Major capsid protein gene sequence analysis of the Santee-Cooper ranaviruses DFV, GV6, and LMBV.

The Santee-Cooper ranaviruses doctor fish virus (DFV), guppy virus 6 (GV6), and largemouth bass virus (LMBV) are members of the genus Ranavirus within the family Iridoviridae. The major capsid protein (MCP) is a main structural protein of iridoviruses and supports the differentiation and classification of ranaviruses. Presently the complete sequence of the MCP gene is known for most ranaviruses with the exception of the Santee-Cooper ranaviruses. In the present study, the complete nucleotide sequence of the MCP gene of DFV, GV6, and LMBV was determined. DFV and GV6 are identical within the MCP gene sequence. The identity compared to the corresponding sequence in LMBV amounts to 99.21%. The MCP gene of DFV, GV6, and LMBV exhibits only approximately 78% identity compared to the respective gene of other ranaviruses. Based on the sequence data obtained in the present study, a Rana MCP polymerase chain reaction (PCR) and subsequent restriction fragment length polymorphism (RFLP) analysis were developed to identify and differentiate ranaviruses, including DFV, GV6, and LMBV.

Ranavirus phylogeny and differentiation based on major capsid protein, DNA polymerase and neurofilament triplet H1-like protein genes.

In this study, we developed new methods for differentiation of ranaviruses based on polymerase chain reaction and restriction enzyme analysis of DNA polymerase and neurofilament triplet H1-like (NF-H1) protein gene. Using these methods, we were able to differentiate the 6 known ranaviruses--Bohle iridovirus (BIV), European catfish virus (ECV), epizootic haematopoietic necrosis virus (EHNV), European sheatfish virus (ESV), frog virus 3 (FV3) and Singapore grouper iridovirus (SGIV)--with 3 less characterised virus isolates: short-finned eel ranavirus (SERV), Rana esculenta virus Italy 282/I02 (REV 282/I02) and pike-perch iridovirus (PPIV). Doctor fish virus (DFV) and guppy virus 6 (GV6) were distinguished as a group from the other viruses. In addition, all 11 isolates were analysed and compared based on nucleotide sequences from 3 different genomic regions: major capsid protein (MCP), DNA polymerase and NF-H1. The partial DNA polymerase gene was sequenced from all analysed viruses. The complete sequence of the MCP and a fragment of the NF-H1 gene were obtained from BIV, ECV, EHNV, ESV, FV3, PPIV, REV 282/I02 and SERV. With the exception of GV6, DFV and SGIV, the sequence analyses showed only a few variations within the analysed viruses. The sequence data suggest that PPIV, REV 282/I02 and SERV are new members of the genus Ranavirus. The methods developed in this study provide tools to differentiate between closely related ranaviruses of different host and geographical origin.

Etiology of respiratory disease in non-vaccinated, non-medicated calves in rearing herds.

The aim of this study was to examine the occurrence of bacterial, mycoplasmal and viral pathogens in the lower respiratory tract of calves in all-in all-out calf-rearing units. According to clinical status, non-medicated calves with and without respiratory disease signs were selected of the 40 herds investigated to analyse the micro-organisms present in healthy and diseased calves. Tracheobronchial lavage (TBL) and paired serum samples were analysed for bacteria, mycoplasmas, respiratory syncytial virus (RSV), parainfluenza virus 3 (PIV3), bovine corona virus (BCV) and bovine adenovirus (BAV). Pasteurella multocida was the most common bacterial pathogen. It was isolated from 34% of the TBL samples in 28 herds and was associated with clinical respiratory disease (p < 0.05) when other pathogenic bacteria or mycoplasma were present in the sample. Mannheimia spp. and Histophilus somni were rarely found. Mycoplasma bovis was not detected at all. Ureaplasma diversum was associated with clinical respiratory disease (p < 0.05). TBL samples from healthy or suspect calves were more often negative in bacterial culture than samples from diseased calves (p < 0.05). No viral infections were detected in six herds, while 16-21 herds had RSV, BCV, BAV or PIV3. In the herds that had calves seroconverted to BCV, respiratory shedding of BCV was more frequently observed than faecal shedding. This study showed that the microbial combinations behind BRD were diverse between herds. M. bovis, an emerging pathogen in many countries, was not detected.

Intravenous immunoglobulin g attenuates pulmonary hypertension but induces local neutrophil influx in meconium aspiration in piglets.

BACKGROUND: Pulmonary hypertension and inflammation are well-identified pathogenetic features in meconium aspiration syndrome of newborns, but current approaches to their treatment or prevention are still often unsatisfactory. OBJECTIVES: To investigate the possible protective effects of human intravenous immunoglobulin G (IVIG) on the hypertensive and inflammatory lung injury in severe neonatal meconium aspiration. METHODS: Eleven newborn (10-12 days old) ventilated and catheterized piglets that received an intratracheal bolus (3 ml/kg) of a 65-mg/ml mixture of human meconium were studied for 6 h. IVIG was infused in 5 piglets 30 min before meconium administration, and 6 piglets served as controls and received the vehicle only. RESULTS: Meconium instillation induced a biphasic pulmonary hypertensive response, which was significantly diminished by IVIG pretreatment. Similarly, IVIG improved the oxygenation of the piglets, but the intrapulmonary shunt fraction or systemic hemodynamic parameters did not differ between the study groups, except of a minor decrease in the mean arterial blood pressure caused by IVIG. The blood leukocyte count was comparable in the 2 groups. The lung tissue ultrastructural and histological changes, number of apoptotic cells and phospholipase A2 activity were similar in the 2 groups. The amount of neutrophil accumulation, assessed by myeloperoxidase activity, was however significantly increased in macroscopically damaged lung tissue after IVIG administration. CONCLUSIONS: Our results thus indicate that IVIG treatment of newborns with severe meconium aspiration significantly diminishes the pulmonary hypertensive response and improves oxygenation, but the effects do not extend to protection of lung cellular injury.

Effectiveness of duct cleaning methods on newly installed duct surfaces.

Two kinds of air duct cleaning methods, mechanical brushing with different brushes and compressed air cleaning, were compared in the laboratory and in newly built buildings. The ducts were contaminated either with test dust or with dust originated from a construction site. The amount of dust on the duct surface was measured with the vacuum test method and estimated visually before and after the cleaning. In addition, the cleaning times of the different techniques were compared and the amount of residual oil in the ducts was measured in the laboratory test. The brushing methods were more efficient in metal ducts, and compressed air cleaning was more efficient in plastic ducts. After the duct cleaning the mean amount of residual dust on the surface of the ducts was

The effect of cleanliness control during installation work on the amount of accumulated dust in ducts of new HVAC installations.

The aim of this study was to evaluate the amount of dust in supply air ducts in recently installed ventilation systems. The samples for the determination of dust accumulation were collected from supply air ducts in 18 new buildings that have been constructed according to two different cleanliness control levels classified as category P1 (low oil residues and protected against contaminations) and category P2, as defined in the Classification of Indoor Climate, Construction and Building Materials. In the ducts installed according to the requirements of cleanliness category P1 the mean amount of accumulated dust was 0.9 g/m2 (0.4-2.9 g/m2), and in the ducts installed according to the cleanliness category P2 it was 2.3 g/m2 (1.2-4.9 g/m2). A significant difference was found in the mean amounts of dust between ducts of categories P1 and P2 (P < 0.008). The cleanliness control procedure in category P1 proved to be a useful and effective tool for preventing dust accumulation in new air ducts during the construction process. Additionally, the ducts without residual oil had lower amounts of accumulated dust indicating that the demand for oil free components in the cleanliness classification is reasonable.

Dexamethasone treatment attenuates pulmonary injury in piglet meconium aspiration.

To investigate the pulmonary effects of steroid treatment in neonates with meconium aspiration, 25 10- to 12-d-old piglets were studied for 6 h after an intratracheal bolus of human meconium. Dexamethasone (0.5 mg/kg) was given in two treatment schedules, either 1 h before (n = 6) or 1 h after meconium instillation (n = 8). Eight piglets served as controls. Three additional piglets were given dexamethasone without meconium instillation. Pulmonary hemodynamics and oxygenation were followed, and lung tissue samples investigated for signs of inflammation and ultrastructural injury, including apoptosis. Pulmonary artery pressure and vascular resistance increased after meconium instillation, but this rise was significantly prevented after prophylactic dexamethasone. This treatment also improved the acutely deteriorated oxygenation of the piglets after meconium insufflation. Prophylactic, but not early, dexamethasone treatment further protected the lungs from the ultrastructural changes caused by meconium instillation. Additionally, the increase of apoptotic epithelial cell deaths was significantly prevented by both dexamethasone treatments. These results show that prophylactic dexamethasone treatment significantly attenuates the early pulmonary hemodynamic deterioration and structural lung damage caused by meconium aspiration. Further studies on the apoptosis-inhibiting effect of dexamethasone administration in neonatal lungs exposed to heavy meconium are warranted.

In vivo detection of vascular adhesion protein-1 in experimental inflammation.

Vascular adhesion protein-1 (VAP-1) is an inflammation-inducible endothelial glycoprotein which mediates leukocyte-endothelial cell interactions. To study the pathogenetic significance of VAP-1 in inflammatory disorders, an in vivo immunodetection method was used to detect the regulation of luminally expressed VAP-1 in experimental skin and joint inflammation in the pig and dog. Moreover, VAP-1 was studied as a potential target to localize inflammation by radioimmunoscintigraphy. Up-regulation of VAP-1 in experimental dermatitis and arthritis could be visualized by specifically targeted immunoscintigraphy. Moreover, the translocation of VAP-1 to the functional position on the endothelial surface was only seen in inflamed tissues. These results suggest that VAP-1 is both an optimal candidate for anti-adhesive therapy and a potential target molecule for imaging inflammation.

Human meconium has high phospholipase A2 activity and induces cellular injury and apoptosis in piglet lungs.

Aspiration of meconium produces an inflammatory reaction resulting in necrotic changes in lung tissue. To further investigate the mechanisms of the meconium-induced early pulmonary injury, twenty 10-12-d-old piglets were studied for lung tissue ultrastructural and apoptotic changes and phospholipase A2 activity. Twelve piglets received an intratracheal bolus (3 mL/kg) of a 20-mg/mL (thin, n = 6) or 65-mg/mL (thick, n = 6) mixture of human meconium, and control piglets (n = 5) received the same amount of intratracheal saline. Three ventilated piglets with no aspiration were also studied. Pulmonary hemodynamics and systemic oxygenation were followed for 6 h after meconium or saline insufflation. In the control groups, the pulmonary tissue showed open alveolar spaces and intact vascular walls, whereas meconium administration resulted in severe pneumonitis, with alveolar spaces filled with inflammatory exudate. Meconium instillation additionally resulted in edematous changes in the vascular walls and alveolar epithelium, whereas type II pneumocytes were intact. The amount of apoptotic cells was increased, especially in the respiratory epithelium, and the catalytic activity of phospholipase A2 in lung tissue samples was significantly elevated after thick meconium instillation. This activity rise proved to be mainly because of human group I phospholipase A2, introduced by meconium. Our data thus show that aspiration of meconium leads to severe lung tissue inflammation with early ultrastructural changes in the pulmonary alveolar walls and is associated with apoptotic cell death in the epithelium, already during the first hours after the insult. These results further suggest that high phospholipase A2 activity, mainly introduced into the lungs within the meconium, may have an important role in the initiation of these alterations in neonatal lungs.

Nitric oxide inhalation inhibits pulmonary apoptosis but not inflammatory injury in porcine meconium aspiration.

To investigate the possible protective effects of nitric oxide (NO) inhalation in newborns with meconium aspiration, 18 10-12-d-old piglets were studied for 6h after an intratracheal bolus (3 ml/kg) of a 65-mg/ml mixture of human meconium. Twelve of the piglets were treated with continuous NO inhalation at a dose of 1 ppm (n = 6) or 10 ppm (n = 6), started 30 min before the insult. Pulmonary haemodynamics and systemic oxygenation were followed, and lung tissue samples were studied for signs of inflammation, evidence of ultrastructural injury and apoptotic cell changes. Inhalation of 10 ppm NO, in contrast to 1 ppm NO, significantly delayed the meconium-induced pulmonary pressure rise and the increase in intrapulmonary shunt fraction, and maintained better oxygenation in the piglets. Histologically and biochemically, treatment with 1 or 10 ppm NO inhalation did not protect the lungs against meconium-induced inflammatory injury. Further, ultrastructural lung tissue analysis revealed a significant amount of alveolar exudate and oedematous alveolar epithelium and endothelium after meconium instillation, also in the lungs treated with NO inhalation. However, the increase in apoptotic epithelial cell deaths, previously shown to be stimulated by intratracheal meconium, was significantly impeded after inhalation of 10 ppm. These results thus show that early continuous NO inhalation controls the rise in pulmonary artery pressure and improves the efficiency of arterial oxygenation, and further prevents the increase in epithelial apoptosis, but does not protect against early inflammatory damage caused by meconium aspiration.

Meconium aspiration induces a concentration-dependent pulmonary hypertensive response in newborn piglets.

To investigate the effects of aspirating different meconium concentrations on the pulmonary circulation in 10- to 12-day-old piglets, 30 catheterized animals were studied. The piglets received an intratracheal bolus of 3 ml/kg of a mixture of human meconium in saline with concentrations of 20 mg/ml (light, n = 7), 40 mg/ml (moderate, n = 6), or 65 mg/ml (thick, n = 10) meconium in saline. Control piglets (n = 7) received 3 ml/kg of intratracheal saline. Pulmonary and systemic pressures were measured and vascular resistances calculated at baseline and serially for 4 hours after instillation. Four of the piglets died early and were excluded from the study. In addition, 23 samples of human meconium-stained amniotic fluid were collected at delivery for determination of their meconium concentration. After an initial rise in pulmonary artery pressure and vascular resistance after meconium and saline instillation, pulmonary artery pressure and resistance increased progressively and concentration-dependently in the meconium groups, but returned to baseline in the control group. The saline and meconium-induced initial increases, and the subsequent meconium-stimulated progressive rise in vascular resistance occurred mainly in the postarterial segment. There were no significant changes in systemic hemodynamics. Mean airway pressure increased and oxygenation deteriorated after meconium instillation. The impairment of oxygenation depended on the meconium concentration in the instilled bolus and persisted throughout the study after moderate and thick meconium instillation. Similarly, the intrapulmonary shunt fraction increased initially and remained elevated in the moderate and thick meconium groups. Meconium concentrations in the human amniotic fluid samples were in the same range as concentrations used in the present experimental study. These results indicate that aspirated meconium at concentrations found in light to moderate meconium-stained human amniotic fluid has significant effects on pulmonary hemodynamic and oxygenation in newborn piglets.

Treatment of post-traumatic oedema in lower legs using intermittent pneumatic compression.

Post-traumatic oedema after 6-12 weeks immobilization in a cast was treated using intermittent pneumatic compression. The study group comprised 18 patients with 19 distal fractures of the lower leg. They were given compression treatment on five consecutive days for about 1 h daily. Reduction in oedema was assessed by measuring leg circumferences at three levels: the malleolus; 20 cm proximally from the malleolus; and the Chopart joint. The untreated control group consisted of five patients. Changes in oedema were followed daily for one week. A significantly greater reduction in oedema occurred in the study group at all three measurement levels (p less than 0.001) as compared with the control group.