Nonredundant Dimethyl Sulfoxide Reductases Influence Salmonella enterica Serotype Typhimurium Anaerobic Growth and Virulence.

Facultative anaerobic enteric pathogens can utilize a diverse array of alternate electron acceptors to support anaerobic metabolism and thrive in the hypoxic conditions within the mammalian gut. Dimethyl sulfoxide (DMSO) is produced by methionine catabolism and can act as an alternate electron acceptor to support anaerobic respiration. The DMSO reductase complex consists of three subunits, DmsA, DmsB, and DmsC, and allows bacteria to grow anaerobically with DMSO as an electron acceptor. The genomes of nontyphoidal Salmonella enterica encode three putative dmsABC operons, but the impact of the apparent genetic redundancy in DMSO reduction on the fitness of nontyphoidal S. enterica during infection remains unknown. We hypothesized that DMSO reduction would be needed for S. enterica serotype Typhimurium to colonize the mammalian gut. We demonstrate that an S. Typhimurium mutant with loss of function in all three putative DMSO reductases (ΔdmsA3) poorly colonizes the mammalian intestine when the microbiota is intact and when inflammation is absent. DMSO reduction enhances anaerobic growth through nonredundant contributions of two of the DMSO reductases. Furthermore, DMSO reduction influences virulence by increasing expression of the type 3 secretion system 2 and reducing expression of the type 3 secretion system 1. Collectively, our data demonstrate that the DMSO reductases of S. Typhimurium are functionally nonredundant and suggest DMSO is a physiologically relevant electron acceptor that supports S. enterica fitness in the gut.

Research Note: Repetitive element-based polymerase chain reaction genotyping improves efficiency of Salmonella surveillance in a model broiler production system.

The genetic relatedness and antimicrobial susceptibility profiles of Salmonella isolated from poultry and their environment were determined. One broiler breeder flock (BBF1) and 2 broiler flocks (BF1 and BF2) were reared over a 1.75-year period on the same poultry research farm. Hatching eggs were obtained from BBF1 to produce BF1 chicks, while BF2 chicks were progeny of a separate, unsampled broiler breeder flock. BF1 and BF2 were reared in the same housing facilities but 6 mo apart. Salmonella isolates were collected via litter sock sampling (BF1), cecal excision (BF1 and BF2), or cloacal swabs (BBF1). Serotyping identified Salmonella enterica subsp. enterica serovar Altona (SA) in BBF1 and S. enterica subsp. enterica serovar Senftenberg (SS) in BF1 and BF2. Genotypic fingerprinting was achieved with Rep-PCR using the (GTG)5 primer and revealed sequence homology among Senftenberg isolates from BF1 and BF2. For each isolate, the minimum inhibitory concentration was determined for 27 antimicrobial agents using Sensititre plates with formularies specific to antimicrobials used in poultry production or those used to control gram negative pathogens. Isolates from the 3 flocks were resistant to clindamycin, erythromycin, novobiocin, penicillin, and tylosin tartrate and demonstrated intermediate resistance to azithromycin, florfenicol, and spectinomycin. These data demonstrated that serovar Altona and Senftenberg were harbored by poultry, the latter appeared to persist in broiler flocks, and both serotypes shared similar patterns of antimicrobial susceptibility in an integrated research operation. In the case of multiple Salmonella isolates, combining genotypic fingerprinting methods with serotyping of representative isolates would reduce the number of samples required for serotyping and more clearly identify relatedness of isolates. These methods facilitate effective surveillance in poultry production systems, thus allowing for implementation of precise Salmonella control measures.

Pathogenesis of Enterococcal Spondylitis Caused by Enterococcus cecorum in Broiler Chickens.

Enterococcal spondylitis (ES) is a disease of commercial broiler chickens, with a worldwide distribution. Symmetrical hind limb paralysis typical of ES results from infection of the free thoracic vertebra (FTV) by pathogenic strains of Enterococcus cecorum . To determine the pathogenesis of ES, birds with natural and experimental ES were studied over time. In natural disease, case birds (n = 150) from an affected farm and control birds (n = 100) from an unaffected farm were evaluated at weeks 1-6. In control birds, intestinal colonization by E. cecorum began at week 3. In case birds, E. cecorum was detected in intestine and spleen at week 1, followed by infection of the FTV beginning at week 3. E. cecorum isolates recovered from intestine, spleen, and FTV of case birds had matching genotypes, confirming that intestinal colonization with pathogenic strains precedes bacteremia and infection of the FTV. Clinical intestinal disease was not required for E. cecorum bacteremia. In 1- to 3-week-old case birds, pathogenic E. cecorum was observed within osteochondrosis dissecans (OCD) lesions in the FTV. To determine whether OCD of the FTV was a risk factor for ES, 214 birds were orally infected with E. cecorum, and the FTV was evaluated histologically at weeks 1-7. Birds without cartilage clefts of OCD in the FTV did not develop ES; while birds with OCD scores ≥3 were susceptible to lesion development. These findings suggest that intestinal colonization, bacteremia, and OCD of the FTV in early life are crucial to the pathogenesis of ES.

Culture methods impact recovery of antibiotic-resistant Enterococci including Enterococcus cecorum from pre- and postharvest chicken.

Pathogenic strains of Enterococcus cecorum (EC) expressing multidrug resistance have emerged. In National Antimicrobial Resistance Monitoring System (NARMS) data, EC is rarely recovered from chickens. Two NARMS methodologies (FDA and USDA) were compared with standard culture (SC) techniques for recovery of EC. NARMS methods failed to detect EC in 58 caecal samples, 20 chicken breast or six whole broiler samples. EC was recovered from 1 of 38 (2·6%) and 2 of 38 (5·2%) preharvest spinal lesions (USDA and FDA method, respectively). In contrast, using the SC method, EC was recovered from 44 of 53 (83%) caecal samples, all 38 (100%) spinal lesions, 14 of 20 (70%) chicken breast samples, and all three spinal lesions identified in whole carcasses. Compared with other Enterococcus spp., EC isolates had a higher prevalence of resistance to macrolides. The NARMS methods significantly affected recovery of enterococcal species other than EC. When the postharvest FDA method was applied to preharvest caecal samples, isolates of Enterococcus faecium were preferentially recovered. All 11 E. faecium isolates were multidrug resistant, including resistance to penicillin, daptomycin and linezolid. These findings confirm that current methodologies may not accurately identify the amount and range of antimicrobial resistance of enterococci from chicken sources. SIGNIFICANCE AND IMPACT OF THE STUDY: Enterococci are an important reservoir for antimicrobial resistance. This study demonstrates how current culture methods underreport resistance to macrolides in enterococci by selecting against strains of Enterococcus cecorum in pre- and postharvest chicken. Further, the application of postharvest surveillance methods to preharvest samples resulted in selective recovery of Enterococcus faecium over Enterococcus faecalis. Isolates of E. faecium recovered exhibited multidrug resistance including penicillin, daptomycin and linezolid resistance. These findings suggest that culture methodology significantly impacts the range and amount of antimicrobial resistance detected in enterococci isolated from chicken.

Zebrafish (Danio rerio) as a screen for attenuation of Lancefield group C streptococci and a model for streptococcal pathogenesis.

Group C streptococci are highly contagious pyogenic bacteria responsible for respiratory tract, lymph node, urogenital tract, and wound infections. Wild-type strains of Streptococcus equi ssp equi (S. equi) and Streptococcus equi ssp zooepidemicus (S. zoo) as well as a commercially available modified live vaccine strain of S. equi were evaluated for virulence in zebrafish. Survival times, histologic lesions, and relative gene expression were compared among groups. Based on the intramuscular route of infection, significantly shorter survival times were observed in fish infected with wild-type strain when compared to modified live vaccine and S. zoo strains. Histologically, S. zoo-infected fish demonstrated a marked increase in inflammatory infiltrates (predominantly macrophages) at the site of infection, as well as increased cellularity in the spleen and renal interstitium. In contrast, minimal cellular immune response was observed in S. equi-injected fish with local tissue necrosis and edema predominating. Based on whole comparative genomic hybridization, increased transcription of positive acute-phase proteins, coagulation factors, and antimicrobial peptides were observed in S. equi-injected fish relative to S. zoo-injected fish, while mediators of cellular inflammation, including CXC chemokines and granulin, were upregulated in S. zoo-injected fish relative to S. equi-injected fish. In a screen of 11 clinical isolates, S. equi strains with a single nucleotide deletion in the upstream region of szp, a known virulence factor of streptococci, were found to be significantly attenuated in zebrafish. These collective findings underscore the value of the zebrafish as a model of streptococcal pathogenesis.

The effect of bursectomy on natural xenoreactive antibodies and vascularized rat cardiac xenograft rejection in the chicken.

The clinical application of xenotransplantation between distantly related species is currently prevented by the occurrence of hyperacute rejection (HAR). Controversy exists over the importance of natural xenoreactive antibody (NAb)-mediated activation of the classical complement pathway vs. direct activation of the alternative C pathway in this process. In order to evaluate HAR of xenografts (Xgs) in the absence of NAb, this study utilized K strain leghorn chickens that were bursectomized (Bx) on day 17 in ovo (n = 18) to prevent B cell development and production of NAb. Aged-matched untreated siblings served as controls (n = 13). Based on pretransplant antibody levels, the Bx chickens were divided into two groups: totally Bx (Total Bx, n = 9) and partially Bx (Part Bx, n = 9). Chickens then underwent heterotopic cardiac xenotransplantation using PVG rats as donors, where the Xg was connected to the circulation of the chicken recipient utilizing cannulae. For the control group, Xg survival was 28 +/- 3 min (mean +/- SEM), while Part Bx prolonged survival to 80 +/- 15 min. Total Bx extended rat Xg survival to 102 +/- 11 min, with 5 of 9 Xgs functioning well at the time of termination of the study (90-120 min). Three chickens in the Total Bx group with rat cardiac Xgs that were functioning at 120 min were given a 1 ml i.v. injection of heat inactivated control chicken serum. This led to loss of Xg function within 10 min, confirming the important role for NAb in HAR in this species combination. Histologic examination of the Xgs following perfusion revealed significant arterial endothelial injury in the control and Part Bx groups but not in the Total Bx group. Conversely, Xgs from the Total Bx group showed marked venous congestion, which was not seen in the other two groups. This study demonstrates that: (1) Bx effectively eliminates NAb; (2) Xg survival is significantly prolonged in the absence of NAb in this rat-to-chicken xenogeneic combination; (3) the presence of NAb is associated with arterial endothelial injury; and (4) in the absence of NAb, marked venous congestion and injury occurs, which is possibly mediated by alternative C pathway activation or other humoral mechanisms.

Expression and kinetics of induced procoagulant activity in bovine pulmonary alveolar macrophages.

Leukocytes, especially macrophages, are important cellular mediators of fibrin deposition and removal at tissue sites of inflammation. Pulmonary fibrin deposition is a prominent feature of bovine acute lung injury; therefore, we studied the resting and stimulated procoagulant responses of bovine pulmonary alveolar macrophages (PAM) and peripheral blood neutrophils (PMN). Freshly isolated normal PAM and PMN expressed negligible procoagulant activity. PAM stimulated with endotoxin lipopolysaccharide (LPS), 4 beta-phorbol 12-myristate 13-acetate (PMA) and bovine recombinant interleukin-1 beta (rBIL-1 beta) exhibited protein synthesis- and dose-dependent enhancement of procoagulant activity in 8-h cultures. Bovine recombinant granulocyte macrophage-colony stimulating factor (rBGM-CSF) and recombinant human gamma-interferon (rHIFN-gamma) did not induce procoagulant activity. The kinetics of LPS- and PMA-enhanced PAM procoagulant activity differed: LPS-induced enhancement developed earlier and more rapidly than PMA-induced enhancement. Pasteurella haemolytica LPS was more potent than Escherichia coli LPS in enhancing PAM procoagulant activity, while dexamethasone decreased both baseline and LPS- or PMA-stimulated activity by approximately 50%. PAM procoagulant activity resulted from tissue factor expression. Bovine PMN produced negligible procoagulant activity when stimulated, and are thus unlikely to be major contributors to procoagulant activity in bovine lung. Activity inhibitory to bovine tissue factor was present in both calf and adult sera, and was partly dependent on the presence of factor X for activity. Rapid induction of bovine PAM procoagulant activity by inflammatory mediators, and subsequent resistance to degradation, may thus combine to promote an alveolar microenvironment permissive to fibrin deposition in bovine acute lung injury.

The role of leukocytes in the pathogenesis of fibrin deposition in bovine acute lung injury.

The peculiarly fibrinous nature of bovine acute lung injury due to infection with Pasteurella haemolytica A1 suggests an imbalance between leukocyte-directed procoagulant and profibrinolytic influences in the inflamed bovine lung. Calves with experimental pneumonia produced by intratracheal inoculation with P. haemolytica A1 developed acute locally extensive cranioventral fibrinopurulent bronchopneumonia. Pulmonary alveolar macrophages (PAM) recovered by segmental lavage from affected lung lobes were 30 times more procoagulant than PAM obtained from unaffected lung lobes and 37-fold more procoagulant than PAM from control calf lungs. Unlike the enhancement of procoagulant activity, profibrinolytic activity (plasminogen activator amidolysis) of total lung leukocytes (PAM and plasminogen activator neutrophils [PMN]) was decreased 23 times in cells obtained from affected lung lobes and also was decreased four times in cells obtained from unaffected lobes of infected animals. This marked imbalance in cellular procoagulant and fibrinolytic activity probably contributes significantly to enhanced fibrin deposition and retarded fibrin removal. In addition, PAM from inflamed lungs were strongly positive for bovine tissue factor antigen as demonstrated by immunocytochemistry. Intensely tissue factor-positive PAM enmeshed in fibrinocellular exudates and positive alveolar walls were situated such that they were likely to have, in concert, initiated extrinsic activation of coagulation in the acutely inflamed lung. These data collectively suggest that enhanced PAM-directed procoagulant activity and diminished PAM- and PMN-directed profibrinolytic activity represent important modifications of local leukocyte function in bovine acute lung injury that are central to the pathogenesis of lesion development with extensive fibrin deposition and retarded fibrin removal.

Endotoxin-mediated bovine alveolar macrophage procoagulant induction is dependent on protein kinase C activation.

The induction of pulmonary alveolar macrophage (PAM) tissue factor-dependent procoagulant activity is central to the deposition of inflammatory fibrin in the pulmonary alveolus. The presence of enhanced tissue factor activity is often associated with pulmonary fibrin deposition, an important pathogenetic event that can delay resolution of pulmonary inflammation and promote the induction of pulmonary fibrosis. Since tissue factor synthesis induction and activation pathways are potential therapeutic targets for modulation of alveolar macrophage tissue factor (procoagulant) activity, we examined the pathways through which endotoxin lipopolysaccharide (LPS) induces bovine PAM tissue factor-dependent procoagulant activity. PAM procoagulant activity was markedly enhanced to 10 times the levels of freshly isolated PAM after 8 h of culture in the presence of either the protein kinase C (PKC) agonist phorbol 12-myristate 13-acetate (PMA) or LPS. Both LPS-(P less than 0.002) and PMA-induced activity (P less than 0.007) was completely ablated by the PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H 7,100 microM) but was unaffected by the cyclic nucleotide-dependent protein kinase inhibitor N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004, 100 microM). The arachidonate cyclooxygenase pathway inhibitor phenylbutazone (10(-4) M) had modest effects that were not statistically significant. The unstimulated increase of procoagulant activity in 8-h cultures was unaffected by the same inhibitory modulations.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium mobilization in C5a-stimulated adult and newborn bovine neutrophils.

Calcium (Ca2+) is an important second messenger central to many neutrophil (PMN) functional activities. Impaired Ca2+ mobilization in newborn PMNs following membrane perturbation could be one of the mechanisms underlying observed abnormalities in neonatal PMN function. To compare Ca2+ mobilization in bovine newborn and adult PMNs, cytosolic Ca2+ responses after stimulation with recombinant human C5a (rHC5a) were measured. PMNs from normal newborn calves (N = 6) and adult cows (N = 5) were loaded with fura-2/AM for 60 min at room temperature and the fluorescence changes monitored following stimulation with 0.1, 1, 10, or 50 nM rHC5a in Ca2(+)-containing buffer. Resting levels of Ca2+ in both newborn (54.6 +/- 1.9 nM) and adult (57.3 +/- 1.8 nM) bovine PMNs were comparable. After stimulation with rHC5a, a rapid rise of cytosolic Ca2+ was observed, which peaked within 20 sec and was followed by a sustained phase of elevated Ca2+ lasting up to 20 min. There were no significant differences (P greater than 0.05) in peak levels of cytosolic Ca2+ obtained by newborn and adult PMNs at 0.1, 10, and 50 nM rHC5a. At 1 nM rHC5a, newborn PMNs reached significantly (P less than 0.05) higher levels of cytosolic Ca2+ (217.9 +/- 21.7 nM) than did adult PMNs (158.7 +/- 7.9 nM). At 1 nM rHC5a, newborn PMNs also sustained higher levels of cytosolic Ca2+ for 3 min following the peak. At all concentrations of rHC5a tested, the time required to reach the peak and the duration of the peak were comparable in both populations. In the absence of extracellular Ca2+ (Ca2(+)-free buffer with 1 mM EGTA), resting levels of cytosolic Ca2+ were lower in both newborn (33.3 +/- 2.9 nM) and adult PMNs (27.9 +/- 2.4 nM) and the magnitude of the peak response to rHC5a was diminished at all concentrations of agonist. Additionally, in the absence of extracellular Ca2+, the return to basal cytosolic Ca2+ levels occurred rapidly and the sustained phase of increased cytosolic Ca2+ seen with rHC5a-stimulated PMNs in Ca2(+)-containing buffer was virtually eliminated. These results indicate that bovine PMNs respond well to rHC5a, that stimulated newborn bovine PMNs can mobilize Ca2+ as efficiently as adult PMNs, and that the sustained cytosolic Ca2+ response to rHC5a in both age groups requires both release of Ca2+ from intracellular stores as well as influx of extracellular Ca2+. Such data suggest that observed functional deficits in newborn bovine PMNs are probably not related to improper mobilization of Ca2+ following stimulation.

Alterations in complement-induced shape change and stimulus-specific superoxide anion generation by neonatal calf neutrophils.

Increased susceptibility of neonates to infection may be related to defects in newborn neutrophil (PMN) functional activities, including altered responses to complement fragments (Cf) and defective microbicidal activity. We therefore compared the kinetics of newborn and adult bovine PMN membrane shape change responses following stimulation with zymosan-activated plasma (ZAP) as a source of Cf. Measurement of PMN membrane shape change was a rapid, sensitive, and reproducible measure of Cf stimulation within a population of PMNs; a maximum of 67-85% of the PMNs exhibited easily detectable membrane ruffling, lamellipodia formation, and polarity within 2 min. Newborn PMNs exhibited significantly increased (P less than 0.01) membrane shape change at 20, 30, 60, 120, and 300 sec after Cf stimulation. A maximum of 85.8 +/- 3.2% of newborn PMNs exhibited such Cf-induced shape changes by 120 sec. which was significantly greater (P less than 0.01) than the maximum stimulation (67.7 +/- 4.3%) attained with adult PMNs. These data indicate enhanced kinetics of induced newborn PMN membrane shape change in response to Cf stimulation. We also compared stimulus-specific superoxide anion (O2-) generation as a measure of respiratory burst activity after incubation of newborn and adult PMNs with soluble (phorbol myristate acetate, PMA) and particulate (opsonized zymosan, OZ) stimuli. When PMA was used as the stimulus, newborn PMNs generated significantly less O2- (9.3 +/- 0.5 nmol O2-/10(6) PMN, P less than 0.05) than did adult PMNs (12.4 +/- 0.3 nmol O2-/10(6) PMN). This finding was reversed when OZ was used as the stimulus; newborn PMNs generated significantly more O2- (7.7 +/- 0.4 nmol O2-/10(6) PMN, P less than 0.05) than did adult PMNs (5.5 +/- 0.5 nmol O2-/10(6) PMN). These findings collectively document biochemical and morphological differences between newborn and adult PMNs as determined by stimulus-specific O2- generation and Cf-induced membrane shape change. Such differences may be important to neonatal disease susceptibility.

Isolation and characterization of equine microvascular endothelial cells in vitro.

The use of cultured tissue has not yet become widespread in research involving equine disease, and this may be attributable in part to the scarcity of published reports concerning tissue culture methods for this species. We report here the isolation of equine microvascular endothelium (EMVE) from fresh omental tissue of horses and ponies. Fresh donor tissue was minced, subjected to collagenase digestion, and filtered. Cells were layered on 5% bovine serum albumin for gravity sedimentation, the bottom layer was collected, and the cells were plated onto fibronectin-coated flasks. Medium consisted of Dulbecco modified Eagle medium with 10% whole fetal bovine serum (wFBS) and 20 micrograms of endothelial cell growth supplement/ml. The EMVE grew readily in culture, had the cobblestone morphologic feature at confluence, stained positively for factor VIII-related antigen, and metabolized acetylated low-density lipoprotein. Fibroblast and smooth muscle cell contamination was minimal in primary cell cultures, which were successfully passed and maintained in culture for 3 to 5 serial passages, using various media and substrates. Preliminary studies were undertaken to determine optimal growth conditions with a range of variables: serum concentration, extracellular matrix components, and growth factors, Optimal conditions were achieved with a minimum of 10% wFBS, and with either fibronectin or laminin as extracellular matrix substrates. The EMVE grew adequately in Dulbecco modified Eagle medium plus 10% wFBS, and the added growth factors or serum supplements did not appear necessary for growth of EMVE.

Ontogeny of inflammatory cell responsiveness: superoxide anion generation by phorbol ester-stimulated fetal, neonatal, and adult bovine neutrophils.

Newborn calves, like human infants, are uniquely susceptible to bacterial infections. Part of this increased susceptibility may be related to defects in newborn polymorphonuclear leukocyte (PMN) defensive functions. It remains unclear whether reported deficits in newborn PMN function represent maturational disorders or are manifestations of some form of perinatal suppression phenomenon. We therefore compared the ability of bovine newborn PMNs (less than 24 h old), newborn PMNs (7-10 days of age), fetal PMNs (210-220 days gestational age), and adult PMNs to generate superoxide anion (O2-) as an indicator of respiratory burst activity. Citrated blood was collected, and PMNs were isolated to greater than 95% purity and 98% viability. O2- generation was measured as the superoxide dismutase-inhibitable (10 micrograms/ml) reduction of ferricytochrome c (2 mg/ml) after activation of PMNs with phorbol myristate acetate (PMA, 2 micrograms/ml) to directly stimulate protein kinase C. The reaction kinetics were measured (37 degrees C, 550 nm) using a spectrophotometer and chart recorder for continuous monitoring. O2- generation was measured for 5 min after the initial lag period and the total nanomoles of O2- generated calculated using the extinction coefficient for ferricytochrome c. Newborn PMNs (N = 10) generated significantly less O2- (5.7 +/- 0.8 nmol O2-/10(6) cells/5 min, P less than 0.01) than did adult PMNs (N = 14) (9.6 +/- 2.1 nmol O2-/10(6) cells/5 min) or fetal PMNs (N = 4) (10.7 +/- 0.7 nmol O2-/10(6) cells/5 min). PMNs from 7- to 10-day-old calves (N = 9) generated almost identical amounts of O2- as newborn PMNs (5.7 +/- 1.6 nmol O2-/10(6) cells/5 min). There was no difference in measured lag time period between newborn and adult PMNs, but fetal PMNs had significantly reduced (P less than 0.01) mean lag time. The data indicated that bovine newborn PMNs have a decreased ability to generate O2- in response to PMA stimulation, which persists for at least 7-10 days, and that this functional decrement may be a manifestation of some form of perinatal PMN suppression phenomenon rather than a developmental abnormality since fetal PMNs produced O2- as well as adult PMNs.

Neonatal viral bronchiolitis and pneumonia induces bronchiolar hypoplasia and alveolar dysplasia in rats.

The objective of this research was to determine the effects of viral bronchiolitis and pneumonia on postnatal bronchiolar and alveolar growth. Neonatal (5-day-old) and weanling (25-day-old) outbred rats were infected with parainfluenza type 1 (Sendai) virus and were studied from 0 to 110 days after inoculation by scanning and transmission electron microscopy, by light microscopic morphometry, and by analysis of airway corrosion casts. Viral infection induced necrotizing bronchiolitis and interstitial pneumonia in both age groups of rats. Viral infection had a much more marked effect on neonatal rats in the proliferative stage of lung growth than on weanlings in the equilibrated stage of lung growth. Viral infection in neonatal rats resulted in delayed or impaired growth of secondary septa into alveolar saccules. The impaired septal ingrowth was multifocal and predominantly centriacinar in distribution and was associated with alveolar enlargement and significant decreases (14 to 26%, p less than 0.01) in alveolar surface density. Total alveolar surface area in rats inoculated with virus as neonates was 22% lower (p less than 0.05) that that in control animals by 110 days after inoculation. Alveolar septa in these rats inoculated as neonates had enlarged interalveolar pores and defects compatible with mild alveolar emphysema. Airway corrosion casts prepared at 30 and 90 days after neonatal viral inoculation had terminal bronchioles that were 11 and 20% smaller in diameter (p less than 0.02), respectively, than those from age-matched control rats. The density of attachments of alveolar septa to bronchiolar walls in viral-inoculated rats at these times was significantly decreased (p less than 0.001). Viral-infected rats had elevated respiratory resistance (p less than 0.005) and decreased dynamic compliance (p less than 0.02) at 39 days after inoculation. The results indicate that viral bronchiolitis and pneumonia during early life in rats induces abnormal alveolar development and bronchiolar hypoplasia that are associated with abnormalities in pulmonary function. Continued postnatal lung growth does not compensate for early virus-induced abnormalities in alveolar and bronchiolar growth.

Amyloplast sedimentation and organelle saltation in living corn columella cells.

Amyloplast sedimentation during gravistimulation and organelle movements was studied in living central rootcap cells of Zea mays L. cv. Merit. Cells from sectioned roots were viewed with a horizontally-mounted videomicroscope. The kinetics of gravity-induced amyloplast sedimentation were comparable to those calculated from experiments using fixed material. Individual amyloplasts fell at an average velocity of 5.5 micrometers min-1; the maximal velocity of fall measured was 18.0 micrometers min-1. Amyloplasts often rotated, sometimes rose in the cytoplasm, and occasionally underwent sudden rapid movements as fast as 58 micrometers min-1. Saltations of other organelles were frequently observed. This appears to be the first report of cytoplasmic streaming in the presumptive statocytes of roots.

Amyloplast sedimentation kinetics in gravistimulated maize roots.

Amyloplast sedimentation in gravistimulated maize (Zea mays L.) roots was measured using the change in angle from the center of the cell to each amyloplast as an index of sedimentation. Using tissue fixed after gravistimulation, the relationship between mean amyloplast angle and the duration of gravistimulation was found to be linear when plotted on a logarithmic time scale. Extrapolated values for the onset of angular change are 5.9 s after the start of gravistimulation for the entire population of amyloplasts and 11.8 s for lead amyloplasts. By multiplying the instantaneous angular velocity (in radians) by the cell center to amyloplast radius, it is possible to calculate the initial sedimentation velocity to be 19.1 micrometers min-1 at 5.9 s. During sedimentation, the mean amyloplast angles surpass the calculated cell corner angle of 123 at 2.2 min for all amyloplasts and at 19 s for lead amyloplasts near the new lower wall. Thus, substantial sedimentation occurs within the presentation time, calculated to be 4.1 min. These kinetics are consistent with several hypotheses of graviperception.

Amyloplast sedimentation kinetics in gravistimulated maize roots.

Amyloplast sedimentation in gravistimulated maize (Zea mays L.) roots was measured using the change in angle from the center of the cell to each amyloplast as an index of sedimentation. Using tissue fixed after gravistimulation, the relationship between mean amyloplast angle and the duration of gravistimulation was found to be linear when plotted on a logarithmic time scale. Extrapolated values for the onset of angular change are 5.9 s after the start of gravistimulation for the entire population of amyloplasts and 11.8 s for lead amyloplasts. By multiplying the instantaneous angular velocity (in radians) by the cell center to amyloplast radius, it is possible to calculate the initial sedimentation velocity to be 19.1 µm min(-1) at 5.9 s. During sedimentation, the mean amyloplast angles surpass the calculated cell corner angle of 123° at 2.2 min for all amyloplasts and at 19 s for lead amyloplasts near the new lower wall. Thus, substantial sedimentation occurs within the presentation time, calculated to be 4.1 min. These kinetics are consistent with several hypotheses of graviperception.

Kinetics of amyloplast sedimentation in gravistimulated maize coleoptiles.

Inner mesophyll cells from coleoptiles of Zea mays L. cv. Merit were fixed after varying periods of gravistimulation. A statistically significant amount (17-21%) of amyloplast sedimentation occurred in these cells after 30 s of gravistimulation. The presentation time is approx. 40 s or less. The accumulation of amyloplasts near the new lower wall shows a linear relationship to the logarithm of the gravistimulation time (r=0.92 or higher). The intercept of this line with the baseline value of amyloplasts in vertical coleoptiles indicates that the number of amyloplasts on the new lower wall begins increasing 11-15 s after the onset of gravistimulation. Direct observations of living cells confirm that many amyloplasts sediment within less than 15-30 s. These rapid kinetics are consistent with the classical statolith hypothesis of graviperception involving the sedimentation of amyloplasts to the vicinity of the new lower wall.