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1.  In vitro exposure of bovine morulae to Ureaplasma diversum. 
Ureaplasma diversum has been associated with infertility in the cow experimentally and in naturally occurring cases. However, the pathogenic mechanism is undetermined. The purpose of this study was to determine whether ureaplasmas are pathogenic for bovine morulae in vitro. Twenty-one morulae were recovered from three superovulated, mature, Holstein cows six or seven days postestrus. The embryos were divided into three groups (A,B,C) and incubated for 16 hours at 37 degrees C in humidified air with 10% CO2. Group A was incubated in embryo culture medium alone, Group B was incubated in culture medium with sterile ureaplasma broth added and Group C was incubated in culture medium containing 1.7 X 10(6) colony forming units Ureaplasma diversum strain 2312. After incubation, the morulae were examined using an electron microscope. Structures morphologically identical to U. diversum were present on the outer surface of the zonae pellucidae of all the morulae exposed to the organism and none were present on the unexposed control embryos. No other morphological differences were observed in either the ureaplasma-exposed embryos or the two groups of control embryos. Ureaplasma diversum was isolated from three of the five embryos incubated in culture medium with sterile ureaplasma broth added. These three embryos were recovered from one donor cow which cultured positive for U. diversum from the vulva and flush fluid. This finding suggests that the contaminating organisms entered the embryo culture wells either in the embryo collection medium or attached to the embryos.(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC1255303  PMID: 3607652
2.  Protein A gold identification of ureaplasmas on the bovine zona pellucida. 
The object of this study was to develop a prefixation protein A gold labelling technique for Ureaplasma diversum and to apply this to bovine embryos. Sixteen hour cultures of Ureaplasma diversum strain 2312 were incubated with either specific antiserum or nonimmune serum, followed by exposure to protein A gold and negative staining. The ureaplasmas which were incubated with specific antiserum were labelled with gold particles while those ureaplasmas which were incubated with nonimmune serum were not labelled. Twenty-three unhatched, day 7 bovine embryos were then incubated in either embryo culture medium (ECM) alone, ECM with sterile ureaplasma broth added or ECM with 1.7 X 10(6) colony forming units of Ureaplasma diversum strain 2312 per embryo. After 16 hours, the embryos were washed twice and incubated with either specific antiserum or nonimmune serum. The embryos were then incubated with medium containing protein A gold and examined by electron microscopy. No ureaplasmas were identified on the zona pellucida of the control embryos. Ureaplasmas were identified on the outer surface of the zona pellucida of 13 of the 17 embryos which had been exposed to the organism. Of these, the embryos which were incubated with specific antiserum had labelled ureaplasmas while the embryos which were incubated with nonimmune serum had unlabelled ureaplasmas on the zona pellucida. It was concluded that the protein A gold method was a suitable technique for the identification of ureaplasmas in EM preparations. The presence of ureaplasmas on the outer surface of the bovine zona pellucida following in vitro exposure to the organism was confirmed.(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC1255543  PMID: 2469532
3.  Ureaplasma diversum infection in vitro alters prostaglandin E2 and prostaglandin F2a production by bovine endometrial cells without affecting cell viability. 
Infection and Immunity  1994;62(5):1528-1533.
Bovine epithelial and stromal cells of the endometrium were inoculated with Ureaplasma diversum, pathogenic strain 2312, at 10(6) or 10(3) color-changing units (ccu)/ml in the presence of 1% fetal bovine serum (depleted of steroids by dextran-charcoal treatment) to assess the effect of infection on prostaglandin biosynthesis. When the inoculum of U. diversum was 10(6) ccu/ml, the concentration of U. diversum in the culture medium decreased with time. U. diversum was found on the epithelial and stromal cell monolayers, increasing in titer 100-fold, indicating that attachment and eventually growth occurred. When the inoculum was 10(3) ccu/ml, the titer of U. diversum remained the same or increased in the supernatant and increased on epithelial and stromal cells. The effect of infection was evaluated by measurement of the primary prostaglandin produced by each cell type, prostaglandin F2a for epithelial cells and prostaglandin E2 for stromal cells. Infection with U. diversum significantly decreased prostaglandin F2a accumulation, by 44.7% +/- 6.0% at 10(6) ccu/ml (P < or = 0.005) and 15.8% +/- 5.3% at 10(3) ccu/ml (P < or = 0.05) in epithelial cells. Prostaglandin E2 accumulation by stromal cells was decreased by 34.0% +/- 4.0% at 10(6) ccu/ml (P < or = 0.001) and by 13.5% +/- 2.7% at 10(3) ccu/ml (P < or = 0.005). Infection with 10(6) ccu/ml did not alter endometrial cell viability, as shown by protein measurement, trypan blue dye exclusion, and cell plating efficiency tests. Thus, alterations in prostaglandin production were not due to cell deterioration. These observations suggest that U. diversum can alter prostaglandin E2 and prostaglandin F2a patterns in primary cultures of bovine endometrial cells without affecting cell viability.
PMCID: PMC186347  PMID: 8168914
4.  The effect of intrauterine inoculation with Ureaplasma diversum on bovine fertility. 
To determine the influence of Ureaplasma diversum on bovine fertility 11 uninfected virgin heifers with normal ovarian cyclic activity were randomly allocated to test or control groups. At a synchronized estrus, five test heifers were given an intrauterine broth inoculum containing 1.09 x 10(8) to 1.4 x 10(9) colony forming units of U. diversum and six control animals were infused with sterile ureaplasma broth medium. All animals were artificially inseminated within one hour of infusion. Pregnancy was diagnosed in one of five test heifers and all of six controls by serum progesterone concentrations measured to 25 days postinsemination. The difference in pregnancy rates between the two groups was statistically significant (p = 0.0152). It was concluded that under the conditions of this experiment U. diversum is capable of causing infertility in cattle.
PMCID: PMC1255361  PMID: 3453263
5.  Invasion of Ureaplasma diversum in bovine spermatozoids 
BMC Research Notes  2011;4:455.
Ureaplasma diversum has been associated with infertility in cows. In bulls, this mollicute colonizes the prepuce and distal portion of the urethra and may infect sperm cells. The aim of this study is to analyze in vitro interaction of U. diversum isolates and ATCC strains with bovine spermatozoids. The interactions were observed by confocal microscopy and the gentamycin internalization assay.
U. diversum were able to adhere to and invade spermatozoids after 30 min of infection. The gentamicin resistance assay confirmed the intracellularity and survival of U. diversum in bovine spermatozoids.
The intracellular nature of bovine ureaplasma identifies a new difficulty to control the reproductive of these animals.
PMCID: PMC3219583  PMID: 22032232
Ureaplasma diversum; bovine spermatozoid; invasion; confocal microscopy
6.  Apoptosis in HEp-2 cells infected with Ureaplasma diversum 
Biological Research  2014;47(1):38.
Bacterial pathogens have many strategies for infecting and persisting in host cells. Adhesion, invasion and intracellular life are important features in the biology of mollicutes. The intracellular location of Ureaplasma diversum may trigger disturbances in the host cell. This includes activation or inhibition of pro and anti-apoptotic factors, which facilitate the development of host damage. The aim of the present study was to associate U. diversum infection in HEp-2 cells and apoptosis induction. Cells were infected for 72hs with four U. diversum clinical isolates and an ATCC strain. The U. diversum invasion was analyzed by Confocal Laser Scanning Microscopy and gentamicin invasion assay. The apoptosis was evaluated using pro-apoptotic and anti-apoptotic gene expression, and FITC Annexin V/Dead Cell Apoptosis Kit.
The number of internalized ureaplasma in HEp-2 cells increased significantly throughout the infection. The flow cytometry analysis with fluorochromes to detect membrane depolarization and gene expression for caspase 2, 3 and 9 increased in infected cells after 24 hours. However, after 72 hours a considerable decrease of apoptotic cells was observed.
The data suggests that apoptosis may be initially induced by some isolates in association with HEp-2 cells, but over time, there was no evidence of apoptosis in the presence of ureaplasma and HEp-2 cells. The initial increase and then decrease in apoptosis could be related to bacterial pathogen-associated molecular pattern (PAMPS). Moreover, the isolates of U. diversum presented differences in the studied parameters for apoptosis. It was also observed that the amount of microorganisms was not proportional to the induction of apoptosis in HEp-2 cells.
PMCID: PMC4167145  PMID: 25299837
Ureaplasma diversum; Invasion; Apoptosis; HEp-2 cells
7.  Immune response of heifers to vaginal submucosal or subcutaneous vaccination and intravaginal challenge with Ureaplasma diversum. 
Twenty beef heifers were randomly assigned to five equal groups and vaccinated: Group 1--in vaginal submucosa (VM) with Ureaplasma diversum ultrasonicated whole cells (WC) in complete Freund's adjuvant (CFA); Group 2--in VM with U. diversum cell membranes (CM) in CFA; Group 3--subcutaneously (SC) with CM in CFA; Group 4--in VM with CM alone; and Group 5--in VM with phosphate buffered saline (PBS) in CFA. A second vaccination with the same antigens in incomplete Freund's adjuvant was given after four weeks, and three weeks later, all heifers were challenged intravaginally with 3.6 x 10(7) colony-forming units (CFU) of U. diversum strain 2312. Immunoglobulins that reacted with U. diversum were measured in serum and cervicovaginal mucus (CVM) by an enzyme-linked-immunosorbent assay. In groups 1 and 2, vaccination by the VM route with WC or CM antigens, stimulated high levels of U. diversum-reactive IgG1 and IgG2 antibodies in serum as well as CVM, but a low IgA response only in CVM. In group 4, VM vaccination with CM (no adjuvant) elicited a minimal IgG1 and IgG2 response in serum and CVM. In group 3, SC vaccination with CM antigen stimulated high IgG1 and IgG2 reactivity in both serum and CVM, but no IgA reactivity. Very little IgM reactivity was detected in the four vaccinated groups. Intravaginal challenge resulted in characteristic granular vulvitis in all vaccinated and control heifers, with all animals remaining culture-positive for the 35 day observation period. The infection stimulated a marked increase in the specific IgA response in CVM of the three groups vaccinated with either, adjuvanted antigen.(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC1263675  PMID: 8004535
8.  Activation of murine macrophages and lymphocytes by Ureaplasma diversum. 
Ureaplasma diversum is a pathogen in the bovine reproductive tract. The objective of the research was to study interactions with macrophages and lymphocytes which might elucidate aspects of pathogenetic mechanisms of this organism. We studied the activation of murine macrophages of C3H/HeN (LPS-responder) and C3H/HeJ (LPS-low-responder) genotype for TNF-alpha, IL-6, IL-1 and nitric oxide production and blastogenic response of C3H/HeJ splenocytes after Ureaplasma diversum stimulation. Live and heat-killed U. diversum induced TNF-alpha, IL-6 and IL-1 in peritoneal macrophage cultures of both C3H/HeN and C3H/HeJ mice in a dose dependent manner. Interferon-gamma modulated the cytokine production, by increasing the production of TNF-alpha, IL-6 and nitric oxide, but IL-1 secretion was only enhanced in C3H/HeJ macrophages stimulated by live ureaplasmas. Supernatant of U. diversum sonicate was mitogenic for murine spleen lymphocytes. The blastogenic response was dose dependent, and stimulation with both U. diversum and Concanavalin A seemed to have an additive effect. These results suggest that U. diversum, similar to other mycoplasmas, activates murine macrophages and lymphoid cells. The studies should be repeated with bovine cells in order to elucidate pathogenetic aspects of inflammation in cattle caused by U. diversum.
PMCID: PMC1263712  PMID: 7889459
9.  Differential contributions of Ng-CAM and N-CAM to cell adhesion in different neural regions 
The Journal of Cell Biology  1986;103(1):145-158.
Individual neurons can express both the neural cell adhesion molecule (N-CAM) and the neuron-glia cell adhesion molecule (Ng-CAM) at their cell surfaces. To determine how the functions of the two molecules may be differentially controlled, we have used specific antibodies to each cell adhesion molecule (CAM) to perturb its function, first in brain membrane vesicle aggregation and then in tissue culture assays testing the fasciculation of neurite outgrowths from cultured dorsal root ganglia, the migration of granule cells in cerebellar explants, and the formation of histological layers in the developing retina. Our strategy was initially to delineate further the binding mechanisms for each CAM. Antibodies to Ng-CAM and N-CAM each inhibited brain membrane vesicle aggregation but the binding mechanisms of the two CAMs differed. As expected from the known homophilic binding mechanism of N-CAM, anti-N- CAM-coated vesicles did not co-aggregate with uncoated vesicles. Anti- Ng-CAM-coated vesicles readily co-aggregated with uncoated vesicles in accord with a postulated heterophilic binding mechanism. It was also shown that N-CAM was not a ligand for Ng-CAM. In contrast to assays with brain membrane vesicles, cellular systems can reveal functional differences for each CAM reflecting its relative amount (prevalence modulation) and location (polarity modulation). Consistent with this, each of the three cellular processes examined in vitro was preferentially inhibited only by anti-N-CAM or by anti-Ng-CAM antibodies. Both neurite fasciculation and the migration of cerebellar granule cells were preferentially inhibited by anti-Ng-CAM antibodies. Anti-N-CAM antibodies inhibited the formation of histological layers in the retina. The data on perturbation by antibodies were correlated with the relative levels of expression of Ng-CAM and N-CAM in each of these different neural regions. Quantitative immunoblotting experiments indicated that the relative Ng-CAM/N-CAM ratios in comparable extracts of brain, dorsal root ganglia, and retina were respectively 0.32, 0.81, and 0.04. During culture of dorsal root ganglia in the presence of nerve growth factor, the Ng-CAM/N-CAM ratio rose to 4.95 in neurite outgrowths and 1.99 in the ganglion proper, reflecting both polarity and prevalence modulation. These results suggest that the relative ability of anti-Ng-CAM and anti-N-CAM antibodies to inhibit cell-cell interactions in different neural tissues is strongly correlated with the local Ng-CAM/N-CAM ratio.(ABSTRACT TRUNCATED AT 400 WORDS)
PMCID: PMC2113806  PMID: 3522601
10.  Adhesion molecules during somitogenesis in the avian embryo 
The Journal of Cell Biology  1987;104(5):1361-1374.
In avian embryos, somites constitute the morphological unit of the metameric pattern. Somites are epithelia formed from a mesenchyme, the segmental plate, and are subsequently reorganized into dermatome, myotome, and sclerotome. In this study, we used somitogenesis as a basis to examine tissue remodeling during early vertebrate morphogenesis. Particular emphasis was put on the distribution and possible complementary roles of adhesion-promoting molecules, neural cell adhesion molecule (N-CAM), N-cadherin, fibronectin, and laminin. Both segmental plate and somitic cells exhibited in vitro calcium- dependent and calcium-independent systems of cell aggregation that could be inhibited respectively by anti-N-cadherin and anti-N-CAM antibodies. In vivo, the spatio-temporal expression of N-cadherin was closely associated with both the formation and local disruption of the somites. In contrast, changes in the prevalence of N-CAM did not strictly accompany the remodeling of the somitic epithelium into dermamyotome and sclerotome. It was also observed that fibronectin and laminin were reorganized secondarily in the extracellular spaces after CAM-mediated contacts were modulated. In an in vitro culture system of somites, N-cadherin was lost on individual cells released from somite explants and was reexpressed when these cells reached confluence and established intercellular contacts. In an assay of tissue dissociation in vitro, antibodies to N-cadherin or medium devoid of calcium strongly and reversibly dissociated explants of segmental plates and somites. Antibodies to N-CAM exhibited a smaller disrupting effect only on segmental plate explants. In contrast, antibodies to fibronectin and laminin did not perturb the cohesion of cells within the explants. These results emphasize the possible role of cell surface modulation of CAMs during the formation and remodeling of some transient embryonic epithelia. It is suggested that N-cadherin plays a major role in the control of tissue remodeling, a process in which N-CAM is also involved but to a lesser extent. The substratum adhesion molecules, fibronectin and laminin, do not appear to play a primary role in the regulation of these processes but may participate in cell positioning and in the stabilization of the epithelial structures.
PMCID: PMC2114487  PMID: 3553211
11.  Invasion of Ureaplasma diversum in Hep-2 cells 
BMC Microbiology  2010;10:83.
Understanding mollicutes is challenging due to their variety and relationship with host cells. Invasion has explained issues related to their opportunistic role. Few studies have been done on the Ureaplasma diversum mollicute, which is detected in healthy or diseased bovine. The invasion in Hep-2 cells of four clinical isolates and two reference strains of their ureaplasma was studied by Confocal Laser Scanning Microscopy and gentamicin invasion assay.
The isolates and strains used were detected inside the cells after infection of one minute without difference in the arrangement for adhesion and invasion. The adhesion was scattered throughout the cells, and after three hours, the invasion of the ureaplasmas surrounded the nuclear region but were not observed inside the nuclei. The gentamicin invasion assay detected that 1% of the ATCC strains were inside the infected Hep-2 cells in contrast to 10% to the clinical isolates. A high level of phospholipase C activity was also detected in all studied ureaplasma.
The results presented herein will help better understand U. diversum infections, aswell as cellular attachment and virulence.
PMCID: PMC2907839  PMID: 20236540
12.  Ureaplasma infection of cell cultures. 
Infection and Immunity  1986;52(2):437-444.
Studies were performed to characterize the effects of ureaplasmas in HeLa, 3T6, and CV-1 cell cultures. The ureaplasmas studied were human Ureaplasma urealyticum T960 (serotype VIII), bovine U. diversum T95, simian strain T167-2, ovine strain 1202, canine strain D1M-C, and feline strains 382 and FT2-B. FT2-B was the only ureaplasma to grow in the cell free culture medium, Dulbecco modified Eagle-Earle medium containing 10% fetal bovine serum. The growth pattern of the ureaplasmas varied in the different cell cultures, but each strain grew in at least two of the cell cultures, suggesting a requirement for a product of the cell culture and for low concentrations of urea. When growth occurred, organisms grew to concentrations that approached, but did not equal, those observed in 10B broth. Most, but not all, ureaplasmas grew quickly, reaching peak titers 2 days after infection. Canine strain D1M-C did not grow in 3T6, but showed rapid growth in HeLa and CV-1 cells, killing both cultures, In some systems, e.g., U. urealyticum T960 and simian strain T167-2, the infection persisted, and ureaplasmas could be recovered from cell cultures four passages after infection, when studies were terminated. The cell culture ureaplasmas grew on T agar, but not on mycoplasma agar medium.
PMCID: PMC261018  PMID: 3699891
13.  Measurement of the cytotoxic effects of different strains of Mycoplasma equigenitalium on the equine uterine tube using a calmodulin assay. 
The cytopathic effects induced by five strains of Mycoplasma equigenitalium for cells of equine uterine tube explants were tested by measuring changes in cellular and extracellular concentrations of calmodulin (CaM). Calmodulin concentrations in samples of total homogenate (TH) and total homogenate supernates (THS) of the infected equine uterine tube explants were significantly lower than respective measurements on noninfected controls. In tissue culture medium fractions (TCM) of some infected explants, CaM concentrations were significantly higher than noninfected controls (p > 0.95). The results suggest that M. equigenitalium colonization on ciliated cells of the equine uterine tube can affect the permeability of the cell membrane leading to leakage or release of CaM during cell breakdown. Measurement of CaM concentrations in samples of TH revealed significant differences in the cytotoxic effects induced by different strains of M. equigenitalium on the equine uterine tube (EUT). The data suggests that some strains of M. equigenitalium may have a role in reproductive failure in the mare. In addition comparisons of the means of the concentrations of CaM in samples of TH or THS in EUT explants from four mares in the follicular and four in the luteal phase of the estrous cycle were found to be not significantly different.
PMCID: PMC1263565  PMID: 1477802
14.  The effects of two Ureaplasma diversum strains on early pregnancy in heifers. 
Two field isolates of Ureaplasma diversum spp. were used to infect heifers at the time of insemination in a preliminary study to observe the effect of infection on early pregnancy. M84-14c-1 was a field isolate from a bull's prepuce typed by immunofluorescence to be similar to U. diversum strain T-44 (Group C). M84-477c-4 was a field isolate from bovine semen typed by immunofluorescence to be similar to U. diversum strain T-288 (Group A). All three heifers infected with M84-477c-4 had a mild granular vulvitis at some time during the trial. None was pregnant when slaughtered 27 days after infection. The result of infection with M84-14c-1, a preputial isolate, was not consistent. One heifer had no infection and a normal pregnancy, one heifer was infected with an abnormal pregnancy, and one heifer was open but ureaplasmas were not detected until day 17 of the trial.
PMCID: PMC1255382  PMID: 3453276
15.  Evidence for autoregulation of camR, which encodes a repressor for the cytochrome P-450cam hydroxylase operon on the Pseudomonas putida CAM plasmid. 
Journal of Bacteriology  1993;175(24):7828-7833.
The regulatory gene camR on the CAM plasmid of Pseudomonas putida (ATCC 17453) negatively controls expression of the cytochrome P-450cam hydroxylase operon (camDCAB) for the camphor degradation pathway and is oriented in a direction opposite to that of the camDCAB operon. In this study, we examined expression of the camR gene by monitoring the beta-galactosidase activity of camR-lacZ translational fusions in P. putida camR and camR+ strains. We found that the camR gene was autogenously regulated by its own product, CamR. To search for an operator site of the camR gene, a cam repressor (CamR)-overproducing plasmid, pHAOV1, was constructed by placing the camR gene under the control of a pL promoter. The translational initiation codon of CamR was changed by site-directed mutagenesis from GTG to ATG to improve translation efficiency. Judging from sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the CamR protein was expressed up to about 10% of the soluble protein of CamR-overproducing Escherichia coli JM83/pHAOV1 cells. Results of DNase I footprinting assays using the cell lysate indicated that the CamR repressor covered a single region between the camR gene and the camDCAB operon. Our findings also suggest that the camR gene autogenously regulates its own expression by binding of the gene product, CamR, to the operator, which also serves as an operator of the camDCAB operon.
PMCID: PMC206958  PMID: 8253671
16.  Altered expression of neuronal cell adhesion molecules induced by nerve injury and repair 
The Journal of Cell Biology  1986;103(3):929-945.
Peripheral nerve injury results in short-term and long-term changes in both neurons and glia. In the present study, immunohistological and immunoblot analyses were used to examine the expression of the neural cell adhesion molecule (N-CAM) and the neuron-glia cell adhesion molecule (Ng-CAM) within different parts of a functionally linked neuromuscular system extending from skeletal muscle to the spinal cord after peripheral nerve injury. Histological samples were taken from 3 to 150 d after crushing or transecting the sciatic nerve in adult chickens and mice. In unperturbed tissues, both N-CAM and Ng-CAM were found on nonmyelinated axons, and to a lesser extent on Schwann cells and myelinated axons. Only N-CAM was found on muscles. After denervation, the following changes were observed: The amount of N-CAM in muscle fibers increased transiently on the surface and in the cytoplasm, and in interstitial spaces between fibers. Restoration of normal N-CAM levels in muscle was dependent on reinnervation; in a chronically denervated state, N-CAM levels remained high. After crushing or cutting the nerve, the amount of both CAMs increased in the area surrounding the lesion, and the predominant form of N-CAM changed from a discrete Mr 140,000 component to the polydisperse high molecular weight embryonic form. Anti-N-CAM antibodies stained neurites, Schwann cells, and the perineurium of the regenerating sciatic nerve. Anti-Ng- CAM antibodies labeled neurites, Schwann cells and the endoneurial tubes in the distal stump. Changes in CAM distribution were observed in dorsal root ganglia and in the spinal cord only after the nerve was cut. The fibers within affected dorsal root ganglia were more intensely labeled for both CAMs, and the motor neurons in the ventral horn of the spinal cord of the affected segments were stained more intensely in a ring pattern by anti-N-CAM and anti-Ng-CAM than their counterparts on the side contralateral to the lesion. Taken together with the previous studies (Rieger, F., M. Grumet, and G. M. Edelman, J. Cell Biol. 101:285-293), these data suggest that local signals between neurons and glia may regulate CAM expression in the spinal cord and nerve during regeneration, and that activity may regulate N-CAM expression in muscle. Correlations of the present observations are made here with established events of nerve degeneration and suggest a number of roles for the CAMs in regenerative events.(ABSTRACT TRUNCATED AT 400 WORDS)
PMCID: PMC2114294  PMID: 2427528
17.  Susceptibilities of Mycoplasma bovis, Mycoplasma dispar, and Ureaplasma diversum strains to antimicrobial agents in vitro. 
The purpose of this study was to determine the susceptibility of various strains of Mycoplasma bovis, Mycoplasma dispar, and Ureaplasma diversum, which are prevalent causes of pneumonia in calves, to 16 antimicrobial agents in vitro. The MICs of the antimicrobial agents were determined by a serial broth dilution method for 16 field strains and the type strain of M. bovis, for 19 field strains and the type strain of M. dispar, and for 17 field strains of U. diversum. Final MICs for M. bovis and M. dispar were read after 7 days and final MICs for U. diversum after 1 to 2 days. All strains tested were susceptible to tylosin, kitasamycin, and tiamulin but were resistant to nifuroquine and streptomycin. Most strains of U. diversum were intermediately susceptible to oxytetracycline but fully susceptible to chlortetracycline; most strains of M. bovis and M. dispar, however, were resistant to both agents. Strains of M. dispar and U. diversum were susceptible to doxycycline and minocycline, but strains of M. bovis were only intermediately susceptible. Susceptibility or resistance to chloramphenicol, spiramycin, spectinomycin, lincomycin, or enrofloxacin depended on the species but was not equal for the three species. The type strains of M. bovis and M. dispar were more susceptible to various antimicrobial agents, including tetracyclines, than the field strains. This finding might indicate that M. bovis and M. dispar strains are becoming resistant to these agents. Antimicrobial agents that are effective in vitro against all three mycoplasma species can be considered for treating mycoplasma infections in pneumonic calves. Therefore, tylosin, kitasamycin, and tiamulin may be preferred over oxytetracycline and chlortetracycline.
PMCID: PMC187660  PMID: 8452363
18.  Mycoplasmas and bovine respiratory disease: studies related to pathogenicity and the immune response--a selective review. 
Three species of mycoplasma have been established as being of importance as causes of pneumonia in housed calves, based on pathogenicity studies and frequency of association with the disease. These three species are Mycoplasma bovis, M. dispar, and Ureaplasma diversum. M. bovis is the most pathogenic of these species but the disease outbreaks with which it is associated are sporadic. M. dispar is regularly isolated from pneumonic calves but is also found causing mild superficial and asymptomatic infections of the respiratory mucosa. The bovine ureaplasmas are serologically complex. They are distinct from ureaplasmas isolated from other non-ruminants by PAGE analysis, G + C content of DNA, and serology. A second species within the genus ureaplasma has been proposed to accommodate the bovine ureaplasmas, U. diversum. Control of mycoplasma respiratory infections of cattle based on immunization might be possible. Calves have been immunized against M. bovis and immunity has been related to antibody in the lung. M. dispar appears less immunogenic in calves than M. bovis and this may contribute to its pathogenicity.
PMCID: PMC2590553  PMID: 6382831
19.  Humoral and secretory antibodies to Ureaplasma diversum in heifers following subcutaneous vaccination and vaginal infection. 
We measured antibody levels in serum and cervicovaginal mucus (CVM) of four heifers vaccinated with two inoculations of killed Ureaplasma diversum strain 2312 in incomplete Freund's adjuvant (IFA) two weeks apart, and six heifers given a placebo. Two weeks later, the vaccinates and four placebo heifers, were challenged by intravaginal inoculation with 6.4 x 10(8) colony-forming units of the homologous U. diversum strain. The remaining two placebo heifers served as unvaccinated, unchallenged controls. Antibody levels in serum and CVM of all heifers were determined by an enzyme-linked immunosorbent assay (ELISA). Vaccination stimulated specific IgG1 and IgG2 responses in serum and CVM but only a slight IgM and no IgA response. In both vaccinate and placebo heifers, subsequent intravaginal challenge resulted in a granular vulvitis (GV) with a predominant IgA response in the CVM. The GV gradually subsided during the 35 day observation period but ureaplasmas were consistently demonstrated by culture. We concluded that subcutaneous vaccination stimulated a specific, albeit nonprotective, IgG response in serum and CVM. In contrast, vaginal infection primarily induced a mucosal IgA response.
PMCID: PMC1263674  PMID: 8004534
20.  The Distribution of Mycoplasmas and Ureaplasmas in the Genital Tract of Normal Artificial Insemination Bulls 
Bull semen is commonly contaminated with mycoplasmas. To determine the source of contamination, semen and the genital tracts of 45 artificial insemination bulls were cultured for these organisms. The results indicate that mycoplasmas colonize the prepuce and the distal part of the urethra. Only rarely were they found in the ampullae or seminal vesicles. In 92% of the bulls with contaminated semen the same Mycoplasma species or Ureaplasma diversum was isolated from the prepuce and urethral orifice as was found in the semen. This suggests that the prepuce and distal urethra is the source of contamination. Colonization of the genital tracts with Mycoplasmas or U. diversum was not associated with histological changes.
PMCID: PMC1680053  PMID: 17422489
Mycoplasma; ureaplasma; genital tract; semen; artificial insemination; microbial colonization
21.  Cell-Deposited Matrix Improves Retinal Pigment Epithelium Survival on Aged Submacular Human Bruch's Membrane 
Resurfacing submacular human Bruch's membrane with a cell-deposited extracellular matrix increases long-term survival of retinal pigment epithelial cells. This effect is most marked in submacular Bruch's membrane of aged Caucasians.
To determine whether resurfacing submacular human Bruch's membrane with a cell-deposited extracellular matrix (ECM) improves retinal pigment epithelial (RPE) survival.
Bovine corneal endothelial (BCE) cells were seeded onto the inner collagenous layer of submacular Bruch's membrane explants of human donor eyes to allow ECM deposition. Control explants from fellow eyes were cultured in medium only. The deposited ECM was exposed by removing BCE. Fetal RPE cells were then cultured on these explants for 1, 14, or 21 days. The explants were analyzed quantitatively by light microscopy and scanning electron microscopy. Surviving RPE cells from explants cultured for 21 days were harvested to compare bestrophin and RPE65 mRNA expression. Mass spectroscopy was performed on BCE-ECM to examine the protein composition.
The BCE-treated explants showed significantly higher RPE nuclear density than did the control explants at all time points. RPE expressed more differentiated features on BCE-treated explants than on untreated explants, but expressed very little mRNA for bestrophin or RPE65. The untreated young (<50 years) and African American submacular Bruch's membrane explants supported significantly higher RPE nuclear densities (NDs) than did the Caucasian explants. These differences were reduced or nonexistent in the BCE-ECM-treated explants. Proteins identified in the BCE-ECM included ECM proteins, ECM-associated proteins, cell membrane proteins, and intracellular proteins.
Increased RPE survival can be achieved on aged submacular human Bruch's membrane by resurfacing the latter with a cell-deposited ECM. Caucasian eyes seem to benefit the most, as cell survival is the worst on submacular Bruch's membrane in these eyes.
PMCID: PMC3101675  PMID: 21398292
22.  Calmodulin stabilization of kinetochore microtubule structure to the effect of nocodazole 
The Journal of Cell Biology  1988;107(6):2243-2251.
To investigate the function of calmodulin (CaM) in the mitotic apparatus, the effect of microinjected CaM and chemically modified CaMs on nocodazole-induced depolymerization of spindle microtubules was examined. When metaphase PtK1 cells were microinjected with CaM or a CaM-TRITC conjugate, kinetochore microtubules (kMTs) were protected from the effect of nocodazole. The ability of microinjected CaM to subsequently protect kMTs from the depolymerizing effect of nocodazole was dose dependent, and was effective for approximately 45 min, with protection decreasing if nocodazole treatment was delayed for more than 60 min after injection of CaM. The CaM-TRITC conjugate, similar to native CaM, displayed the ability to activate bovine brain CaM- dependent adenylate cyclase in a Ca++-dependent manner and showed a Ca++-dependent mobility shift when subjected to PAGE. A heat-altered CaM-TRITC conjugate also protected kMTs from the effect of nocodazole. However, this modified CaM was not able to activate adenylate cyclase nor did it display a Ca++-dependent mobility shift when electrophoresed. In a permeabilized cell model system, both CaM analogs were observed to bind to the spindle in a Ca++-independent manner. In contrast, a performic acid-oxidized CaM did not have a protective effect on spindle structure when microinjected into metaphase cells before nocodazole treatment. The oxidized CaM did not activate adenylate cyclase and did not exhibit Ca++-dependent mobility on polyacrylamide gels. These results are interpreted as supporting the hypothesis that CaM binds to the mitotic spindle in a Ca++-independent manner and that CaM may serve in the spindle, at least in part, to stabilize kMTs.
PMCID: PMC2115647  PMID: 3198685
23.  Isolation of Ureaplasma diversum and mycoplasmas from genital tracts of beef and dairy cattle in Saskatchewan 
We report herein a survey in which cultures of bovine reproductive tracts for Ureaplasma diversum and mycoplasmas were carried out in order to better understand the role of these organisms in granular vulvitis (GV). Samples cultured were vulvar swabs from clinically normal cows or ones with GV, preputial swabs or raw semen from bulls, and abomasal contents of aborted fetuses.
Ureaplasma diversum was isolated from 104 (43.3%) of 240 dairy cows, 32 (27.1%) of 118 beef cows, 43 (47.2%) of 91 beef heifers, 23 (67.6%) of 34 beef bulls, and three (60%) of five dairy bulls. Mycoplasmas were isolated from 18 (7.5%) dairy cows, two (1.6%) beef cows, three (8.8%) beef bulls, and one dairy bull. No isolation was made from 97 aborted fetuses. For 65 dairy cows and 30 beef heifers with vulvar lesions, the isolation rates for ureaplasmas of 62.5% and 69.7%, respectively, were significantly higher (X2) than those for normal animals (37.5% and 30.3%). On immunofluorescent serotyping of 137 of the 205 isolates, there were 66 in serogroup C (strain T44), 18 in serogroup B (strain D48), eight in serogroup A (strain A417 or strain 2312), 14 cross-reacting, and 31 that were not identified. It was concluded that U. diversum is commonly present in the lower reproductive tract of beef/dairy cattle in Saskatchewan and is associated with granular vulvitis.
PMCID: PMC1481172  PMID: 17423929
24.  Rapid identification of mycoplasmas by indirect immunoperoxidase test using small square filter paper. 
The indirect immunoperoxidase test using small, square filter paper was used for rapid identification of mycoplasmas. Colonies of type strains of 22 mycoplasma species, 3 acholeplasma species, and three Ureaplasma diversum serogroups were stained by this test with high sensitivity and specificity. All of 49 isolates from bovine materials and cell cultures were easily identified by this test, and the results agreed with those obtained by growth inhibition test. Use of filter paper made it possible to add different kinds of antisera or conjugates to the same agar plate simultaneously and also to save antiserum and conjugate. This test proved to be a simple and useful technique for rapid identification of many mycoplasma species grown on agar medium.
PMCID: PMC265805  PMID: 3539989
The Journal of Cell Biology  1971;50(3):598-615.
Administration of estrogen (E) to immature chicks triggers the cytodifferentiation of tubular gland cells in the magnum portion of the oviduct epithelium; these cells synthesize the major egg-white protein, ovalbumin. Electron microscopy and immunoprecipitation of ovalbumin from oviduct explants labeled with radioactive amino acids in tissue culture were used to follow and measure the degree of tubular gland cell cytodifferentiation. Ovalbumin is undetectable in the unstimulated chick oviduct and in oviducts of chicks treated with progesterone (P) for up to 5 days. Ovalbumin synthesis is first detected 24 hr after E administration, and by 5 days it accounts for 35% of the soluble protein being synthesized. Tubular gland cells begin to synthesize ovalbumin before gland formation which commences after 36 hr of E treatment. When E + P are administered together there is initially a synergistic effect on ovalbumin synthesis, however, after 2 days ovalbumin synthesis slows and by 5 days there is only 1/20th as much ovalbumin per magnum as in the E-treated controls. Whereas the magnum wet weight doubles about every 21 hr with E alone, growth stops after 3 days of E + P treatment. Histological and ultrastructural observations show that the partially differentiated tubular gland cells resulting from E + P treatment never invade the stroma and form definitive glands, as they would with E alone. Instead, these cells appear to transform into other cell types—some with cilia and some with unusual flocculent granules. We present a model of tubular gland cell cytodifferentiation and suggest that a distinct protodifferentiated stage exists. P appears to interfere with the normal transition from the protodifferentiated state to the mature tubular gland cell.
PMCID: PMC2108294  PMID: 4329151

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