McCoy Cells Research Articles

Detection of aerolysin gene in Aeromonas strains isolated from drinking water, fish and foods by the polymerase chain reaction

A polymerase chain reaction (PCR) technique was used to assay the presence of the aerolysin gene in a total of 89 Aeromonas hydrophila and A. sobria strains isolated from drinking water, fish and foods. These strains were also characterized for the production of virulence factors such as haemolysin, protease and cytotoxin. The primers used in the PCR targeted a 209-bp fragment of the aer gene coding for the β-haemolysin and detected template DNA only in haemolytic A. hydrophila strains. The cell-free culture supernatants of these aerolysin-positive A. hydrophila strains were also cytotoxic to the HeLa and McCoy cells. The haemolytic A. sobria and non-haemolytic A. hydrophila were consistently negative in the PCR assay. Primer specificity was determined in the PCR by using a control haemolytic Escherichia coli, Streptococcus pyogenes and a restriction endonuclease assay. The PCR clearly identified the aerolysin-producing strains of A. hydrophila and may have application as a rapid species-specific virulence test.

Detection of Chlamydia trachomatis in semen by antibody-enzyme immunoassay compared with polymerase chain reaction, antigenenzyme immunoassay, and urethral cell culture

To compare the results obtained by four different techniques for the detection of Chlamydia trachomatis in the male genital tract. Prospective study. Andrology unit of a university hospital. Male infertility patients. Analysis of semen samples and urethral swabs for the presence of C. trachomatis by recombinant antibody-enzyme-linked immunosorbent assay (rELISA), polymerase chain reaction (PCR), antigen-enzyme immunoassay (EIA) and McCoy cell culture. Detection of C. trachomatis. In 57 of 205 semen samples (27.8%) immunoglobulin A-antibodies against C. trachomatis were found. In contrast, only 1 of 56 semen samples (1.8%) was positive for C. trachomatis-DNA by PCR, only 1 of 139 semen samples (0.7%) was positive by antigen-EIA, and only 4 of 173 urethral swabs (2.3%) grew C. trachomatis in cell culture. The discrepancy of positive results found by the antibody-rELISA and direct methods for the detection of C. trachomatis indicates successful eradication of the microorganism in > 90% of antibody-positive men. Therefore, detection of antibodies against C. trachomatis in seminal plasma appears to be of limited diagnostic value.

Detection of Chlamydia psittaci (Ovis) antigen in tissue sections and McCoy cells using streptavidin-biotin and the imagen ™ staining method

Two procedures for identifying chlamydial antigen in infected McCoy cells and tissue sections are described. Both the streptavidin-biotin method and the IMAGEN test clearly detected chlamydial antigen in ovine placental tissue sections and in infected McCoy cells before they could be detected by conventional stains. The streptavidin-biotin method is lengthy, but specific and sensitive, and the slides can be kept indefinitely for later examination. By contrast, the IMAGEN test is a single step procedure which requires fluorescent microscopy and slides can only be kept for up to 1 week before examination.

Comparison of the ToxA test with cytotoxicity assay and culture for the detection of Clostridium difficile-associated diarrhoeal disease

Stool samples (355 from 350 patients) were examined in a new commercial assay, the ToxA test, for the rapid diagnosis of Clostridium difficile-associated diarrhoea. The results were compared with direct assay of cytotoxin in McCoy cell tissue culture, and detection of toxigenic C. difficile by culture and cytotoxin testing of isolates. Discordant results were resolved by consultation of clinical records. Test sensitivities were 84.6% for the ToxA test, 78.5% for the direct cytotoxicity assay and 95.4% for culture. The specificity was 100% for all the tests. The ToxA test is a rapid and reliable alternative for the detection of toxigenic C. difficile where tissue culture facilities are unavailable.

Selective translocation of annexins III, IV, and V during intracellular redistribution of Chlamydia trachomatis serovar L2 in HeLa and McCoy cells.

Selective translocation of annexins III, IV, and V during intracellular redistribution of Chlamydia trachomatis serovar L2 in HeLa and McCoy cells.

In vitro activity of dirithromycin against Chlamydia trachomatis

Dirithromycin is a new macrolide antibiotic with an active metabolite, erythromycylamine. We evaluated the in vitro activities of both drugs against 16 isolates of Chlamydia trachomatis and compared them with that of doxycycline. In vitro susceptibility testing was performed with McCoy cell monolayers. The MIC was defined as the lowest concentration of antibiotic without inclusions. The MBC was defined as the lowest concentration of antibiotic yielding no inclusions after passage onto 24-h-old antibiotic-free McCoy cell monolayers. Dirithromycin and erythromycylamine appeared to be equally effective against these 16 strains of C. trachomatis (MIC for 90% of strains tested, 1 mg/ml; MBC for 90% of strains tested, 2 micrograms/ml). Both were less active than doxycycline (MIC for 90% of strains tested, 0.06 micrograms/ml; MBC for 90% of strains tested, 0.12 micrograms/ml). The combination of dirithromycin and erythromycylamine appeared to be additive.

Lipopolysaccharide in cells infected by Chlamydia trachomatis.

In view of the controversy concerning the expression of chlamydial lipopolysaccharide (LPS) in the eukaryotic host cell, the presence of this molecule was examined in three cell types which were experimentally infected with Chlamydia trachomatis serotype E. LPS was detected in the McCoy cell line, human endometrial epithelium and human amniotic epithelium with two monoclonal antibodies. The appearance and distribution of LPS at the host cell surface during the chlamydial developmental cycle and its transmission to neighbouring cells were examined by immunofluorescence microscopy after air drying of the host cells. LPS distribution was not uniform; it was first observed on regions of the cell surface in close proximity to the chlamydial endosome (inclusion). Soon after, the antigen was also detected at points of contact with neighbouring uninfected cells. Immunofluorescent plaques of host cells contaminated with LPS were thus formed in the vicinity of infected cells. These plaques increased in size over 2 d before becoming smaller as host cell lysis occurred. The major outer-membrane protein (MOMP) was not visualized on the host cell surface after air drying. No cell-surface LPS antigen was observed in live cells or those fixed in formaldehyde without air drying. Conventional methanol fixation and immunolocalization of LPS and MOMP in parallel infected cultures stained these antigens within inclusions in the expected fashion. Radio-immunoassays were used to quantify LPS in confluent McCoy cell monolayers during the chlamydial developmental cycle. Cell-surface-associated and inclusion-associated LPS, measured by direct binding of 125I-labelled anti-LPS monoclonal antibodies to air-dried or methanol-fixed monolayers respectively, increased for up to 3 d then declined.(ABSTRACT TRUNCATED AT 250 WORDS)

Chlamydia trachomatis genitourinary infections: laboratory diagnosis and therapeutic aspects. Evaluation of in vitro and in vivo effectiveness of azithromycin.

Chlamydia trachomatis (C.t.) cell culture represents a sensitive method for the diagnosis of chlamydial infection and the only one which makes it possible to determine the susceptibility of an isolate to antibiotics so that an appropriate drug can be selected for individual treatment. In 11 patients, affected by urethroprostatitis and suspected of treatment failure with standard drug regimens, either due to lack of compliance with therapy or antibiotic resistance, C.t. was isolated in McCoy cell culture from urethral swabs, after prostatic massage. The in vitro activity of azithromycin against these isolates and the in vivo efficacy of the drug in the patients treated with a single 1 g dose have been evaluated. All the C.t. strains tested were susceptible to the action of azithromycin (MIC range 0.125-1.0 microgram/ml). Bactericidal values were one dilution higher (MBC range 0.25-2.0 microgram/ml). These in vitro results are consistent with clinical observations as all the patients treated had negative culture at a 4-week follow-up visit.

Expression of recombinant DNA introduced into Chlamydia trachomatis by electroporation

Electroporation was used to introduce DNA into the elementary bodies of the obligate parasitic bacterium Chlamydia trachomatis. The source of DNA for these experiments was the chimeric plasmid pPBW100, which was constructed from the well-characterized 7.5-kb plasmid of C. trachomatis and the Escherichia coli plasmid pBGS9. To select directly for C. trachomatis carrying pPBW100, an in-frame gene fusion between the chlamydial promoter P7248 and a promoterless chloramphenicol acetyltransferase (cat) cassette was incorporated into the plasmid. After infection of McCoy cells with electroporated elementary bodies containing pPBW100, the following were observed: (i) the plasmid DNA was detected inside the chloramphenicol-resistant chlamydial inclusions by in situ and Southern hybridization analyses; (ii) both physical and biochemical evidence showed that chloramphenicol acetyltransferase was synthesized by the electroporated C. trachomatis; (iii) expression of P7248::cat was developmentally regulated and occurred during the early stages of chlamydial reticulate body development; and (iv) although the expression from P7248::cat was mainly transient, there were rare instances where chloramphenicol-resistant C. trachomatis were observed after four passages.

Re-examination of the McCoy cell line for confirmation of its mouse origin: karyotyping, electron microscopy and reverse transcriptase assay for endogenous retrovirus

Background: The McCoy cell line originally derived from human synovial fluid in 1955, has been later found useful for cultivation of Chlamydia trachomatis. This cell line has been subcultured and exchanged between laboratories for many years. In recent years, the McCoy cell line has been widely used in many clinical diagnostic laboratories and has been supplied through commercial companies for the isolation and identification of Chlamydia trachomatis from clinical specimens. Objectives: Since retrovirus-like particles have been observed in McCoy cells and the species of origin of the currently used cell line has not been adequately documented, further characterization of McCoy cell line obtained from commercial sources was carried out. Study design: This study includes karyotypes analysis using G-banding for the confirmation of species origin of McCoy cells, electron microscopy for examination of virus particles associated with the cells and biochemical assay for reverse transcriptase activity for detection of retrovirus. Results: Our results showed by karyotype analysis that McCoy cells are of mouse origin. Electron microscopic examination revealed the presence of endogenous retrovirus type-A and type-C virions. Biochemical assays of culture supernatant fluids from McCoy cells detected reverse transcriptase activity which required Mg 2+ ions. Conclusions: The present study has confirmed that McCoy cells currently used by many laboratories are mouse cells, not the original McCoy cells derived from human cells. Laboratory workers should be aware of the presence of endogenous murine retrovirus in this cell line and appropriate precautions should be taken.

The treatment and follow up of adult chlamydial ophthalmia.

Sixty two patients diagnosed as having adult chlamydial ophthalmia were treated with oral doxycycline and roxithromycin in association and tetracycline eye wash for 2 weeks. Chlamydial ophthalmia was diagnosed by laboratory detection of the micro-organism in ocular specimens using direct immunofluorescent monoclonal antibody staining for Chlamydia trachomatis, chlamydial culture in cycloheximide treated McCoy cells, and Giemsa staining. An immunoenzymatic method for detection of specific IgG and IgA in patients' serum was used as an additional test to confirm the diagnosis. All patients were reexamined 3 weeks after completing their course of antibiotics and in the case of persistent infection a further course of treatment was given. With this treatment regimen 48 out of 62 patients (77.4%) were cured after three courses. Because of the risks of an inadequate response to therapy, we recommend a proper post-treatment follow up in all patients with chlamydial eye infections.

A randomized, prospective trial comparing amoxicillin and erythromycin for the treatment of Chlamydia trachomatis in pregnancy

OBJECTIVE: Our purpose was to evaluate the efficacy of amoxicillin as an alternative therapy to erythromycin for the treatment of cervical chlamydial infections during pregnancy. STUDY DESIGN: A randomized, prospective trial of two treatment regimens for Chlamydia trachomatis was performed in a cohort of pregnant women enrolled for care in an inner-city, university-based prenatal clinic, with an alternate-therapy crossover arm for primary treatment failures. Pregnant women diagnosed with chlamydial infection by McCoy cell culture of cervical swabs were assigned to receive either 500 mg of amoxicillin three times daily or 500 mg of erythromycin four times daily for 7 days. Patients' partners were treated with doxycycline. Compliance information was obtained by a standardized questionnaire at a posttherapy follow-up visit. Patients with positive follow-up cultures were crossed over into the alternate treatment arm and recultured at a later visit. RESULTS: During the study period 74 women consented to participate in this trial; 36 were treated with amoxicillin and 38 with erythromycin. Initial cure rates of 82.3% (28/34) for the amoxicillin group and 84.6% (27/32) for erythromycin were obtained before crossover (p = 0.91); four patients in each group were lost to follow-up. Overall cure rates after crossover were 84.6% (33/39) for amoxicillin and 84.2% (32/38) for erythromycin (p = 0.83). In the amoxicillin group 12.8% of patients reported side effects compared with 31.6% treated with erythromycin (p = 0.09), although seven erythromycin-treated patients compared with none of those in the amoxicillin arm stopped therapy because of side effects (p = 0.02). CONCLUSION: Amoxicillin offers a reasonable alternative to erythromycin for the treatment of Chlamydia trachomatis in pregnancy, on the basis of both cure rates and patient compliance. (AM J OBSTET GYNECOL 1994;170:829-32.)

Antichlamydial activity of cervical secretion in different phases of the menstrual cycle and influence of hormonal contraceptives

Cervical secretion from three groups of asymptomatic women, either being oral contraceptive (OC) non-users or users (ethinylestradiol plus desogestrel or levonorgestrel), was tested for its capacity to prevent Chlamydia trachomatis, serotype I, from forming inclusions in cycloheximide-treated McCoy cells. The non-user groups were comprised of 12 women from whom cervical secretion was collected twice weekly during the menstrual cycle and 15 women from whom cervical secretion was collected once or twice weekly. The OC users included 66 women from whom cervical secretion was collected once or twice during their menstrual cycle. Cervical secretion from the nonusers produced a decrease in the chlamydial inclusion count by 70%–90% during the first 3 weeks of the menstrual cycle, as compared with the fourth and fifth week when the reduction was 56%–68% (p < 0.001). Secretion from the OC users showed a more effective decrease in the inclusion count during the first 3 weeks of the menstrual cycle, as compared with samples obtained at the fourth and fifth weeks, i.e. 15%–35% vs. 20%–25% (p < 0.001). Cervical secretion of the non-users as compared to the users produced a significant decrease in the inclusion count, viz. 70%–90% vs. 15%–35% (p < 0.001) during the first 3 weeks as compared with 56%–68% vs. 20%–25% (p < 0.001) in the fourth or fifth week. The study suggests that natural resistance to genital chlamydial infection can differ during the menstrual cycle and it may be influenced by oral contraceptive use.

Detection of Chlamydia trachomatis by the polymerase chain reaction in the cervices of women with acute salpingitis

OBJECTIVE: Our objective was to determine whether an increased prevalence of Chlamydia trachomatis could be detected by the polymerase chain reaction as opposed to culture in the cervices of women with acute salpingitis. STUDY DESIGN: Endocervical samples from 15 women with laparoscopy-verified acute salpingitis and 20 women seeking medical help for conditions other than pelvic pain were tested for Chlamydia trachomatis with the polymerase chain reaction. The oligonucleotide primer pairs used were specific for a 144 bp region of the major outer membrane protein that contained a single Eco RI endonuclease cleavage site. The detection of a 144 bp band that was cleaved by Eco RI to a 103 bp band denoted a Chlamydia trachomatis -positive sample. Cervical samples were cultured for Chlamydia trachomatis with the use of McCoy cells. The lymphocyte proliferative response to Chlamydia trachomatis elementary bodies was also determined. RESULTS: Nine of the 15 women (60%) with salpingitis had positive results when tested with the polymerase chain reaction for cervical Chlamydia trachomatis . Only two of these women (13%), both of whom had positive results when tested with the polymerase chain reaction, had cultures that were positive for Chlamydia trachomatis ( p Chlamydia . Those women plus two women with unexplained recurrent abortions had positive polymerase chain reaction test results for Chlamydia trachomatis . A lymphocyte proliferative response to Chlamydia trachomatis was detected in five of eight women with salpingitis, as well as three of the other four patients, all of whom had positive polymerase chain reaction test results; lymphocytes from the remaining women were unresponsive. Follow-up cervical samples were obtained 4 to 6 months after treatment from six of the patients with salpingitis who had positive polymerase chain reaction test results; at that time five had negative polymerase chain reaction test results for Chlamydia trachomatis . CONCLUSION: The polymerase chain reaction appeared to be more sensitive and more rapid than culture in detecting Chlamydia trachomatis in the cervices of women with acute salpingitis. This assay may be of value for the early diagnosis of chlamydial infections.

Clinical evaluation of an automated enzyme-linked fluorescent assay for the detection of chlamydial antigen in specimens from high-risk patients

A fully automated enzyme-linked fluorescent assay (ELFA) on the Vitek Immunodiagnostic Assay System (VIDAS CHL) was evaluated for the detection of chlamydial antigen in specimens from symptomatic and asymptomatic high-risk patients. The results were compared with those from McCoy cell culture and Chlamydiazyme with a blocking assay. False-positive VIDAS specimens were centrifuged and the pellet stained with direct fluorescent antibody (DFA). Of the 158 urine specimens, 52 (33%) were infected by Chlamydia trachomatis. The sensitivity and specificity of the VIDAS when compared with cell culture, DFA, and Chlamydiazyme were 75% and 96%, respectively, for urine specimens while the predictive value of a positive (PVP) and a negative (PVN) were 91% and 88%, respectively. Of the 245 urethral swabs, 75 (31%) were considered positive. The sensitivity and specificity were 71% and 92%, respectively, and the PVP and PVN were 80% and 87%, respectively. The sensitivity and specificity on the 108 cervical swabs, 22 of which were positive, were 95% and 95%, respectively, and PVP and PVN were 88% and 99%, respectively. Compared with Chlamydiazyme, the VIDAS was more sensitive on specimens from female patients and urine specimens, but less sensitive on urethral specimens from male patients.

Selective translocation of annexins during intracellular redistribution of Chlamydia trachomatis in HeLa and McCoy cells

When Chlamydia trachomatis elementary bodies enter epithelial cells, they occupy membrane-bound vesicles that aggregate with each other in a calcium-dependent manner but that do not fuse with lysosomes. As members of the annexin family of calcium- and membrane-binding proteins have been implicated in mediating calcium-regulated membrane traffic during endo- and exocytosis, we examined the intracellular localization of certain annexins following invasion of HeLa and McCoy cells by C. trachomatis serovar L2. Immunofluorescence staining with a panel of polyclonal antibodies against five human annexins revealed that annexins III, IV, and V translocate within the cytoplasm to the proximity of intracellular chlamydiae whereas the distribution of annexins I and VI was unaffected. The distinct distribution of annexins I and III was further analyzed by confocal microscopy, which revealed an intimate association between chlamydial aggregates or inclusions and annexin III. Confocal microscopy also confirmed the nonassociation of annexin I with chlamydial aggregates. Depletion of intracellular Ca2+ did not prevent association of annexin III with individual elementary body-containing endosomes but did prevent formation of chlamydial aggregates and translocation of annexin III. Furthermore, chloramphenicol-treated cells also showed association between chlamydial aggregates and annexin III, indicating that the annexins are of host cell origin. These data suggest that certain cytosolic annexins may be involved in the Ca(2+)-dependent aggregation and fusion of chlamydia-containing vesicles. The fact that these Ca(2+)-binding proteins differ in their ability to associate with chlamydia-containing vesicles and inclusions implies that the factors that regulate the interaction of annexin I and annexin III with membrane are different and suggests a selective regulatory mechanism for endosome aggregation and avoiding lysosome fusion during chlamydia infection.

The LPS localization might explain the lack of protection of LPS-specific antibodies in abortion-causing Chlamydia psittaci infections

Four monoclonal antibodies against chlamydial lipopolysaccharide (LPS) were used to study their localization and distribution in the Chlamydia psittaci AB7 abortion-causing strain by immunoelectron microscopy. A non-embedding technique on whole chlamydiae, together with a post-embedding technique on McCoy cells infected with the strain, were performed. Immunogold labelling was observed on the surface of reticular bodies (RB), but not on elementary bodies (EB). Immunolabelling was observed in ultrathin sections on both sides of the external chlamydial membrane, mainly on the inner side of EB and on the outer side of RB. Immunogold density was higher in EB than in RB; however, the absolute number of gold particles was higher in RB than EB, suggesting a loss of immunolabelling during the transformation of RB into EB. Specific labelling of LPS was also found in electrodense and adielectronic vacuoles near the surface of the cytoplasmic membrane of infected McCoy cells. These results suggest that the lack of protection against some chlamydial strains, despite the presence of anti-LPS specific antibodies, is due to the localization of LPS on the inner side of the external membrane of EB.

Comparative Diagnosis of Neonatal Chlamydial Conjunctivitis by Polymerase Chain Reaction and McCoy Cell Culture

Cervical and ocular swabs from 100 mother/newborn pairs delivering on the clinic service were assayed for Chlamydia trachomatis with standard McCoy cell culture and with standard and biotinylated polymerase chain reaction techniques, using primers directed against the major outer membrane protein gene and C. trachomatis-specific cryptic plasmid, respectively. Using the polymerase chain reaction, 20 (20%) mothers and seven (7%) neonates were positive for Chlamydia. All neonates positive by polymerase chain reaction were from mothers positive by polymerase chain reaction, yielding a 35% transmission rate. Only five of 20 (25%) mothers and two of seven (28%) neonates positive by polymerase chain reaction were positive by cell culture. All cell culture samples were positive by polymerase chain reaction testing. Culture and polymerase chain reaction analysis two weeks after treatment with oral erythromycin were negative. The polymerase chain reaction assay appears to be equally specific and more sensitive than McCoy cell culture for the detection of C. trachomatis from ocular specimens.

Antichlamydial activity of cervical secretion

SummarySummaryCervical secretion from women with and without symptoms and signs of genital infection were analysed for their ability to inhibit inclusion formation of Chlamydia trachomatis in McCoy cell cultures. Of 173 cervical samples, 115 (66 per cent) had antichlamydial activity. The inhibition was concentration-dependent and the inhibitory factor had a molecular weight of < 10 000 Da and was heat labile. C. trachomatis was isolated by culture from six (3 per cent) of the 173 women. Those who were culture-positive for C. trachomatis had higher levels of antichlamydial activity than other age matched women. There was no relation between the antichlamydial activity and the antichlamydial antibody titre either in cervical secretion or in sera, as measured by micro-immunofluorescence tests and an enzyme-linked immunosorbent assay.

Clinical evaluation of a new polymerase chain reaction assay for detection of Chlamydia trachomatis in endocervical specimens

A clinical evaluation of the Amplicor polymerase chain reaction (PCR) assay for the detection of Chlamydia trachomatis in endocervical swabs (Roche Molecular Systems, Branchburg, N.J.) is described. This new clinical system used one-step sample preparation, amplification with biotinylated cryptic plasmid primer pairs (CP24-CP27), uracil-N-glycosylase (AmpErase), and a microtiter format for amplicon capture and detection. Culture with McCoy cells in duplicate 1-dram (3.697-ml) vials with fluorescent immunostaining was the reference system. Endocervical swab samples from 945 women provided 74 culture-positive specimens, of which PCR detected 71. The initial PCR result was positive for 12 additional specimens. Arbitration of the PCR-positive, culture-negative samples by PCR with major outer membrane protein primers, duplicate culture, elementary body direct fluorescent-antibody staining, and DNA extraction PCR showed that all 12 samples were positive for chlamydia, raising the number of truly positive samples from 74 to 86. After arbitration the true sensitivities of PCR and culture were 96.5 and 86%, respectively (P = 0.02). Specificities for both were 100%. For PCR, the positive and negative predictive values were 100 and 99.7%, respectively. Total test efficiency was 99.7%. A high-test-volume (121 samples) timing study with all items included in the College of American Pathologists work load method indicated that this PCR format took approximately 3 min per sample. Because of the high sensitivity, specificity, and improved ease of handling, we found PCR to be a good alternative to culture for detection of C. trachomatis.