Mycoplasma Organisms Research Articles

Epidemiologic analysis of Mycoplasma spp isolated from bulk-tank milk samples obtained from dairy herds that were members of a milk cooperative

Objective To determine the prevalence of Mycoplasma spp in herds that were members of a milk cooperative. Design Epidemiologic study. Sample Population 267 dairy herds that were members of a milk cooperative. Procedure Bulk-tank milk samples were collected monthly during a 6-year period from all dairies in the cooperative. Samples were submitted to the cooperative's laboratory for bacterial culture for Mycoplasma spp, using direct plating. Milk samples positive for Mycoplasma organisms were speciated. Results Prevalence of positive samples varied from 1.8 to 5.8% for all species of Mycoplasma and from 1.2 to 3.1% for Mycoplasma spp known to be mastitis pathogens. One mycoplasmal species was isolated initially on 99 of 198 (50.0%) dairies, but 68 of 198 (34.3%) dairies had 2 species isolated. Mycoplasma bovis, M californicum, and M bovigenitalium were consistently isolated, but M bovis (243/499; 48.6%) was the most commonly isolated species. Acholeplasma laidlawii was more prevalent in 1989 and 1995 than other years. Mycoplasma bovigenitalium and M californicum had a seasonal distribution. Less than 50 colonies per plate were isolated for most (317/500; 63.4%) bulk-tank samples. Of the milk samples with > 100 colonies/plate, Mycoplasma bovis was isolated most frequently (73/243; 30.0%). Clinical Implications Distribution of Mycoplasma spp varied by year, number of colonies isolated per sample, season, and herd. Therefore, it may be necessary to routinely sample bulk-tank milk, and all isolates should be speciated. Culture results from milk cooperatives should be used with other monitoring information to determine the Mycoplasma status of herds. (J Am Vet Med Assoc 1997;211:1036–1038)

Atypical Pneumonia in Active Patients

Atypical pneumonias can affect brief young, otherwise healthy individuals who have close contact with one another, such as athletes in team sports. Symptoms, which often progress gradually, may mimic an upper respiratory tract infection. Mycoplasma, chlamydia, and legionella organisms, along with certain viruses, are the usual atypical pneumonia agents, and antimicrobial therapies are recommended. Because complications, though rare, can be protracted, affect athletic performance, and result in sudden death, return-to- play guidelines should be cautious and patient- and sport-specific.

Effects of interleukin-2 and cyclosporin A on pathologic features in Mycoplasma pneumonia.

To elucidate the immunopathologic mechanisms of Mycoplasma pneumonia, the effects of interleukin-2 (IL-2) and cyclosporin A (CYA) on Mycoplasma pneumonia in mice were investigated. Mice were intranasally inoculated with Mycoplasma pulmonis (M. pul) and treated with IL-2, CYA, or minocycline (MINO) every day between Days 3 and 9. They were killed at Days 7, 14, or 21 after the inoculation. Cell-mediated immunity (CMI) of the host was assessed by delayed-type hypersensitivity (DTH) to sheep red blood cells (SRBC). Peribronchial and perivascular lymphocyte cuffing and the accumulation of macrophages at the ends of bronchioles were exacerbated (p < 0.05) in IL-2-treated mice at Day 14 and reduced (p < 0.05) in CYA-treated mice at Day 7. Although the DTH responses to SRBC of saline-inoculated, IL-2-treated mice were increased at Days 7 (p < 0.01) and 14 (p < 0.05), those of M. pul-inoculated, IL-2-treated mice at Day 7 could not recover to the control level. The CMI levels of M. pul-inoculated, CYA-treated mice were decreased at Days 7, 14 (p < 0.05), and 21 (p < 0.01). Mycoplasma organisms in the lung showed the greatest decrease (p < 0.05) in MINO- and IL-2-treated mice, but increased at Day 21 (p < 0.05) in mice treated with IL-2 alone. These results suggest that the pathologic features of Mycoplasma pneumonia could be modified by the degree of host CMI.

Immunohistochemical and ultrastructural characteristics of [formula omitted] subsp. [formula omitted] in caprine abortion: A case report

Described in this study are the immunohistochemical and ultrastructural findings in a case of caprine abortion due to the experimental infection of the dam with strain GM13 of Mycoplasma capricolum subsp. capricolum . Mycoplasma antigens were seen mainly in choriallantoic trophoblasts and in the lumen of blood vessels in the allantoic membrane. Examination with an electron microscope showed that the chorioallantoic trophoblasts were filled with typical mycoplasma organisms. No other bacteria were observed in any of the samples. Our results confirm by immunohistochemical and electron microscopic techniques that Mycoplasma capricolum subsp. capricolum can cause caprine abortion and that the process can occur without premonitory signs.

A simple preparation of mycoplasmal DNA template for PCR from biological samples using effective surfactants.

To prepare mycoplasmal DNA template for PCR from biological samples rapidly and easily, surfactants which can solubilize cell membranes effectively were investigated. 3-[3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) was considered an effective surfactant. This surfactant could solubilize mycoplasma cell membrane without suppressing the polymerase reaction. In addition, proteinase K treatment played an important role in preparing mycoplasmal DNA template from a simulated biological sample. It was therefore considered that a combination of proteinase K- and CHAPS- added lysis buffer would be more useful in preparing mycoplasmal DNA template. We could detect PCR products by using the lysis buffer with a simulated lung emulsion sample containing mycoplasma organisms at 10(4) CFU per gram.

Occurrence of keratoconjunctivitis apparently caused by Mycoplasma gallisepticum in layer chickens.

Natural cases of keratoconjunctivitis, apparently caused by Mycoplasma gallisepticum (MG), in layer chickens are described. The disease occurred in a commercial flock consisting of 36,000 pullets (Babcock), first appearing around 30 days of age. Clinically, affected chickens showed unilateral or bilateral swelling of the facial skin and the eyelids, increased lacrimation, congestion of conjunctival vessels, and respiratory rales. Some of the severely affected chickens closed their eyes. The morbidity reached 27.8%, and it was estimated that approximately 10% died from reduced feed intake due to impaired vision. Ten 70-day-old chickens with clinical diseases were examined for lesions. There was acute to subacute keratoconjunctivitis in all of the chickens, and some exhibited laryngitis. Adherence of mycoplasma organisms to epithelial cells of the conjunctiva, cornea, and larynx was frequently observed. These organisms had an ultrastructure characteristic of MG and showed a positive reaction with rabbit polyclonal antibodies against the S6 strain of MG by immunohistochemical analysis. MG was isolated from tissue homogenates of the eyelids and tracheas of the affected chickens. Many of the chickens had atrophic bursae, and infectious bursal disease virus antigens were detected in necrotic bursal follicles by immunostaining. Therefore, immunosuppression due to infectious bursal disease was implicated in the pathogenesis of keratoconjunctivitis in the present cases.

Ureaplasma urealyticum and chronic lung disease.

Neonatal lower respiratory tract colonisation with mycoplasma organisms was examined for an association with chronic lung disease. Ureaplasma urealyticum colonised 9/70 (13%) infants less than 1500 g. Seven (78%) colonised and 33 (54%) non-colonised infants developed chronic lung disease. Logistic regression analyses revealed early gestation, but not mycoplasma colonisation, was independently associated with chronic lung disease.

Species-specific oligonucleotide probes complementary to 16S rRNA of Mycoplasma gallisepticum and Mycoplasma synoviae

Mycoplasma gallisepticum and M synoviae are important avian pathogens causing respiratory diseases which result in great economic losses in poultry farming. Two oligonucleotide probes, complementary to the variable region V8 of 165 rRNA from the avian mycoplasmas M gallisepticum and M synoviae have, therefore, been designed and used in direct filter hybridisation experiments. Both probes gave strong hybridisation signals with their homologous targets, whereas no cross-hybridisations were obtained with any of the other avian mycoplasmas tested. It was possible to detect 2–3 × 10 4 mycoplasma organisms by direct filter hybridisation experiments with radiolabelled probes. The probes were also used to analyse several laboratory strains and field isolates of M gallisepticum and M synoviae with complete agreement between the probe technique and the other methods used for species determination. Atypical M gallisepticum strains also gave strong hybridisation signals with the M gallisepticum specific probe.

Community-acquired pneumonia

The optimal approach to management of community-acquired pneumonias is evolving. Newer organisms are recognized as common etiologic agents while advanced molecular techniques are being applied successfully to microbiologic diagnosis. Newer antimicrobial agents with activity against both intracellular pathogens (Legionella, Chlamydia, and Mycoplasma organisms) and typical pathogens (pneumococci, and Haemophilus organisms) are being evaluated. The numerous research reports published in the past year show that major advances can be anticipated for improved diagnosis and treatment of community-acquired pneumonia.

Detection of mycoplasma by DNA staining at the ultrastructural level

The Feulgen-like reaction with the osmium ammine-B complex was employed for the detection of mycoplasma infection in cell cultures used for research and biotechnological purposes. General morphology of mycoplasma in ultrathin sections and after negative staining were also investigated. The ultrastructure of the various mycoplasmas exhibited characteristic features previously described by other authors. Sometimes it was difficult to distinguish mycoplasma organisms from cell surface projections. In sections of culture cells infected by mycoplasmas, stained with osmium ammine-B, only cell nuclei DNA and mycoplasma DNA were intensely contrasted. DNA in mycoplasma could be distinguished from cellular DNA because of its filamentous aspect; the latter appears in granular form. The difference of DNA aggregation in prokaryotic and eukaryotic chromatins could be due to the relative ratio of protein to DNA, which in prokaryotes is about 10 times less than in eukaryotes. The presence of mycoplasma DNA inside the cytoplasm of cultured cells has not been observed in samples studied during this work. Application of Feulgen-like osmium ammine-B reaction for specific DNA staining at the ultrastructural level is a sensitive and relatively rapid technique for direct examination of mycoplasmas in cell cultures and tissues infected by these microorganisms.

Granular vulvovaginitis (GVV) in Sheep Experimentally Induced with Mycoplasma ovine/caprine Serogroup 11

Kumar D., P. P. Gupta, J. S. Rana, H. S. Banga: Granular Vulvovaginitis (GVV) in Sheep Experimentally Induced with Mycopiasma Ovine/Caprine Serogroup 11. Acta vet. Brno, 61, 1992: 241-249. To have insight into the pathogenicity of Mycoplasma ovine/caprine serogroup 11 for female genital system of sheep, 8 apparently healthy lambs were taken. Two ml of M. ovine/caprine serogroup 11 (2-D) culture containing 6 x 108 CFU (colony forming units) ml-l was inoculated into lightly scarified vulvovaginal canal of 7 lambs, while 2 ml of sterile mycoplasma broth was similarly inoculated in one lamb, which served as control. All the infected lambs exhibited swelling of vulva along with mucus discharge from next day post-inoculation (DPI), and this persisted up to 10-12 DPI. The experiment was of 70 d duration during which one lamb was sacrificed at every 10 d interval. The control lamb was also sacrificed on 70th d. Grossly, in the vulva and vagina, congestion, oedema and fine nodules were discernible by 10 DPI. Microscopically, all the infected lambs developed lymphocytic vulvitis, vaginitis, cervicitis, and endometritis, which aggravated with the increase in DPI. M. ovine/caprine serogroup 11 could be re-isolated from vaginal swabs of all the infected lambs up to 70th DPI. The findings reported here indicate that M. ovine/ /caprine serogroup 11 is pathogenic for female genital tract of sheep as it causes granular vulvovaginitis (GVV), and it is the first description of the pathologic features of GVV in sheep induced by this organism. Vulva, vagina, cervix, uterus, lymphoid follicles, GVV, sheep Mycoplasma ovine/caprine serogroup 11 was first isolated from an outbreak of granular vulvovaginitis (GVV) in sheep in Australia (Cottew et aI. 1974). Subsequently, this organism was isolated from spontaneous cases of GVV in sheep in India (Tiwana 1982). Despite its association with spontaneous cases of vulvovaginitis in sheep, its pathogenicity to female genital tract and its role in producing GVV has not been proven experimentally. Therefore, the present work was undertaken to assess its pathogenicity and to study the sequential pathological changes in. the female genital tract of sheep infected with this organism. Materials and Methods Experimental animals Eight female lambs aged 6-8 months were kept under observation for 1 week before starting the experiment. During this period, no mycoplasma organisms or any grossly visible vulvar/vaginal lesions were detected and all the animals were adjudged healthy.

The Fluoroquinolones

The fluoroquinolone class of antibiotics promises to become as diverse and as important as beta-lactam agents. The fluoroquinolones inhibit bacterial DNA gyrase and are bactericidal. All fluoroquinolones have potent activity against most gram-negative bacteria; ciprofloxacin is the most active against Pseudomonas aeruginosa. Activity against gram-positive organisms is variable; methicillin-resistant Staphylococcus aureus has acquired resistance to the fluoroquinolones at an alarming rate. Currently available quinolones do not have, but new quinolone agents likely will have, substantial activity against anaerobic bacteria. Some quinolones are also active against Mycobacterium, Chlamydia, and Mycoplasma organisms. All fluoroquinolones have excellent absorption after oral administration; however, this process can be impaired by the presence of aluminum- or magnesium-containing antacids and by zinc, iron, or calcium supplements. Ciprofloxacin is also available for intravenous use. Although most fluoroquinolones do not achieve adequate cerebrospinal fluid levels, penetration into other tissues is excellent. Dosage adjustments for renal and hepatic dysfunction vary among the quinolones. Although side effects are rare, concomitant use of caffeine or theophylline with some quinolones may cause toxicity to the central nervous system. Because they may affect the development of cartilage, all fluoroquinolones are contraindicated in children, adolescents, and pregnant or breast-feeding women.

Identification of Mycoplasma incognitus infection in patients with AIDS: an immunohistochemical, in situ hybridization and ultrastructural study.

Monoclonal antibodies (Mabs) were developed against antigens from a pure culture of Mycoplasma incognitus grown in modified SP-4 medium. All the Mabs obtained were shown to react only with M. incognitus, and not with other species of human mycoplasma. The Mabs identified M. incognitus immunohistologically in thymus, liver, spleen, lymph node, or brain from 22 patients with AIDS, as well as in 2 placentas delivered by patients with AIDS. Using an 35S-labeled DNA probe specific for M. incognitus and in situ hybridization technique, we also identified M. incognitus-specific genetic material in these tissues. Furthermore, ultrastructural studies of the specific areas of tissues which were highly positive for M. incognitus antigens revealed characteristic structures of mycoplasma organisms. These mycoplasma-like particles could be identified intracellularly and extracellularly. Histopathology of the tissues infected by M. incognitus varied from no pathological changes to fulminant necrosis with or without an associated inflammatory reaction. M. incognitus, a novel pathogenic mycoplasma, was cytopathic and cytocidal.

Short-term Exposure to Nitrogen Dioxide Enhances Susceptibility to Murine Respiratory Mycoplasmosis and Decreases Intrapulmonary Killing ofMycoplasma pulmonis

In C57BL/6N and C3H/HeN mice known to be free of all murine pathogens and matched for age, sex, microbiologic, and environmental factors, exposure to NO2 for 4 h prior to exposure to infectious aerosols of Mycoplasma pulmonis resulted in potentiation of murine respiratory mycoplasmosis (MRM). In the C57BL/6N mice, NO2 increased the incidence of death, incidence of gross lung lesions, and incidence of microscopic lung lesions, but did not increase the incidence of infection in the lungs. Nitrogen dioxide affected the C3H/HeN mice (a strain known to be more susceptible than the C57BL/6N strain to MRM) similarly, with the exception that the incidence of death and microscopic lesions were not affected in this strain at the concentrations of M. pulmonis used. Exposure to the oxidant also increased the severity of microscopic lesions and the numbers of Mycoplasma organisms in the lungs of both mouse strains. Thus, NO2 appeared to affect host lung defense mechanisms responsible for limiting the extent of infection. The NO2 exposure level required to produce potentiation varied with the genetic background of the host, the number of Mycoplasma organisms administered, and the end point measured. In further experiments in C57BL/6N mice, exposure to 5 or 10 ppm of NO2 for 4 h prior to infection with aerosolized, radiolabeled M. pulmonis reduced clearance of these organisms from the lungs over a 72-h time period. Nitrogen dioxide exposure did not change the rate of physical removal of Mycoplasma organisms from the lung. Reduced clearance was due to impaired intrapulmonary killing of Mycoplasma organisms in NO2-exposed mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of tiamulin on antipyrine kinetics in chickens.

Tiamulin {14-deoxy-14[(2-diethylaminoethyl)-mercaptoacetoxy]-mutilin} hydrogen fuma-rate is a semisynthetic antibiotic. It is clinically effective against pathogenic mycoplasma organisms, e.g. Treponema hyodysenteriae, a large number of Gram-positive and some Gram-negative organisms (Drews et al., 1975). Tiamulin has been shown, under controlled laboratory conditions (Laber & Schutze, 1975; Baughn et al., 1978) and against natural infection under field conditions (Stipkovits et al., 1977), to control respiratory tract infections effectively involving Mycoplasma galliseptkum in chickens and turkeys. It has also been demonstrated that tiamulin could be used as an aid in the control of avian coccidiosis produced by Eimeria acervulina and E. tenella in broilers chickens (Cruthers et al., 1980).

Relevance of soluble cytotoxic factors generated by mycoplasma-contaminated targets to natural killer cell-mediated killing

The possible involvement of soluble cytotoxic factors (SCF) in the lytic mechanism of natural killer (NK) cell-mediated cytotoxicity (CMC) was investigated. Tumor target cells (TC) were examined for their ability to stimulate the release of SCF from human peripheral blood lymphocytes (PBL). SCF were detected when effector cells (EC) were stimulated with mycoplasma-contaminated (MC)-TC and with concanavalin A (Con A), but mycoplasma-free (MF)-TC were unable to induce SCF. Pretreatment of EC with beta-interferon augmented the production of SCF with MC-K562 and Con A, but not with MK-K562. However, MF-K562 once infected with the culture supernatant from MC-K562, as well as mycoplasmas concentrated from culture supernatant, were able to induce SCF. Only Con A was capable of generating cytotoxic supernatants from EC that had been inactivated with MF-K562 and from EC depleted of Leu 11b positive cells. Moreover, when EC were evaluated for NK activity after coculture with MF-K562, their ability to kill fresh MF-K562 in the standard NK-CMC assay was greatly reduced. This loss of lytic potential was not accompanied by SCF release. Collectively, these data suggest that mycoplasma organisms play a crucial role in the induction of human SCF, and the implication that SCF, including NK cytotoxic factors (NKCF), are the lytic mediators in NK-CMC needs to be re-evaluated.

Indirect visualization of hematopoietic colony-forming stem cell (CFUs) as a possible target cell for Mycoplasma arthritidis.

Utilizing specific rabbit antiserum against M. arthritidis together with complement, a portion of the marrow 7-day CFUs population of CBA mice infected with live mycoplasma organisms 1 day previously was shown to be inactivated. These cells might therefore be considered as candidate target cells for M. arthritidis. 11-day CFUs were unaffected by similar treatment.

A "lymphokine-like" soluble product that induces proliferation and maturation of B cells appears in the serum-free supernatant of a T cell hybridoma as a consequence of mycoplasmal contamination.

The serum-free supernatant of a cloned murine T cell hybridoma supports the proliferation and maturation to Ig secretion of purified B cells (mu+ cells) from BALB/c nu/nu mice, but has no effect on the proliferation of nylon wool-selected BALB/c nu/+ splenic T cells. Although the supernatant activates B cells without co-stimulation, it synergizes with anti-mu for the proliferative response. The induction of B cell proliferation and maturation to Ig secretion is directly related to contamination of the hybridoma by Mycoplasma hyorhinis. Hybridoma cells freed of mycoplasma by detergent treatment fail to produce active supernatant, and reinfection of the treated cells reconstitutes the activity. Furthermore, deliberate infection of a mycoplasma-free unrelated T cell hybridoma, as well as the monocytic cell line P388D1, results in the production of supernatants with B cell proliferating activity. Mycoplasma organisms isolated from the supernatant induce B cell proliferation without subsequent maturation to Ig secretion. Gel filtration chromatography of the supernatant from mycoplasma-contaminated hybridoma cells yields two peaks of activity. The first peak, found at the exclusion limit of the gel, results in B cell proliferation without maturation and may be attributed to mycoplasma organisms. The second peak (average m.w. 90,000) results in B cell proliferation as well as differentiation to Ig secretion. A "lymphokine-like" soluble product released by Mycoplasma hyorhinis is most likely responsible for this B cell activation, because fractionation of the supernatant from deliberately contaminated P388D1 cells gives essentially the same results, and gel filtration of mycoplasma-free supernatants does not generate any active fractions. The possibility should be considered that mycoplasma-derived soluble products may be among the many factors controlling in vitro B cell growth and maturation.

Acridine orange staining for diagnosis of Mycoplasma bovis infection in cow milk

Mycoplasma organisms were readily recognized in samples of milk or udder secretions from cows with clinical Mycoplasma bovis mastitis when these samples were stained with 0.01% acridine orange at pH 3.0. Samples could be stored at -4 degrees C for several days or subjected to repeated freezing and thawing without loss of staining or fluorescence properties. Use of this procedure in diagnostic laboratories on suspect samples from cows with clinical mastitis could hasten inauguration of control measures against this highly contagious disease by several days; however, definitive diagnosis still requires standard culture methods.

Induction of NKCF-like activity in mixed lymphocyte-tumor cell culture: direct involvement of mycoplasma infection of tumor cells.

Co-culture of CBA/J spleen cells and certain lines of YAC-1 stimulators resulted in the appearance of NKCF-like activity in 24- to 48-hr supernatants. Numerous other in vitro cell lines were effective stimulators of this splenic cytotoxic factor (SCF). The cells participating in SCF production were absent from normal thymocytes and were present in BALB/c nu/nu spleen, were nonadherent, asialo GM1+, and bore low levels of Thy-1.2. SCF could mediate lysis of certain NK-sensitive tumor targets in an 18-hr 51Cr-release assay. However, the induction of SCF was not correlated with the ability of a particular cell line to be lysed by NK cells, but showed an absolute correlation with the presence of mycoplasma contamination in cultured tumor cell lines. Mycoplasma negative cell lines, including an uninfected but NK-sensitive subline of YAC-1, were unable to induce SCF. Decontamination of mycoplasma-infected lines with antibiotics or by passage through syngeneic mice abrogated the ability of infected tumor cells to stimulate SCF. The ability to induce SCF could be restored by reinfection with mycoplasma. Tumor cell-free supernatants from contaminated cultures were mitogenic for CBA spleen cells and could themselves induce SCF activity in spleen cell supernatants. SCF production and the agent responsible could be removed by passing such supernatants through 0.1-micron filters. The organism apparently responsible for SCF induction from CBA spleen cells was typed and found to be Mycoplasma orale, a nonfermentative, arginine-dependent, common tissue culture contaminant. About 50 to 60% of SCF activity could be removed by 0.1-micron filters, suggesting that SCF is composed of two components: mycoplasma organisms themselves and a soluble cytotoxic factor produced in response to mycoplasma.