Experimental pathogenicity of mollicutes from bovines with reproductive disorders in rabbit fallopian tube organ culture.

Mollicutes (10) belonging to Mycoplasma and Acholeplasma isolated from various reproductive disorders were tested in rabbit fallopian tube (FT) organ culture. Parameter for describing pathogenic status of Mollicutes in rabbit FT organ culture included multiplication of organisms, and its effect on ciliary activity along with histopathological changes in FT explants. M. mycoides (LC, Y-Goat), M. bovoculi, M. bovigenitalium, Mycoplasma sp. and A. oculi were categorized as pathogenic; A. axanthum and A. laidlawii as mildly pathogenic; and M. bovis, M. arginini. and A. granularum, as nonpathogenic to rabbit FT organ culture. Thus, rabbit FT organ culture is recommended for use as a suitable and economical in vitro model to assess the pathogenicity of Mollicutes of reproductive tract origin.     

The Effect of Porcine Mycoplasmas on Pig Tracheal Organ Cultures

S ummary : Tracheal organ cultures were made from 2–3 week old pigs. Tracheal rings were used to assess the pathogenicity of Mycoplasma hyopneumoniae, M. hyorhinis, M. granularum and M. hyosynoviae. Both serum-resistant and serum-sensitive strains of M. hyorhinis had a marked effect on ciliary activity and caused necrosis of the epithelium. Mycoplasma granularum and M. hyosynoviae caused cessation of ciliary movement, but to a less extent, and necrosis did not occur. With M. hyopneumoniae , the infected organ cultures survived longer than the control cultures.     

Promotion of Mycoplasma pulmonis growth in rat tracheal organ cultures by ammonium chloride

During exacerbation of respiratory mycoplasmosis in rats by environmental ammonia, numbers of Mycoplasma pulmonis organisms in the respiratory tract are increased. To test whether or not exposure of respiratory epithelium to ammonia in vitro promotes growth of the organism, rat tracheal organ cultures were treated with 50 mM ammonium chloride, inoculated with M. pulmonis, and quantitatively cultured. After 48 hours, treated tracheas harbored almost 10 times more M. pulmonis colony-forming units than control tracheas. Cellular lesions in the epithelium of treated tracheas resembled those in the nasal passages of rats exposed to gaseous ammonia. To determine whether or not growth-modifying factors were released from tracheal epithelium exposed to ammonium chloride, M. pulmonis growth was assessed in medium collected from ammonium chloride-treated and control tracheas. Growth in medium from treated tracheas was greater than that in medium from untreated tracheas.     

Intracellular levels of adenosine triphosphate in hamster trachea organ cultures exposed to Mycoplasma pneumoniae cells or membranes

The amount of adenosine triphosphate (ATP) in hamster trachea organ cultures was determined with a technique based on light emission from a luciferin/luciferase/ATP reaction. The amount of ATP, expressed as ng per mg dry weight, was consistent in tracheal explants prepared from various animals and changed negligibly when explants were cultivated in vitro for several days. The amount of ATP was related directly to cellular activity and integrity in the epithelium since inactivation by heat or freeze-thaw rapidly depleted measurable ATP, and ciliary activity and ATP content were related directly. When tracheal explants were infected with 10(5) to 10(7) CFU of virulent Mycoplasma pneumoniae cells, both ciliary activity and ATP content in the tissue dropped dramatically after approximately 5 to 8 days (up to 85% and 60% decreases, respectively). Exposure of explants to 50 to 200 microgram per ml of purified M. pneumoniae membranes also caused significant decreases in ciliary activity and ATP. When explants were infected with attenuated or nonvirulent mycoplasmas, ciliary activity was only slightly decreased, while ATP values often rose slightly. The technology associated with the determination of ATP levels in tracheal explants should prove useful as a new, objective, analytical approach to cell viability in organ cultures.     

Adherence of clinical isolates ofPseudomonas aeruginosa to hamster trachael epithelium in vitro

The adherence to hamster tracheal epithelium, of mucoid and nonmucoid clinical isolates ofPseudomonas aeruginosa from cystic fibrosis patients, was studied using tracheal organ cultures. Tracheal cultures were infected with 10⁷ colony-forming units per ml of either mucoid or nonmucoid clinical isolates ofP aeruginosa. The tracheal explants were rinsed at various time intervals to remove nonadherent bacteria, fixed, and prepared for transmission-and scanning-electron microscopy. Mucoid isolates were seen adhering to the ciliated epithelium as early as 4 h after initiation of infection, whereas nonmucoid isolates were only observed adhering at 6 to 8 h after infection. Mucoid organisms were found as clusters of bacteria embedded in an extensive extracellular matrix. The nonmucoid isolates were generally found as single organisms with no evidence of an extracellular matrix. These results suggest that the prevalence of mucoid isolates ofP. aeruginosa in cystic fibrosis may be due to adherent properties of the mucoid organism.     

Intracellular levels of adenosine triphosphate in hamster trachea organ cultures exposed toMycoplasma pneumoniae cells or membranes

Summary The amount of adenosine triphosphate (ATP) in hamster trachea organ cultures was determined with a technique based on light emission from a luciferin/luciferase/ATP reaction. The amount of ATP, expressed as ng per mg dry weight, was consistent in tracheal explants prepared from various animals and changed negligibly when explants were cultivated in vitro for several days. The amount of ATP was related directly to cellular activity and integrity in the epithelium since inactivation by heat or freeze-thaw rapidly depleted measurable ATP, and ciliary activity and ATP content were related directly. When tracheal explants were infected with 10⁵ to 10⁷ CFU of virulentMycoplasma pneumoniae cells, both ciliary activity and ATP content in the tissue dropped dramatically after approximately 5 to 8 days (up to 85% and 60% decreases, respectively). Exposure of explants to 50 to 200 μg per ml of purifiedM. pneumoniae membranes also caused significant decreases in ciliary activity and ATP. When explants were infected with attenuated or nonvirulent mycoplasmas, ciliary activity was only slightly decreased, while ATP values often rose slightly. The technology associated with the determination of ATP levels in tracheal explants should prove useful as a new, objective, analytical approach to cell viability in organ cultures. This investigation was supported in part by the National Institutes of Health (PHS Grant AI 12559), by a Biomedical Sciences Support Grant made to the University of Illinois School of Life Sciences, and by the University Research Board.     

In vitro Demonstration of Lactogenic Activity in the Mammalian Placenta

As part of a comparative study of the occurrence of placentalprolactins among mammals, studies were done to see if the placentaeof a variety of mammals produce a prolactin, and if it is produced,during what period of gestation it is demonstrable. The lactogenicactivity of baboon, sheep, chinchilla, hamster, mouse, rat,and rabbit placentae at different stages of pregnancy were examinedby organ co-culture of placental explants with prelactatingmammary tissue, as well as by the addition of placental extractsto mammary organ cultures. The lactogenic activity of humanplacental lactogen (hPL) was also examined. Mammary tissue fromnulliparous (day 11 or 12) pregnant BALB/c Crgl mice were culturedin Waymouth's synthetic medium supplimented with insulin (5µg/ml) and aldosterone (1 µg/ml). The lactogenicactivity of the baboon, sheep, chinchilla, hamster, rat, andmouse placentae was clearly demonstrable. In the rabbit, thelactogenic activity was not detectable.     

Large-Quantity Production of Chicken Embryo Tracheal Organ Cultures and Use in Virus and Mycoplasma Studies

Chicken tracheal organ cultures were made from embryos which were 19 to 20 days old. Transversely cut rings of trachea were placed in screw-capped tissue-culture tubes with Eagle's-N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) medium and incubated in roller drums. The method had advantages over other organ culture systems in that these cultures were prepared in numbers similar to conventional tissue cultures, ciliary activity was quickly and accurately evaluated, and contamination occurred less frequently than with organ cultures in petri dishes. Ciliary activity persisted for at least 1 month when the medium was changed at 5-to 7-day intervals and for 10 to 15 days without a change. Infectious bronchitis virus stopped ciliary movement, and this effect was used as a basis for titrating the virus and for determining the neutralizing capacity of immune mouse ascitic fluid. Twenty-four Mycoplasma strains were tested. Organisms of 17 strains, both avian and mammalian, multiplied in the organ cultures, and 7 strains, belonging to the species M. gallisepticum and M. mycoides var. capri, inhibited ciliary activity.     

Comparison of Ciliostasis by Mycoplasmas in Mouse and Chicken Tracheal Organ Cultures

The cilia-stopping effect of mycoplasmas of human and various animal origin in mouse and chicken tracheal organ cultures was studied. From the results in mouse tracheal organ cultures, the mycoplasma strains tested were divided into three groups: Mycoplasma pulmonis m53, M. pulmonis JB, M. pulmonis OK, M. mycoides subsp. mycoides PG1 and M. gallisepticum S6 showed a strong cilia-stopping effect; M. pulmonis PG22, M. mycoides subsp. capri PG3, M. meleagridis 19729, M. neurolyticum Type A and M. arthritidis PG6 showed a mild effect; and M. pneumoniae FH, M. salivarium Hup, M. hominis type 1-C and M. orale N-C of human origin and Acholeplasma laidlawii PG8 showed a weak effect. On the other hand, in chicken tracheal organ cultures, only M. gallisepticum S6 showed a strong effect, M. meleagridis 19729 was affected to a lesser degree, and other mycoplasma strains showed a weak or no effect. The results indicate that some murine and poultry mycoplasmas showed a cilia-stopping tendency in mouse and chicken tracheal organ cultures, respectively, while human mycoplasmas showed weak or little effect in both organ cultures. In mouse tracheal organ cultures, M. pulmonis m53 treated with heat, trypsin or formaldehyde, and the sterile filtrate of an m53 broth culture showed no cilia-stopping effect. The relationship of the pathogenicity of mycoplasmas for their natural hosts to that for cultured respiratory cells is discussed.     

Animal infectivity of Encephalitozoon cuniculi.

Rabbits, mice, rats and Rhesus monkeys were infected experimentally with a rabbit isolate of the mammalian microsporidan Encephalitozoon cuniculi. The lesions produced were typical of those occurring in spontaneous encephalitozoonosis in rabbits, mice, and rats, respectively. Viable E. cuniculi were recovered from tissues of injected animals with and without lesions. Titration of rabbit, mouse, and hamster isolates of E. cuniculi in mice and in rabbit choroid plexus cell cultures showed that the rabbit isolate was equally infectious for mice and cell cultures. Mouse and hamster isolates were less infectious for cell cultures than for mice. The results provide further evidence that the mouse, hamster, and rabbit isolates of E. cuniculi are identical.     

Mycoplasma gallopavonis in eastern wild turkeys.

Serum samples and tracheal cultures were collected from eastern wild turkeys (Meleagris gallopavo sylvestris) trapped for relocation in South Carolina (USA) during 1985 to 1990. Sera were tested for Mycoplasma gallisepticum and M. synoviae by the rapid plate agglutination and hemagglutination inhibition tests and were found to be negative. Tracheal cultures were negative for all pathogenic Mycoplasma spp., including M. gallisepticum, M. synoviae, M. meleagridis, and M. iowae. However, M. gallopavonis was isolated from every group of wild turkeys tested in 1986 to 1990. These data suggest that M. gallopavonis, which is generally considered nonpathogenic, may be a common microorganism in eastern wild turkeys.     

A tracheal culture model of respiratory tract infection withPseudomonas Aeruginosa

Summary The pathogenesis ofPseudomonas aeruginosa for the respiratory tract has been examined using hamster tracheal organ cultures. Tracheal rings prepared from male Syrian hamsters, strain LSH/LAK, were infected withP. aeruginosa for 4 h and processed at 4-h intervals for 24 h for examination by light- and electron microscopy. Tissue destruction was observed within 8 h after infection with 10⁸ colony-forming units (cfu)/ml and within 12 h after infection with 10⁴ or 10⁶ cfu/ml. Ciliated cells that contained abnormal subcellular organelles were expelled from the epithelium. By 20 h the epithelial borders were composed primarily of nonciliated cells. Transmission- and scanning electron microscopy revealed details of the cellular destruction and attachment ofP. aeruginosa to the ciliated epithelium.Pseudomonas aeruginosa causes a rapid destruction of the epithelium of hamster trachea in cultures. Hamster tracheal organ cultures have been shown to be useful in studying the pathogenesis ofP. aeruginosa for the respiratory tract. This work was supported by Grants G-430B and G-431B from the Cystic Fibrosis Foundation.     

Functional and ultrastructural effects of nontypeable haemophilus influenzae in a hamster trachea organ culture system

Summary A hamster trachea organ culture system was utilized to evaluate quantitatively the effects of a strain of nontypeableHaemophilus influenzae (NTHI) and culture supernatants of the same strain on ciliary activity. Tracheal explants were maintained in organ culture for 96 to 144 h and ciliary activity was observed daily with an inverted microscope. Explants continuously exposed to a strain of NTHI had a progressive decline in ciliary activity which was significantly lower than uninfected controls evaluated concomitantly by 48 h of exposure and thereafter. Histologic studies revealed a progressive degeneration of mucosal cells and exfoliation of ciliated cells. Scanning electron microscopy showed little adherence of NTHI to the mucosal surface. Sterile broth cultures of NTHI and supernatants of organ cultures infected with the same NTHI strain had no adverse effect on ciliary activity. Infected tracheal explants treated with ampicillin 24, 48, or 72 h after continuous bacterial challenge had no significant decline in ciliary activity compared to controls. The lack of adherence and the histologic changes observed when hamster trachea cultures were infected with NTHI suggested a toxin might mediate the damage observed. Broth and organ culture supernatants, however, produced no damage. Therefore, further studies are needed to determine the role, if any, of a toxin in the production of damage to hamster tracheal explants by NTHI. This work was supported by a Merit Review grant from the Veterans Administration and by Grant AI-19641 from the National Institute of Allergy and Infectious Diseases.     

Adsorption of Mycoplasma pneumoniae to Neuraminic Acid Receptors of Various Cells and Possible Role in Virulence

Monkey, rat, and chicken tracheal epithelial cells, as well as monkey, rat, guinea pig, and chicken erythrocytes, adsorbed firmly to colonies of Mycoplasma pneumoniae and M. gallisepticum. Colonies of M. pulmonis also adsorbed erythrocytes but with less avidity than M. pneumoniae or M. gallisepticum; unlike the latter organisms, M. pulmonis did not adsorb tracheal epithelial cells. Colonies of M. orale type 1 and M. orale type 3 adsorbed only chicken red cells. Other mycoplasma species tested, including four of human origin and one of animal origin, did not adsorb red cells or epithelial cells. M. pneumoniae and M. gallisepticum appeared to attach to erythrocytes or tracheal epithelial cells by neuraminic acid receptors on these cells, whereas M. orale types 1 and 3 and M. pulmonis seemed to utilize another type or other types of receptors. Pretreatment of red cells or tracheal epithelial cells with receptor-destroying enzyme, neuraminidase, or influenza B virus removed the adsorption receptors for M. pneumoniae. Similarly, pretreatment of M. pneumoniae colonies with neuraminic acid-containing materials prevented adsorption of erythrocytes or respiratory tract cells. The adsorption sites on M. pneumoniae were specifically blocked by homologous but not heterologous antisera. This property made it possible to study the nature of the mycoplasma adsorption sites by testing the capacity of different fractions of the organism to block the action of adsorption-inhibiting antibodies. Such studies suggested that the mycoplasma binding sites were probably lipid or lipoprotein in nature. The glycerophospholipid hapten was implicated as one such site, since this serologically active hapten blocked the action of hemadsorption-inhibiting antibodies in M. pneumoniae rabbit antiserum. The affinity of M. pneumoniae for respiratory tract epithelium, unique among the mycoplasmas that infect man, may play a role in virulence, since this type of attachment provides an unusual opportunity for peroxide, secreted by the organism, to attack the tissue cell membrane without being rapidly destroyed by catalase or peroxidase present in extracellular body fluids.     

Electron microscopic observations on ciliated epithelium of tracheal organ cultures infected with Bordetella bronchiseptica

Using mouse tracheal organ cultures, the pathogenic effect of Bordetella bronchiseptica to epithelial cells was studied by electron microscopy. The ultrastructure of epithelial cells in uninfected tracheal rings was preserved well for longer than 3 days. In mouse tracheal rings infected with graded doses (3 x 10(5) to 10(7) CFU/ml) of phase I B. bronchiseptica, the colonization in the interciliary spaces of ciliated epithelial cells was observed after a 20-hr infection period. The infected tracheal rings showed swelling of nonciliated cells as well as ciliated cells, rupture of cell membrane of cilia, swelling and disappearance of cilia, and atrophic cytomorphosis of epithelial cells. The severity of these changes occurred depending on the infection doses. These changes were essentially similar to those observed previously in the tracheal epithelia of the B. bronchiseptica-infected mice. The usefulness of this in vitro model was suggested for studying the pathogenesis of Bordetella infection.     

Mycoplasmal PID: a review of natural and experimental infections

The present report is a review of data assuming an etiological relationship between pelvic inflammatory disease (PID) and Mycoplasma hominis. Thus the organism can be isolated from the vagina/cervix more frequently in PID patients than in any other clinical group, i.e., in half to three-fourths of all such cases. One-fourth of PID patients develop a significant antibody response to M. hominis during the course of the disease. The antibody response can be detected by indirect hemagglutination tests. Grivet monkeys infected experimentally with M. hominis develop PID, predominantly parametritis; the infection seems to spread via lymphatics to the parametria. These animals develop a significant antibody response. The animals, like naturally infected women, develop a marked increase in the serum level of IgM. In tissue cell cultures of human fallopian tubes experimentally infected with M. hominis, a decrease of the mucociliary wave activity occurs. So far, few clinical data support an etiological role for Ureaplasma urealyticum in PID. In grivet monkeys, the organism does not produce PID.     

Detection of Mycoplasma ovipneumoniae and M. arginini in bighorn sheep using enrichment culture coupled with genus- and species-specific polymerase chain reaction

Mycoplasma species are of interest as possible primary pathogens in the pneumonia complex of bighorn sheep (Ovis canadensis). Previous investigations have not commonly detected low frequencies of Mycoplasma spp. from free-ranging bighorn sheep, possibly due to the fastidious and slow growth of these organisms. We developed a culture protocol that employed an average initial 3-day enrichment culture in liquid Hayflick broth in a CO(2)-enhanced atmosphere. The broth was plated to solid Hayflick medium and the cultures observed for growth for up to 30 days. Polymerase chain reaction (PCR) was performed on DNA isolated from the enrichment broth and on isolates obtained from culture using Mycoplasma genus-specific PCR assays and species-specific PCR assays for M. arginini and M. ovipneumoniae. Some cultures that grew on Hayflick plates were picked as single colonies but were mixed because two organisms may grow together and appear as a single colony. Culture and PCR tests produced similar results for M. arginini, but for M. ovipneumoniae, culture alone was less accurate than PCR. Use of genus-specific primers also may allow detection of other species in samples negative for M. arginini and M. ovipneumoniae. Two methods of transport from field to laboratory (Port-a-Cul? tubes, cryoprotectant in liquid N(2) and Fisher Transport System) gave similar results under our study conditions.     

Experimental pathogenicity of Mollicutes of bovine udder origin in hamster tracheal ring organ culture.

Hamster tracheal-ring organ culture was employed to examine pathogenic effects of 8 isolates of Mollicutes of bovine udder origin. The tested Mollicutes could be categorized into two groups: (i) Mycoplasma F-38, M. mycoides var. capri, M. bovigenitalium mixed with M. bovirhinis, and M. bovigenitalium mixed with M. bovirhinis and Mycoplasma F-38 produced significant ciliostatic effect and infiltration of neutrophils and lymphocytes in lamina propria/subepithelium, hyperplasia and desquamation of epithelial lining cells and loss of cilia; and (ii) A. laidlawii, A. axanthum, an unidentified Acholeplasma and a mixed isolate of M. bovis, M. bovigenitalium, Mycoplasma F-38 and A. laidlawii showed insignificant ciliostatic effects and produced mild histopathological lesions. This correlates with the disease causing potentials of the strains.     

Mycoplasma hominis and Trichomonas vaginalis symbiosis: multiplicity of infection and transmissibility of M. hominis to human cells

We recently reported that most Trichomonas vaginalis isolates cultured in vitro are infected by Mycoplasma hominis. In this work, we have characterized some aspects of the relationships between the two microorganisms. PCR, cultivation, and immunological methods revealed that the number of M. hominis organisms carried by T. vaginalis in culture varied from isolate to isolate, suggesting a specific multiplicity of infection. Moreover, infected T. vaginalis isolates were able to pass bacteria not only to M. hominis-free protozoa, but also to human-derived epithelial cells. The in vitro transmission of the bacterium from T. vaginalis to both uninfected parasite isolates and human epithelial cells suggests a role for T. vaginalis as a carrier of the M. hominis infection in vivo.     

Scanning electron microscopy of Mycoplasma pneumoniae on the membrane of individual ciliated tracheal cells

Cell monolayer cultures were prepared from hamster tracheal explants by a collagenase exposure and subsequent incubation in Waymouth's MAB 87/3 medium. The epithelial outgrowth occurred on glass cover slips. Cilia on the monolayers continued to beat normally after the "parent" explant was removed. Monolayer cultures infected with Mycoplasma pneumoniae had significant amounts of attachment. A morphological analysis of the attachment was conducted with scanning electron microscopy. Clusters, cocci, and filaments of M. pneumoniae all attached to the epithelial cells, but the filaments were especially common. Mycoplasmas were seen in association with both ciliated and nonciliated cell membranes. On ciliated cells, mycoplasmas were on the ciliary strands and on the cell membrane. When located immediately adjacent to or in between cilia, mycoplasmas were oriented vertically with the constricted attachment tip oriented down toward the host cell membrane. When located more than a micron away from the ciliary fibers, mycoplasmas lay horizontally along the epithelial cell membrane. The photographic data suggest that clusters or "sperules" of mycoplasmas may liberate individual mycoplasmas that attach to the cell membrane. It appears that the receptor sites for M. pneumoniae are rather uniformly distributed along the ciliated cell membrane, and are not restricted to the interciliary areas.