses on the CCP, IMP and BMP systems after 2 years of stocking at 576, 398 and 264 kg N ha^?1, respectively, compared with 27 kg N ha^?1 on the arable control. These large N surpluses are likely to exacerbate nitrate leaching losses in following seasons and make a contribution to the N requirement of future crops.     

First report and histological features of Chlamydia pecorum encephalitis in calves in New Zealand

CASE HISTORY: Between September and October 2013, 40 of 150 crossbred Friesian dairy calves on a farm in the Manawatu region of New Zealand developed neurological signs when between 1 and 3 months of age. Calves were grazed in multiple mobs and calves from each mob were affected. A variable response was observed to initial treatment with thiamine, fluoroquinolone antibiotics and non-steroidal anti-inflammatory drugs.

CLINICAL AND PATHOLOGICAL FINDINGS: Affected calves exhibited a range of neurological signs that included generalised depression, hind limb ataxia with a stiff gait, and knuckling of the fetlocks. In advanced cases, calves became recumbent with opisthotonous. Over a 4-week period, 13 calves died or were subject to euthanasia and a thorough necropsy was performed on three of these calves. Necropsy findings included fibrinous peritonitis, pleuritis and pericarditis, with no gross abnormalities visible in the brain or joints. Histology of the brain was possible in seven of the affected calves, with lesions ranging from lymphocytic and histiocytic vasculitis and meningoencephalitis, to extensive thrombosis and neutrophilic inflammation. Immunohistochemistry using an anti-chlamydial lipopolysaccharide antibody revealed positive immuno-staining in all seven cases, with no brain samples exhibiting immunostaining for Histophilus somni. DNA was extracted from a sample of fresh brain from one case and chlamydial DNA sequences were amplified by PCR and found to be identical to Chlamydia pecorum. PCR was also performed on formalin-fixed brain tissue from three of the other cases, but no chlamydial DNA was amplified.

DIAGNOSIS: Chlamydia pecorum meningoencephalomyelitis (sporadic bovine encephalomyelitis).

CLINICAL RELEVANCE: This is the first time that C. pecorum has been confirmed as a cause of clinical disease in New Zealand. Practitioners should be aware of this disease as a differential in calves with neurological signs, and submit samples of formalin-fixed brain as well as fresh brain to enable confirmation of suspected cases using PCR analysis. Furthermore, these cases illustrate that the histological lesions in the brains of calves with C. pecorum are more variable than previously reported, and pathologists should be aware that histological features may overlap with those traditionally ascribed to other organisms, such as H. somni.     

Immunomodulatory Effects of Domoic Acid Differ Between In vivo and In vitro Exposure in Mice

The immunotoxic potential of domoic acid (DA), a well-characterized neurotoxin, has not been fully investigated. Phagocytosis and lymphocyte proliferation were evaluated following in vitro and in vivo exposure to assay direct vs indirect effects. Mice were injected intraperitoneally with a single dose of DA (2.5 μg/g b.w.) and sampled after 12, 24, or 48 hr. In a separate experiment, leukocytes and splenocytes were exposed in vitro to 0, 1, 10, or 100 μM DA. In vivo exposure resulted in a significant increase in monocyte phagocytosis (12-hr), a significant decrease in neutrophil phagocytosis (24-hr), a significant decrease in monocyte phagocytosis (48-hr), and a significant reduction in T-cell mitogen-induced lymphocyte proliferation (24-hr). In vitro exposure significantly reduced neutrophil and monocyte phagocytosis at 1 μM. B- and T-cell mitogen-induced lymphocyte proliferation were both significantly increased at 1 and 10 μM, and significantly decreased at 100 μM. Differences between in vitro and in vivo results suggest that DA may exert its immunotoxic effects both directly and indirectly. Modulation of cytosolic calcium suggests that DA exerts its effects through ionotropic glutamate subtype surface receptors at least on monocytes. This study is the first to identify DA as an immunotoxic chemical in a mammalian species.