Detection of bacterial and mycoplasma contamination in cell cultures by polymerase chain reaction.

A fast and simple method to detect bacterial and especially mycoplasma contamination in tissue culture by means of polymerase chain reaction (PCR) amplification is described. In a first step the universal primer pairs P1/P2 (190-bp fragment) and P3/P4 (120-bp fragment) directed to different conserved parts of the prokaryotic 16S rRNA gene are used. A positive signal after amplification on cell culture DNA with these primers provides an indication of bacterial infection. Using the internal primers IP1, IP3 and IP'3 complementary to a part of the V4 and V8 variable regions of the 16S rRNA gene, in combination with a universal primer, cultures contaminated with mycoplasma could be identified. Six mycoplasma species, typical contaminants in tissue cultures, were investigated: Mycoplasma orale, M. fermentans, M. arginini, M. hyorhinis, M. hominis and Aeromonas laidlawii. This mycoplasma test is an easy, specific and sensitive assay which should be extremely useful in any tissue culture setting.

Evaluation of several adjuvants as alternatives to the use of Freund's adjuvant in rabbits.

In three experiments we evaluated several types of adjuvants as an alternative to Freund's adjuvant (FA). In the first experiment three adjuvant preparations (a water-in-oil emulsion (Specol), a combination preparation of monophosphoryl lipid A + trehalose dimycolate + cell wall skeleton and a non-ionic block polymer surfactant (TiterMax)) were evaluated. The adjuvants were combined with three different types of weak immunogenic antigens (synthetic peptide, glycolipid and particulate antigen) and administered following the intramuscular and subcutaneous route. The evaluation was based on clinical, pathological and immunological parameters. The animals did not appear to be severely or chronically impaired by the experiment. After injection of the RIBI adjuvant, side effects of the same severity as with FA were induced, while low antibody titers were produced. TiterMax caused few side effects, while antibody responses were very low. In comparing Specol and FA, Specol had far fewer adverse effects than FA. However, Specol had immunostimulating properties of the same level as FA. In the second experiment, the effect of injected volume of FA on side effects and antibody titer was studied. Immunization of rabbits with a total of 0.5 ml FA at different sites does not seem to increase the immune response when compared with the immune response seen after injection of 0.5 ml FA at one site. However side effects were seen in all the animals. In the third experiment, the side effects following intradermal (i.d.) injection of the adjuvants were studied. After i.d. injection of FA or RIBI, undesirable effects were found. No side effects occurred after i.d. injection of Specol or TiterMax. From the studies it is concluded that Specol is an alternative to FA for hyperactivation of the immune response in rabbits.

Clostridium difficile-associated typhlitis in specific pathogen free guineapigs in the absence of antimicrobial treatment.

Clostridium difficile (toxin) associated typhlitis was diagnosed in untreated barrier-maintained specific pathogen free guineapigs. It resembled the pathological lesions of antibiotic induced enterocolitis. The possible role of limited colonization resistance to C. difficile provided by mouse enteric microflora in the pathogenesis of the disease is discussed.

Selective decontamination of the digestive tract of Syrian hamsters.

Conventional Syrian hamsters colonized with aerobic gram-negative bacteria such as Pasteurella pneumotropica and various Enterobacteriaceae species were successfully and permanently freed from these microorganisms by oral treatment for 4 weeks with dihydrostreptomycin and 'Orabase' premixed with appropriate antibiotics. Concomitant oral treatment with dimetridazol for the elimination of intestinal flagellates was unsuccessful. During treatment the animals were maintained under germ-free isolation conditions.

Isolation of Acholeplasmatales from rabbit faeces.

Acholeplasma laidlawii was isolated from the faeces of 23.5% and 24% of groups of 51 conventional and 45 specified-pathogen-free (SPF) rabbits respectively. Isolation of the organism from individual animals could often be repeated, suggesting that infection was not merely transient. Two further acholeplasmas were isolated from two SPF rabbits. One was serologically related to Acholeplasma modicum. The other could not be identified and may be a new species.

Naturally occurring genital mycoplasmosis in mice.

The first isolations of Mycoplasma pulmonis were made from inflamed ovaries of 2 C3H/F1 mice. Investigation of cultures from a further 110 apparently healthy mice revealed 14 cases of M. pulmonis localized in the ovaries and associated with oophoritis.

An alternative method for the decontamination of rats carrying Mycoplasma pulmonis without the use of germfree isolators.

Two strains of Lewis rat were successfully freed from Mycoplasma pulmonis infection by using a combination of oral treatment with oxytetracycline hydrochloride and obtaining young by hysterectomy. Laminar flow cabinets were used to perform hysterectomies on donor animals and for rearing hysterectomy-derived animals. After thorough microbiological examination the rats were brought to the breeding colony of the Laboratory Animal Centre. Periodic laboratory tests using both cultural and enzyme-linked immunosorbent assay methods showed that the animals have remained free from M. pulmonis for the last 3 years.

Acute diarrhoeal disease in rabbit: bacteriological diagnosis and efficacy of oral rehydration in combination with loperamide hydrochloride.

Acute outbreaks of diarrhoea with high mortality rates are frequently observed in rabbits. Amongst various aetiological factors Escherichia coli or its toxins have been found to be commonly incriminated. Sulphonamides or antibiotics are used to treat rabbits with bacterial diarrhoea. The result of the antibiotic treatment is moderately successful. We had good results using oral rehydration treatment in combination with loperamide hydrochloride (Immodium) in a colony of rabbits with E. coli diarrhoea.

[Ureaplasma urealyticum in tracheal aspirate of ventilated premature infants: report of 6 cases]. / Ureaplasma urealyticum in trachea aspiraat van beademde preterme neonaten: een bespreking van 6 casus.

In the Netherlands, up till now no reports have appeared describing neonatal colonisation of mechanically ventilated preterm neonates with Ureaplasma urealyticum. The present, prospective study was designed to assess the incidence of U. ureaplasma infections in a group of preterm newborns with prolonged ventilatory support because of respiratory failure. In 1989 110 preterm newborns with hyaline membrane disease (HMD) were mechanically ventilated; 23 for more than 7 days because of pulmonary abnormalities. Six of them (26%) had positive cultures of endotracheal aspirate for U. urealyticum. Gestational age at birth ranged from 26 to 33 5/7 weeks, birth weight from 790 to 2545 gram. Other bacteria or viruses were not present. Although all patients with positive cultures for U. urealyticum were treated with erythromycin and U. urealyticum was eradicated, no clinical effect was seen. Five of the 6 patients (83%) with U. urealyticum developed bronchopulmonary dysplasia (BPD), 2 of them (33%) died. Of the 17 neonates without U. urealyticum 9 (53%) developed BPD, whereas one (6%) died. Differences were not significant (Chi 2 test/Fisher exact test). Also in the Netherlands U. urealyticum can be demonstrated in endotracheal aspirate of ventilated preterm newborns. The hypothesis, that early diagnosis and treatment of U. urealyticum in this group of patients might decrease the morbidity and mortality caused by HMD and BPD, needs further study.

Discovering an epidemic before it has reached a certain level of prevalence.

In this communication we calculate the probability of discovering a simple epidemic in a large population before the epidemic has reached a given level of prevalence, by regularly taking a small random sample from the population for microbiological screening. Apart from the general formula which has to be evaluated numerically, we derive various simple approximation formulae which shed light on the properties of various monitoring regimes. These formulae are, moreover, rather robust against deviations from the model specifications. The results are applied to the evaluation of the efficiency of an infection-monitoring program in an animal breeding centre.

Application of a Mycoplasma group-specific PCR for monitoring decontamination of Mycoplasma-infected Chlamydia sp. strains.

Mycoplasma contamination of biological materials remains a major problem. Most contaminations are caused by the use of Mycoplasma-contaminated cell lines. We adapted a Mycoplasma group-specific PCR to detect Mycoplasma contamination in cell lines and demonstrate its use in monitoring decontamination procedures with Mycoplasma-contaminated suspensions of Chlamydia spp. Three different methods were investigated: the use of Mycoplasma-specific antiserum in cell culture, physical separation by the combined use of enzymatic treatment and differential centrifugation, and the use of detergents. With these methods only incubation with Triton X-100 resulted in decontamination of Mycoplasma-contaminated suspensions of several laboratory strains of Chlamydia pneumoniae, C. pecorum, and C. trachomatis. Only one C. pneumoniae strain, UZG-1, was sensitive to Triton X-100 treatment. Since 39 of 40 throat swabs from patients with symptoms of an upper respiratory tract infection had positive reactions in the Mycoplasma group-specific PCR, this procedure could also have clinical significance in attempts to propagate C. pneumoniae strains from clinical specimens.

Acholeplasma multilocale sp. nov., isolated from a horse and a rabbit.

Acholeplasma strains were isolated from the nasopharynx of a horse (strain PN525T [T = type strain]) and the feces of a rabbit (strain B1). One clone of strain PN525T and one clone of strain B1 were examined in detail. These clones were indistinguishable from each other and were serologically distinct from the previously described Acholeplasma and Mycoplasma spp. The strains had the following properties: guanine-plus-cytosine content of 31 mol%; sterol was not required for growth, which occurred under both aerobic and anaerobic conditions; glucose was metabolized; and arginine was hydrolyzed. Strain PN525 (= NCTC 11723) is the type strain of a new species, Acholeplasma multilocale.

Survival of Mycoplasma hyorhinis in trypsin solutions.

The survival of four strains of Mycoplasma hyorhinis in stock solutions of trypsin was tested at 22, 4 and -15 degrees C. Low (10(4)-10(5) cfu/ml) and high (10(6)-10(7) cfu/ml) initial concentrations of each strain were used, each was tested three times. A regular decrease of low and high concentrations (1 log in 10 and 20 min, respectively) was seen at 22 degrees C. At 4 degrees C the low concentrations showed a reduction of about 1 log/h, while apart from one strain high concentrations hardly decreased during the first 6 h and the survival time ranged from 24 to more than 30 h at the end of which there was a reduction of 4 logs. At -15 degrees C low concentrations survived up to 1 week in only one of the three tests, high concentrations survived for more than 12 weeks (reduction 3 logs). These latter results suggest that mycoplasmas may be present in trypsin as clumps, which deteriorate very slowly. A study was also performed to compare the sensitivity of different cultural procedures for detecting mycoplasmas.

Monoclonal antibodies against the K99 antigen of Escherichia coli for diagnostic purposes.

Hybridomas secreting monoclonal antibodies against the K99 antigen of Escherichia coli were produced by the fusion of spleen cells from immunized BALB/c mice with P3/X63-Ag8.653 myeloma cells. The seven hybridomas which produced the highest antibody titers in vitro, as detected by enzyme-linked immunosorbent assay (ELISA) and Perma slide agglutination test (PSAT), were chosen for antibody production in vivo. No cross reaction was observed with K88ab, F41 and P987 antigens in the ELISA. The titer of each ascitic fluid was established by the ELISA and the slide agglutination (SAT) tests. The two ascitic fluids with the highest titer in the SAT were incorporated into the set of antisera used for serotyping at our laboratory. The results were satisfactory both in terms of stability and specificity.

Pefloxacin compared with cefotaxime for treating men with uncomplicated gonococcal urethritis.

In a randomized comparative study, 83 male patients suffering from acute uncomplicated gonococcal urethritis were treated with a single dose of either 0.8 g pefloxacin, given orally, or 1.0 g cefotaxime, given intramuscularly. The cure rates were 100% in both treatment groups four to seven days and 21 to 31 days, respectively, after therapy. The MICs of the isolated Neisseria gonorrhoeae ranged from 0.008 to 0.06 mg/l for pefloxacin and from 0.0005 to 0.03 mg/l for cefotaxime. Postgonococcal urethritis was found in 9% of the patients treated with pefloxacin and in 20% of the patients treated with cefotaxime. Chlamydia trachomatis, Mycoplasma hominis and Ureaplasma urealyticum were isolated from 15%, 7% and 22% of the patients, respectively, before therapy and from 22%, 11% and 20% of the patients, respectively, 21 to 31 days after therapy. Both antibiotics had no effect on the presence of these microorganisms. No side effects were recorded in either groups of patients except that 46% of the patients treated with cefotaxime reported mild pain at the injection site. In conclusion, pefloxacin and cefotaxime are safe and effective agents in the treatment of uncomplicated gonococcal urethritis in men.

Acholeplasma vituli sp. nov., from bovine serum and cell cultures.

Organisms isolated from commercial foetal bovine serum and from cell culture lines containing such serum supplements were found to consist of non-helical, non-motile, pleomorphic coccoid forms. One strain (FC 097-2T) cultivated directly from foetal bovine serum was selected for characterization. In ultrastructural examination, individual round cells lacked cell wall structures and cells varied in size, with a mean diameter of about 700 nm. However, variable numbers of cells were filterable through membranes of 300 nm. Optimum growth occurred between 30 and 37 degrees C. The organism fermented glucose, fructose and mannose, but did not hydrolyse arginine. The strain was insensitive to 500 U penicillin ml(-1) and was capable of growing in the absence of serum or cholesterol. The organism was serologically distinct from all 13 currently described species in the genus Acholeplasma and from other sterol-requiring species in the genus Mycoplasma, using growth inhibition, immunoperoxidase and immunofluorescence tests. Strain FC 097-2T was found to have a DNA G+C composition between 37.6 +/- 1 mol% and 38.3 +/- 1 mol%. The genome size was determined to be 2095 kbp. The 16S rDNA sequence of strain FC 097-2T was compared to 16S rDNA sequences of other mollicutes in nucleotide databases. No deposited sequence was found to be identical; the closest relatives were several members of the genus Acholeplasma. On the basis of these findings and other similarities to acholeplasmas in morphology and growth, the absence of a sterol requirement for growth, and similar genomic characteristics, the organism was assigned to the genus Acholeplasma. Strain FC 097-2T is designated the type strain (ATCC 700667T) of a new species, Acholeplasma vituli.

Comparison of PCR, culture, and serological tests for diagnosis of Mycoplasma pneumoniae respiratory tract infection in children.

For diagnosis of Mycoplasma pneumoniae infection we compared two rapid tests, PCR and the immunoglobulin M immunofluorescence assay (IgM IFA), with culture and the complement fixation test (CFT), in a prospective study among 92 children with respiratory tract infection and 74 controls. Based on positivity of culture and/or CFT as the diagnostic criterion, nine patients (10%) were diagnosed with M. pneumoniae infection. All patients positive by culture were also positive by PCR. In all controls cultures, PCRs, and serological assays were negative, except in one with a positive IgM IFA. The IgM IFA had a low positive predictive value of 50%. Only a combination of PCR (seven patients) and CFT (seven patients) allowed diagnosis of all cases.

Genus- and species-specific identification of mycoplasmas by 16S rRNA amplification.

Systematic computer alignment of mycoplasmal 16S rRNA sequences allowed the identification of variable regions with both genus- and species-specific sequences. Species-specific sequences of Mycoplasma collis were elucidated by asymmetric amplification and dideoxynucleotide sequencing of variable regions, using primers complementary to conserved regions of 16S rRNA. Primers selected for Mycoplasma pneumoniae, M. hominis, M. fermentans, Ureaplasma urealyticum, M. pulmonis, M. arthritidis, M. neurolyticum, M. muris, and M. collis proved to be species specific in the polymerase chain reaction. The genus-specific primers reacted with all mycoplasmal species investigated as well as with members of the genera Ureaplasma, Spiroplasma, and Acholeplasma. No cross-reaction was observed with members of the closely related genera Streptococcus, Lactobacillus, Bacillus, and Clostridium or with any other microorganism tested. On the basis of the high copy number of rRNA, a highly sensitive polymerase chain reaction assay was developed in which the nucleic acid content equivalent to a single organism could be detected.