Effects of the synthetic selectin inhibitor TBC1269 on tissue damage during acute Mannheimia haemolytica-induced pneumonia in neonatal calves

OBJECTIVE: To determine effects of the selectin inhibitor TBC1269 on neutrophil-mediated pulmonary damage during acute Mannheimia haemolytica-induced pneumonia in newborn calves. ANIMALS: Eighteen 1- to 3-day-old colostrum-deprived calves. PROCEDURE: Mannheimia haemolytica or saline (0.9% NaCl) solution was inoculated in both cranial lung lobes of 12 and 6 calves, respectively. Calves were euthanatized 2 (saline, n = 3; M haemolytica, n = 4) or 6 hours (saline, n = 3; M haemolytica, n = 8) after inoculation. Four M haemolytica-inoculated calves euthanatized at 6 hours also received TBC1269 (25 mg/kg, IV) 30 minutes before and 2 hours after inoculation. Conjugated diene (CD) concentrations, inducible nitric oxide synthase (iNOS) expression, and apoptotic cell counts were determined in lung specimens collected during necropsy. RESULTS: Conjugated diene concentrations were significantly increased in all M haemolytica-inoculated groups, compared with saline-inoculated groups. Calves treated with TBC1269 had decreased concentrations of CD, compared with untreated calves, although the difference was not significant. Number of apoptotic neutrophils and macrophages increased significantly inTBC1269-treated calves, compared with untreated calves. Inducible nitric oxide synthase was expressed by epithelial cells and leukocytes. However, iNOS was less abundant in airway epithelial cells associated with inflammatory exudates. Degree of iNOS expression was similar between TBC1269-treated and untreated calves. CONCLUSIONS: Mannheimia haemolytica infection in neonatal calves resulted in pulmonary tissue damage and decreased epithelial cell iNOS expression. The selectin inhibitor TCB1269 altered, but did not completely inhibit, neutrophil-mediated pulmonary damage.     

Transduction of Vero cells and bovine monocytes with a herpes simplex virus-1 based amplicon carrying the gene for the bovine herpesvirus-1 Circ protein

Herpes simplex virus-1 (HSV-1) based amplicon vectors are promising gene delivery vehicles because they have a large transgene capacity and can efficiently transduce many different cell types, including non-dividing cells, of various animal species. The Circ protein of bovine herpesvirus-1 (BHV-1) is a myristylated virion component of unknown function. Preliminary experiments with a circ gene deletion mutant indicated that Circ may influence the host's immune response by downregulating MHC-II expression in bovine monocytes. To get more insight into the function of Circ, amplicon vectors were constructed with various open reading frames (ORFs) under the control of the HSV-1 IE4/5 promoter: (i) the Circ ORF alone, (ii) a fusion ORF encoding an N-terminal Circ fused to the enhanced green fluorescent protein (eGFP), (iii) the eGFP ORF alone, and (iv) the Circ ORF in the inverted orientation. Upon helpervirus-free packaging into HSV-1 amplicon particles and transduction of Vero cells, both Circ alone and the Circ-eGFP fusion protein produced a punctate pattern within the cytoplasm, suggesting membrane association of the myristylated protein. In contrast, eGFP alone was evenly distributed over the cytoplasm of transduced cells. Upon infection of bovine buffy-coat cells, it was observed that cells of the monocyte lineage but not lymphocytes were transduced. Transgene expression reached a peak around 20h after transduction and lasted for at least 90h. Transduced monocytes underwent specific morphological changes, which may be attributed to Circ synthesis.     

Early epithelial invasion by Salmonella enterica serovar Typhimurium DT104 in the swine ileum

Salmonella enterica serovar Typhimurium is an important intestinal pathogen in swine. This study was performed to document the early cellular invasion of Salmonella serovar Typhimurium in swine ileum. Ileal gut-loops were surgically prepared in ten 4- to 5-week-old mixed-breed pigs and inoculated for 0-60 minutes. Loops were harvested and prepared for both scanning and transmission electron microscopy (SEM and TEM, respectively). Preferential bacterial adherence to microfold cells (M cells) was seen within 5 minutes, and by 10 minutes bacterial invasion of the apical membrane was seen in M cells, goblet cells, and enterocytes. This multicellular invasion was observed throughout the course of infection. In addition, SEM revealed a specific affinity of Salmonella serovar Typhimurium to sites of cell extrusion. Using TEM, bacteria in these areas were focused in the crevices formed by the extruding cell and the adjacent cells and in the cytoplasm immediately beneath the extruding cell. Our results suggest that early cellular invasion by Salmonella serovar Typhimurium is nonspecific and rapid in swine. Furthermore, the combination of SEM and TEM data suggests that Salmonella serovar Typhimurium may use sites of cell extrusion as an additional mechanism for early invasion.     

Sperm Morphology in the Domestic Cat, and its Relation with Fertility: A Retrospective Study

Knowledge about normal ranges in semen quality and the association between sperm morphology and fertility in felids is limited. The aims of this retrospective study were to (1) define a normal spermiogram in cats; (2) evaluate possible effects of season, age and breed on sperm morphology; and (3) evaluate the relationship between sperm morphology and fertility. Semen samples collected by electroejaculation from 52 cats were evaluated for sperm morphology. The cats constituted two groups: a general population of cats (n = 48) and cats examined because of poor breeding records (n = 4). The general population was divided into household (n = 20), pedigree (n = 19) and colony cats (n = 9) and into three age classes, <12 months, 12-59 months and >or=60 months. The median percentage of normal spermatozoa in the general population was 44.0% (range 1.0-91.0%). Criteria were tentatively set for what was considered a normal spermiogram. The mean percentage of normal spermatozoa was higher during February to July than during August to January (p < 0.05). Pedigree cats had a lower mean percentage of normal spermatozoa than did household cats (p < 0.05). Age had no effect on the percentage of normal spermatozoa but was positively correlated with the percentage of proximal droplets. Of the cats with <40% normal spermatozoa (n = 19), all those with known breeding records (n = 11) had produced litters. The four cats examined because of poor breeding results had higher percentages of different sperm abnormalities than tentatively stipulated for the normal spermiogram. In two of these cats both sperm morphology and fertility changed over time.     

Management of inappetant sheep during export by sea

In the first of 2 experiments, a simulated voyage was conducted to examine the effects of various treatments on bodyweight change and feeding frequency of inappetant sheep at the end of lot-feeding (non-feeders). The treatments, applied during simulated shipping, were: normal quantities of feed and length of troughs; extra trough length; extra feed. Adult Merino wethers (n = 108) were used in each treatment. A voyage to the Middle East was then conducted to establish whether shipboard mortality could be reduced by separating non-feeders (n = 305) from feeders (n = 5,620) late in the feedlot hase and housing the groups separately aboard ship. A control group of non-feeders (n = 215) mixed with feeders (n = 5,732) was used for comparison. Bars (marker bars), containing a coloured dye, were attached to feed troughs to mark sheep that fed. Most non-feeders (82%) began eating when placed in shipping pens in both studies. However, there was no significant difference in percentage of sheep that fed between non-feeders given extra trough length or extra feed compared with non-feeders given standard management at any stage of simulated shipping. There was no significant difference in mean bodyweights between treatment groups on days 1, 8 and 15 of simulated shipping. Differences in bodyweight on d 22 were probably associated with different levels of gut fill. Death rates were not significantly different in separated and control groups (1.1%, 0.9%, P = 0.6) in the voyage of 14 d to the Middle East.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Oil Overlay on Inhibition Potential of Roscovitine in Sheep Cumulus‐Oocyte Complexes

Inhibitors of cyclin‐dependent kinases, as roscovitine, have been used to prevent the spontaneous resumption of meiosis in vitro and to improve the oocyte developmental competence. In this study, the interference of oil overlay on the reversible arrest capacity of roscovitine in sheep oocytes as well as its effects on cumulus expansion was evaluated. For this, cumulus‐oocyte complexes (COCs) were cultured for 20 h in TCM 199 with 10% foetal bovine serum (Control) containing 75 μm roscovitine (Rosco). Subsequently, they were in vitro matured (IVM) for further 18 h in inhibitor‐free medium with LH and FSH. The culture was performed in Petri dishes under mineral oil (+) or in 96 well plates without oil overlay (?) at 38.5°C and 5% CO₂. At 20 and 38 h, the cumulus expansion and nuclear maturation were evaluated under stereomicroscope and by Hoechst 33342 staining, respectively. No group presented cumulus expansion at 20 h. After additional culture with gonadotrophins, a significant rate of COCs from both Control groups (+/?) exhibited total expansion while in both Rosco groups (+/?) the partial expansion prevailed. Among the oocytes treated with roscovitine, 65.2% were kept at GV in the absence of oil overlay while 40.6% of them reached MII under oil cover (p < 0.05). This meiotic arrest was reversible, and proper meiosis progression also occurred in the Control groups (+/?). So, the culture system without oil overlay improved the meiotic inhibition promoted by roscovitine without affecting the cumulus expansion rate or the subsequent meiosis progression.