Change of porcine circovirus 2 target cells in pigs during development from fetal to early postnatal life

Change of porcine circovirus 2 (PCV2) target cells during development from fetal to postnatal life in pigs was examined. PCV2 inoculation was performed in fetuses in utero at either 57, 75 or 92 gestational days and in piglets at 1 day of age. Twenty-one days after virus inoculation, PCV2-infected cells in the heart, lungs, liver, spleen and inguinal lymph nodes were localized and immuno-phenotyped by double-immunofluorescence labeling using different cell markers and PCV2-antibodies. During fetal life, viral antigens were detected in cardiomyocytes, hepatocytes and macrophages and infected cell numbers decreased with increasing fetal age at inoculation. The heart contained the highest number of infected cells and cardiomyocytes were the main target cell. Postnatally, macrophages were the only target cell type in different organs and infected cell numbers were similar to those of fetuses inoculated at 92 days of gestation. One piglet showed exceptionally high number of infected cells in different organs with values 13-513-fold higher compared to littermates. In this piglet, the majority of infected cells in lymphoid tissues could not be typed. This study reveals that PCV2 target cells change from cardiomyocytes, hepatocytes and macrophages during fetal life to only macrophages postnatally.     

Isolation and identification of Mycobacterium kansasii from pleural fluid of a dog with persistent pleural effusion

A 3-year-old spayed female Whippet was examined for cough and respiratory distress. Lung lobe torsion with pleural effusion was diagnosed, and lung lobectomy was performed. Pleural effusion recurred during the following 27 months; conventional bacteriologic cultures of pleural effusion did not result in bacterial growth. A second lung lobectomy, pleuroperitoneal shunt placement. and pericardectomy were subsequently performed. Mycobacterium kansasii was eventually isolated from pleural fluid and identified by polymerase chain reaction amplification and DNA sequencing. The dog was euthanatized before therapeutic response could be evaluated. To our knowledge, this is the first report of M. kansasii infection in a dog. Additionally, this is the first report of mycobacterial isolation from pleural fluid, and one of few reports of antemortem mycobacterial isolation from a body fluid, as opposed to identification in specimens during histologic examination. Routine bacteriologic culture methods are insufficient to isolate mycobacterial agents, and special methods are indicated in dogs with persistent pleural effusion.     

Characterization of Clostridium difficile isolates from foals with diarrhea: 28 cases (1993-1997)

OBJECTIVE: To determine molecular characteristics of Clostridium difficile isolates from foals with diarrhea and identify clinical abnormalities in affected foals. DESIGN: Retrospective study. ANIMALS: 28 foals with C difficile-associated diarrhea. PROCEDURE: Toxigenicity, molecular fingerprinting, and antibiotic susceptibility patterns were determined. Information on signalment, clinical findings, results of clinicopathologic testing, whether antimicrobials had been administered prior to development of diarrhea, and outcome was obtained from the medical records. RESULTS: Twenty-three (82%) foals survived. Toxin A and B gene sequences were detected in isolates from 24 of 27 foals, whereas the toxin B gene alone was detected in the isolate from 1 foal. Results of an ELISA for toxin A were positive for fecal samples from only 8 of 20 (40%) foals. Ten of 23 (43%) isolates were resistant to metronidazole. Molecular fingerprinting revealed marked heterogeneity among isolates, except for the metronidazole-resistant isolates. Sixteen foals had tachypnea. Hematologic abnormalities were indicative of inflammation. Common serum biochemical abnormalities included metabolic acidosis, hyponatremia, hypocalcemia, azotemia, hypoproteinemia, hyperglycemia, and high enzyme activities. Passive transfer of maternal antibodies was adequate in all 12 foals evaluated. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that a large percentage of C difficile isolates from foals with diarrhea will have the toxin A and B gene sequences. Because of the possibility that isolates will be resistant to metronidazole, susceptibility testing is warranted. Clostridium difficile isolates from foals may have a substantial amount of molecular heterogeneity. Clinical and hematologic findings in affected foals are similar to those for foals with diarrhea caused by other pathogens.     

Effects of gossypol from cottonseed meal and dietary vitamin E on the reproductive characteristics of superovulated beef heifers

Superovulated Hereford-Angus crossbred heifers (average 397 kg BW) were used to test the effect of feeding cottonseed meal (gossypol) and vitamin E on embryo quality and ovarian characteristics. Twenty-four heifers were assigned randomly to four treatments with six heifers per treatment. Treatments were the following dietary supplements: 1) SBM (soybean meal + 30 IU vitamin E/kg of diet DM); 2) SBM+E (soybean meal + 4,000 IU vitamin E x animal(-1) x d(-1)); 3) CSM (cottonseed meal + 30 IU vitamin E/kg of diet DM); and 4) CSM+E (cottonseed meal + 4,000 IU vitamin E x animal(-1) x d(-1)). Supplements based on cottonseed meal provided 43.5 g of total gossypol/d (37% negative isomer (-) and 63% positive isomer (+)). Blood samples were collected at the start of the experiment and every 3 wk thereafter up to 12 wk. Plasma a-tocopherol (alpha-T) concentration was affected by treatments (P < 0.05). Heifers supplemented with cottonseed meal had greater (P < 0.05) alpha-T concentration in plasma than heifers supplemented with soybean meal at each concentration of vitamin E. Supplementation at 4,000 IU vitamin E x animal(-1) d(-1) increased (P < 0.05) the concentration of a-T in plasma. Weight gain, hemoglobin and hematocrit were not affected by treatment. Erythrocyte osmotic fragility (EOF) increased (P < 0.05) in cottonseed meal-fed animals; however, EOF was lowered (P < 0.05) with vitamin E supplementation. Heifers fed CSM and CSM+E supplements had greater (P < 0.01) concentrations of (-)-, (+)-, and total-gossypol in plasma, corpora lutea (CL), liver, and endometrium than heifers fed SBM and SBM+E supplements. Tissue alpha-T concentration increased with increased dietary supplemental vitamin E, particularly in great amounts in the CL. Because there was no adverse effect of gossypol on superovulation response or embryo development despite concentrations of gossypol in endometrium that are toxic to embryos, it is likely that systems exist in the reproductive tract to limit gossypol toxicity.     

Pyogranulomatous myocarditis due to Staphylococcus aureus septicaemia in two harbour porpoises (Phocoena phocoena)

Staphylococcus aureus septicaemia was diagnosed in a dead, stranded harbour porpoise from the German Baltic Sea and in a live harbour porpoise by-caught in inner Danish waters and taken into captivity. Lesions included pyogranulomatous myocarditis, necrotising suppurative bronchopneumonia, pyelonephritis, osteomyelitis and leptomeningitis, and abscesses in lymph nodes and skeletal muscles. The captive animal had fibrinous suppurative epicarditis and pyogranulomatous myocarditis with abscesses. In both animals the organism was suspected to have entered through skin lesions or via the respiratory tract.     

Quantification of bioavailable chlortetracycline in pig feces using a bacterial whole-cell biosensor

Bacterial whole-cell biosensors were used to measure the concentration of chlortetracycline (CTC) in the feces of pigs. In this study, the Escherichia coli biosensor used has a detection limit of 0.03 mg/kg CTC in pig feces. The tetracycline concentration was correlated with the appearance and maintenance of fecal coliform bacteria resistant to tetracycline. Initially, large quantities of water-extractable CTC were excreted from the pigs and measurable amounts were detected even at 30 days after treatment cessation. This led to a sharp rise in the number of tetracycline resistant coliform bacteria in the feces, to within the same order of magnitude as the total coliform count. The high level of tetracycline resistance was maintained in spite of the declining concentration of tetracycline.     

Evaluation of L-selectin expression and assessment of protein tyrosine phosphorylation in bovine polymorphonuclear neutrophil leukocytes around parturition

Impaired polymorphonuclear neutrophil leukocyte (PMN) function around parturition has been associated with increased clinical mastitis in dairy cows. Rolling and attachment of PMN to the endothelium is the first step in the recruitment process and is accomplished by interaction between L-selectin on PMN and its ligand on endothelial cells. Furthermore, tyrosine phosphorylation is involved in the initiation of many PMN functions. The objective of this work was to determine changes in expression of L-selectin and tyrosine phosphorylation in the perinatal period. Eight clinically healthy Holstein cows were used as PMN donors at d-21, -14, -7,0 (calving), +1, +2, +7, +14, +28. Evaluation of L-selectin expression was carried out on activated and resting PMN. Anti-bovine L-selectin monoclonal antibody (MAB) and flow cytometric analysis were used to measure the percentage of PMN fluorescing and receptor expression (log mean fluorescent channel, LMFC). Activated and resting PMN showed similar trends in % PMN fluorescence and LM FC. The percentage of PMN fluorescing tended to decrease at parturition, followed by a significant increase at d +14 and +28 (P < 0.02). For LMFC a decrease was observed on d +1 followed by an increase through d +28 (P < 0.01). Protein tyrosine phosphorylation of lysates prepared from PMN isolated throughout the study was detected by electrophoresis and western blotting using anti-phosphotyrosine MAB. Several protein bands were tyrosine phosphorylated. Two of these bands (42-44 kDa and 90 kDa) varied in intensity over time. The intensity of the 42-44 kDa band gradually increased from d -7, peaked at d +7 (P < 0.03), and steadily decreased to d +28 (P < 0.02). Antibody to activated mitogen protein kinase reacted with the 42-44 kDa band. Reduced PMN function during the periparturient period could be related to reduced L-selectin adhesion molecules on the cell surface, and to modulation in the phosphorylation of functionally important molecules.     

Antibody-induced internalization of viral glycoproteins in pseudorabies virus-infected monocytes and role of the cytoskeleton: a confocal study

Addition of pseudorabies virus (PrV)-specific polyclonal immunoglobulins to PrV-infected monocytes induces internalization of plasma membrane anchored viral glycoproteins. This process may interfere with antibody-dependent cell lysis and resembles the well-studied physiological endocytosis process. A confocal study was designed to investigate whether the major cellular components, involved in physiological endocytosis (clathrin, actin, dynein and microtubules), play a role in this virological internalization process. In order to visualize the interaction of endosomes, which contain the internalized viral glycoproteins, with clathrin, actin, dynein and microtubules, a double labeling of viral glycoproteins and different cellular proteins was performed. Porcine monocytes were inoculated with the PrV-strain 89V87 at a multiplicity of infection of 50 for 13h. After the addition of FITC-labeled porcine polyclonal PrV-specific antibodies, cells were fixed with para-formaldehyde at different time points and afterwards permeabilized. The different cellular components were visualized with monoclonal antibodies and a Texas Red-conjugate, with the exception of actin, which was stained with phalloidin-Texas Red. The cells were analyzed by confocal microscopy. A clear co-localization was observed between the viral glycoproteins and clathrin and dynein during the internalization process. The microtubules were in close contact with the internalized vesicles. For actin no co-localization could be observed. It can be stated that clathrin, dynein and microtubules, important components during physiological endocytosis, are also of importance during the antibody-induced internalization of viral glycoproteins.