Mycoplasma 16S Research Articles

Investigation of the prevalence of Mycoplasma ovipneumoniae in Southern Xinjiang, China

IntroductionIt is very important to monitor the infection of Mycoplasma ovipneumoniae as a potential threat to the sheep industry. Southern Xinjiang is a major sheep breeding base in China, however, there is no relevant information concerning the infection of the region’s ovine stock with this bacteria at present. This study aimed to address this knowledge gap.Material and MethodsA total of 824 nasal swabs and the lungs of six sheep that died of pneumonia were collected in four regions between 2018 and 2020. Primers specific for M. ovipneumoniae and universal ones for the genus were used for PCR. Sequencing was undertaken of 159 universal primer-positive samples (153 nasal swabs and 6 lungs) and of 84 specific primer-positive samples (80 nasal swabs, 20 per region; and 4 lungs, 1 per region). The lungs were also sampled for the isolation of M. ovipneumoniae. A phylogenetic tree based on partial sequences of the Mycoplasma 16S rRNA gene was built.ResultsThe overall nasal swab positive rate for M. ovipneumoniae was 40.78%; the rate of animals older than 12 months was significantly different to those of younger sheep (< 3 months, 53.39%; 3 – 12 months, 46.01%; >12 months, 31.76%). Four strains of M. ovipneumoniae were isolated from six lungs. Phylogenetic analysis indicated their origin outside southern Xinjiang. Two other species were also detected: M. arginine and M. conjunctivae.ConclusionOur survey indicated that a high level of M. ovipneumoniae asymptomatic colonisation in sheep, especially in lambs, affects southern Xinjiang and also confirmed the existence of M. conjunctivae and M. arginine. Our results showed that the health of sheep in southern Xinjiang is facing a great threat, and relevant prevention and control measures should be strengthened.

Identification and Genetic Characterization of Mycoplasma Species Affecting Respiratory System in Egyptian Cattle

Abstract: Mycoplasma species are the main important causative agents causing pneumonia in cattle, there are about 200 species or more. The current work aimed to investigate various mycoplasma spp. isolated from the respiratory tract of cattle by microbial culture, conventional polymerase chain reaction technique, and target gene sequencing with phylogenetic analysis. An initial screening was done to confirm the presence of mycoplasma spp. by culture on PPLO’s agar, digitonin sensitivity and biochemical tests. 129 isolates were characterized by fried egg with depressed center colonies, digitonin sensitive and negative to glucose fermentation and arginine utilization test. Out of 305 samples, 20 samples were selected for amplification by PCR technique using Mycoplasma 16S rRNA primer. Seven samples were positive to Mycoplasma species and gave amplified band at 1013 bp. subsequently, the seven isolates were sequencing. Four sequenced isolates (EGS1, EGL6, EGL1 and EGS2) were closely related to each other and very close to Mycoplasma bovis strains and far from M. leachii 99/014/6. Two sequenced isolates (EGL2 and EGL5) were closely related to each other and very close to Mycoplasma bovirhinis strains and far from M. leachii 99/014/6. One sequenced isolate (EGL3) was very close to Mycoplasma arginini strain and far from M. bovis 99/014/6. From these results, we conclude that conventional culture methods for diagnosis either by isolation and identification of Mycoplasma is a time-consuming method to diagnose mycoplasma infection So, these methods can be replaced by PCR and genome analysis technology.

Immune-mediated haemolytic anaemia secondary to haemotrophic mycoplasma infection in a pet ewe

Haemotrophic Mycosplasma species (haemoplasmas) are adapted to adhere to erythrocyte cell membranes of hosts,1 resulting in a range of clinical presentations. Clinical signs of Mycoplasma ovis (previously known as Eperythrozoon...

First Report of Candidatus Mycoplasma haemohominis Infection in Australia Causing Persistent Fever in an Animal Carer

Hemotropic mycoplasmas (hemoplasmas) infect animals and humans and can lead to clinical syndromes mainly characterized by hemolytic anemia. A novel pathogen, Candidatus Mycoplasma haemohominis, was recently associated with a case of human hemoplasmosis in Europe. Here we report the first detection of this pathogen in an Australian patient exhibiting persistent fever, hemolytic anemia, and pancytopenia over a 10-month period. After exhaustive negative testing for human infectious diseases, whole genome sequencing (WGS) was performed on the patient's bone marrow aspirate, using an Illumina NextSeq500 platform. Conventional polymerase chain reaction (PCR), followed by Sanger sequencing, was then performed on blood samples using novel Mycoplasma-specific primers targeting the 16S ribosomal RNA gene. In addition, a Mycoplasma-specific fluorescence in situ hybridization (FISH) assay was developed to differentiate Mycoplasma cells from other erythrocyte inclusions (eg, Pappenheimer and Howell-Jolly bodies) which are morphologically similar to bacterial cocci by light microscopy. WGS analysis revealed that approximately 0.04% of the total number of unmapped reads to human genome corresponded to Mycoplasma species. A 1-kb Mycoplasma 16S fragment was successfully amplified by conventional PCR, and sequence analyses revealed 100% identity with Candidatus Mycoplasma haemohominis. FISH confirmed that several (approximately 2%) epierythrocytic inclusions initially observed by light microscopy corresponded to Mycoplasma cells. This represents the second report of hemolytic anemia associated with hemoplasma infection in a human, and the first report of human hemoplasmosis in Australia. This study highlights the importance of new and emerging diagnostic approaches and need for further investigations on the epidemiology of Candidatus Mycoplasma haemohominis in Australia.

Assessing mycoplasma contamination of cell cultures by qPCR using a set of universal primer pairs targeting a 1.5 kb fragment of 16S rRNA genes.

Mycoplasmas (a generic name for Mollicutes) are a predominant bacterial contaminant of cell culture and cell derived products including viruses. This prokaryote class is characterized by very small size and lack of a cell wall. Consequently, mycoplasmas escape ultrafiltration and visualization under routine microscopic examination, hence the ease with which cells in culture can be contaminated, with routinely more than 10% of cell lines being contaminated. Mycoplasma are a formidable threat both in fundamental research by perverting a whole range of cell properties and functions and in the pharmacological use of cells and cell derived products. Although many methods have been developed, there is still a need for a sensitive, universal assay. Here is reported the development and validation of a quantitative polymerase chain reaction (qPCR) based on the amplification of a 1.5 kb fragment covering the 16S rDNA of the Mollicute class by real-time PCR using universal U1 and U8 degenerate primers. The method includes the addition of a DNA loading probe to each sample to monitor DNA extraction and the absence of PCR inhibitors in the extracted DNA, a positive mycoplasma 16S rDNA traceable reference sample to exclude any accidental contamination of an unknown sample with this reference DNA, an analysis procedure based on the examination of the melting curve and the size of the PCR amplicon, followed by quantification of the number of 16S rDNA copies (with a lower limit of 19 copies) when relevant, and, if useful, the identification of the contaminating prokaryote by sequencing. The method was validated on a collection of mycoplasma strains and by testing over 100 samples of unknown contamination status including stocks of viruses requiring biosafety level 2, 3 or 4 containments. When compared to four established methods, the m16S_qPCR technique exhibits the highest sensitivity in detecting mycoplasma contamination.

Detection and prevalence of four different hemotropic Mycoplasma spp. in Eastern North Carolina American black bears (Ursus americanus).

Hemotropic Mycoplasma spp. are globally emerging, obligate parasitic, epierythrocytic bacteria that infect many vertebrates, including humans. Hemoplasma infection can cause acute life-threatening symptoms or lead to a chronic sub-clinical carrier state. Hemotropic Mycoplasma spp. transmission, prevalence, and host specificity are uncertain. The purpose of this study was to determine the molecular prevalence of Mycoplasma species in blood from 68 free-ranging black bears from the eastern coast of North Carolina. DNA amplification of Mycoplasma 16S rRNA gene identified four distinct species infecting 34/68 (50%) of the black bear blood samples, including Candidatus M. haematoparvum. The high prevalence of hemotropic Mycoplasma infection in this wildlife species highlights the importance of understanding intra and inter species transmission. Black bears may play a role in the transmission of hemotropic Mycoplasma spp. between animals, arthropod vectors, and humans. Further studies are needed to elucidate black bears as a potential reservoir for hemotropic Mycoplasma infections.

Colonization of paediatric lower respiratory tract with genital Mycoplasma species

Recently, much attention has been given to the possible role played by pathogens that colonize neonatal or paediatric airway and their potential involvement in chronic respiratory disease. The goal of the current study was to evaluate the prevalence of Mycoplasma organisms in the BAL fluid of paediatric patients suffering from a variety of chronic respiratory diseases to determine if there was any clear disease association with bacterial presence. We examined 319 paediatric BAL samples for the presence of M.genitalium, M.hominis, U.urealyticum, U.parvum and M.pneumoniae DNA with species-specific PCR. Mycoplasma DNA was found in 32.6% (104/319) of patient samples; 10% (32/319) for M.pneumoniae, 8.8% for U.parvum, 2.8% for U.urealyticum; 4.7% for M.hominis and 9.1% for M.genitalium. There were no significant clinical and laboratory differences except serum IgE in asthmatics according to Mycoplasma colonization or not. Elevated levels of IgE were found more often in Mycoplasma DNA-negative patients than patients with bacterial DNA, 85/109 versus 24/109 respectively (P<0.0001). There was no difference in the frequency of Mycoplasmas between the asthmatics and the non-asthmatics; 30.6% (69/225) versus 37.2% (35/94) for Mycoplasma 16S DNA, and 8% versus 14.9% for M.pneumoniae, respectively. Our data indicate that in addition to M.pneumoniae, urogenital Mycoplasma species may colonize the airway of patients with chronic respiratory diseases. There was, however, no association between chronic asthma diagnosis and Mycoplasma colonization in this study.

Amniotic Fluid Interleukin-1 Beta and Interleukin-6, but not Interleukin-8 Correlate with Microbial Invasion of the Amniotic Cavity in Preterm Labor

We compared the frequency of intra-amniotic infection in preterm labor (PL) with women not in labor, and correlated infection with amniotic fluid (AF) cytokines. Detailed identification of species, especially mycoplasmata, was tried to improve our understanding of the pathogenesis of PL. AF from 20 women with PL and 20 controls were evaluated. Infection was detected by PCR for Mycoplasma hominis, Ureaplasma urealyticum and 16S rRNA bacterial gene, which was cloned and sequenced for bacterial identification. Interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor (TNF)-α levels were measured by ELISA. Frequency of intra-amniotic infection is higher in PL (40.0%). Sequencing-based method identified Bacteroides fragilis, Prevotella bivia and Leptotrichia amnionii, in addition to Mycoplasma species detected by PCR. AF infection correlated with increased IL-1β and IL-6 levels. The frequency of intra-amniotic infection, especially M. hominis, in PL women who delivered with 7 days, is high and correlates with high IL-1β and IL-6 levels, but not IL-8.

Infectious Chlamydia pneumoniae is Associated With Elevated Interleukin-8 and Airway Neutrophilia in Children With Refractory Asthma

Neutrophilic asthma is thought to be less responsive than eosinophilic asthma to anti-inflammatory therapies including corticosteroids. Chlamydia pneumoniae has been implicated in asthma, possibly by induction of interleukin (IL-8). We hypothesized that IL-8 is increased in the bronchoalveolar lavage (BAL) fluid from children with asthma and C. pneumoniae. BAL fluid was analyzed for C. pneumoniae and IL-8 using polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay from 2 asthma patient populations in the Bronx, NY and Massachusetts with an average age of 8 and 8.7 years old, respectively. For comparison, samples were also analyzed for C. trachomatis and Mycoplasma 16s DNA. Of 18 Bronx samples analyzed, 6 (33%) were PCR-positive for C. pneumoniae, 10 (56%) for C. trachomatis, and 8 (44%) for Mycoplasma 16s DNA. IL-8 from C. pneumoniae-positive samples was 3.3-fold higher compared with negative samples (P = 0.003). There was no difference between patients tested for C. trachomatis or Mycoplasma. Of 84 Massachusetts samples analyzed, 42 (50%) were PCR-positive for C. pneumoniae, 42 (50%) for C. trachomatis, and 13 (16%) for Mycoplasma. IL-8 concentration from C. pneumoniae-positive samples was 10.49-fold higher compared with negative samples (P = 0.0001). As in the Bronx cohort, there were no differences between patients tested for C. trachomatis or Mycoplasma. Lastly, BAL neutrophilia predicted the presence of C. pneumoniae but not Mycoplasma or C. trachomatis. Children with asthma who were PCR-positive for C. pneumoniae demonstrated elevated concentrations of IL-8 and neutrophils in BAL fluid compared with similar patients who were positive for C. trachomatis or Mycoplasma organisms, but PCR-negative for C. pneumoniae. Undiagnosed C. pneumoniae infection in children may therefore contribute to poorly controlled asthma via induction of IL-8.

Absence of Mycoplasma-specific DNA sequence in brain, blood and CSF of patients with multiple sclerosis (MS): A study by PCR and real-time PCR

Mycoplasmas are the smallest of the known self-replicating organisms. They lack cell walls and are associated with numerous diseases in humans and animals. We are exploring the possibility that infection by Mycoplasma may induce the inflammatory demyelinating disease of the central nervous system (CNS) that is MS. The presence of specific Mycoplasma species DNA was sought in brain, serum and cerebrospinal fluid (CSF) of patients diagnosed with multiple sclerosis (MS) and other neurological diseases (OND) including inflammatory disorders. The MS samples from patients with active and progressive MS, as well as in remission, a variety of other neurological disease controls, including inflammatory CNS diseases such as meningitis, cryptococcal meningitis and encephalitis and other neurological disorders such as migraine were also examined. Clinical samples were provided by the National Neurological Research Specimen Bank and the Human Brain and Spinal Fluid Resource Centre, Los Angeles. Analysis was carried out by conventional PCR using Mycoplasma-specific primers (McAuliffe et al., 2005) that target the 16S rDNA gene in Mycoplasma species. The Mycoplasma-specific primers could detect 102 Mycoplasma species. In this study, 30 samples of human brain and 57 pairs of serum and CSF and were examined. No Mycoplasma-specific nucleic acid sequence was detected, and the consistent observation of an endogenous gene, human serum albumin (HSA), as a positive control documented the adequacy of the method. Real-time PCR analysis of serum and CSF was done also targeting utilizing the Mycoplasma 16S rDNA gene, and this also demonstrated the lack of Mycoplasma in these samples. The presence of Mycoplasma at extraneural sites in MS patients is now being explored.

A NOVEL MYCOPLASMA DETECTED IN ASSOCIATION WITH UPPER RESPIRATORY DISEASE SYNDROME IN FREE-RANGING EASTERN BOX TURTLES (TERRAPENE CAROLINA CAROLINA) IN VIRGINIA

Clinical signs of upper respiratory tract disease-like syndrome (URTD-LS) were observed in free-ranging eastern box turtles (Terrapene carolina carolina) from Virginia, USA (May 2001-August 2003), some of which also had aural abscesses. After a Mycoplasma sp. was detected by polymerase chain reaction (PCR), a study was undertaken to better define the range of clinical signs of disease and to distinguish mycoplasma-associated URTD-LS from other suspected causes of URTD-LS and aural abscessation in box turtles. Nasal and/or ocular swabs (from turtles possessing URTD-LS) or nasal washes (from asymptomatic turtles) were collected from turtles May 2001-August 2003; samples were assayed for Mycoplasma spp., chelonian herpesvirus, and iridoviruses by PCR testing. A partial DNA sequence (933 bases) of the small ribosomal subunit (16S rRNA) of the box turtle Mycoplasma sp. was analyzed to determine its phylogenetic relatedness to other Mycoplasma spp. of veterinary interest. Mycoplasma sp. was detected in seven (six with clinical signs of URTD-LS; one asymptomatic) of 23 fortuitously collected animals from six of 11 Virginia counties. Clinical signs in Mycoplasma sp.-infected animals included unilateral to bilateral serous to mucopurulent nasal discharge, epiphora, ocular edema, and conjunctival injection. Five Mycoplasma sp.-positive animals possessed aural abscesses; two did not. Analysis of the mycoplasma 16S rRNA gene sequence from one asymptomatic and three symptomatic animals representing four counties revealed a consensus Mycoplasma sp. sequence closely related to, but distinct from, M. agassizii. None of the samples collected contained viral DNA of chelonian herpesviruses or invertebrate and vertebrate (including FV3) iridoviruses. In conclusion, a new Mycoplasma sp. was associated with URTD-LS in native box turtles from Virginia that was not codetected with other suspected causes of chelonian upper respiratory disease; there was no proof of a direct relationship between aural abscessation and the Mycoplasma sp.

Experimental evidence of indirect transmission of Mycoplasma synoviae

The aim of the study was to analyse experimental transmission of Mycoplasma synoviae, an avian pathogen. Three experiments using specific pathogen-free day-old chicks placed in isolators were conducted. In the first experiment, the birds were introduced in an isolator previously contaminated with a M. synoviae broth culture. After 34 days, these birds were eliminated and, for the second trial, the chicks were introduced in the same isolator without disinfecting. In the third assay, the chicks were placed in an isolator containing a mixture of food, feathers and dust collected less than an hour earlier from a M. synoviae infected laying hen flock. In the second and third experiments in order to exacerbate the M. synoviae infection, the birds were inoculated with infectious bronchitis (IB) virus. The presence of M. synoviae in the environment and in tracheal swabs was monitored by culture, a multiplex PCR (mPCR) detecting M. synoviae and Mycoplasma 16S rDNA and a multiplex RT-PCR (mRT-PCR) detecting the M. synoviae mRNA coding for a membrane protein and Mycoplasma 16S rRNA. In in vitro experimental conditions, M. synoviae mRNA and 16S rRNA were detected up to 20 min and 23 h respectively after mycoplasma death. In the first assay, the first infected bird was detected on the 13th day. In the second trial, culturable M. synoviae or viable M. synoviae were detected in the isolator for 3 or 4 to 5 days respectively after depopulation of the birds of the first assay whereas the first culture positive tracheal swabs were detected on the 33rd day, after IB inoculation. In the third experiment, the first infected birds were detected on the 54th day. Thus, the different assays showed that M. synoviae contaminated material (dust, feathers and food) can infect chicks, sometimes after remarkably long silent periods.

Rapid detection of Mycoplasma dispar and M. bovirhinis using allele specific polymerase chain reaction protocols

We describe an allele specific PCR based approach for the rapid detection of two bovine Mycoplasma species associated with respiratory disease. Specific and universal oligonucleotides were used in combination to detect the presence of single nucleotide polymorphisms within the 16S ribosomal DNA sequence. Presence of Mycoplasma 16S rDNA is indicated by the production of a single control fragment, whilst positive samples generate an alternative smaller specific product over the same region. This technique provides a reliable and sensitive method which, although widely used in human genetic screening, has not been documented for diagnosis of bacterial infection.

Taxonomy of the canine Mollicutes by 16S rRNA gene and 16S/23S rRNA intergenic spacer region sequence comparison.

The taxonomy of canine Mollicutes is described, based on phylogenetic analysis of 16S rRNA gene and 16S/23S rRNA intergenic spacer (IGS) region sequences. The nucleotide sequences of the 16S rRNA gene of two untyped mycoplasmas and the IGS region of 11 Mycoplasma species were determined and used for phylogenetic analysis. The two untyped Mycoplasma strains, HRC 689 and VJC 358, were found to be distinct from all known canine mycoplasmas and all published mycoplasma 16S rRNA gene sequences.

A reverse transcription-PCR assay to detect viable Mycoplasma synoviae in poultry environmental samples

In order to study horizontal transmission of Mycoplasma synoviae an avian pathogen, a reverse transcription-polymerase chain reaction (RT-PCR) assay was developed to detect viable Mycoplasma in environment. The test was based on the RT-PCR of the 16S ribosomal RNA (rRNA) of Mycoplasma genus. Results showed that Mycoplasma 16S rRNA was stable up to 23 h after cell death. Therefore, the test allowed detection of viable or very recently (less than 23 h) dead mycoplasmas. M. synoviae survival in artificially contaminated water, food and soil and in the environment of M. synoviae experimentally infected turkeys was estimated by culture and RT-PCR. The RT-PCR method was then applied in a naturally infected laying hen farm showing problems of recurrent mycoplasmosis in the hens. Results confirmed the usefulness of RT-PCR in checking the efficiency of biosecurity measures and in improving cleaning and disinfection protocols.

Detection of mycoplasma contamination in viral stocks by a polymerase chain reaction technique

A polymerase chain reaction (PCR) amplification procedure is described for the detection of mycoplasma contaminants in virus stocks. The method uses primers based upon the DNA coding sequence for mycoplasma 16S and 23S ribosomal RNA. The primers evaluated amplified DNA from commonly encountered mycoplasma contaminants to levels readily detectable on ethidium-stained electrophoresis gels. The same primers did not yield detectable DNA bands when human DNA, mouse DNA, and DNA from related genera of bacteria were amplified. The assay is specific and sensitive and useful in testing a wide range of viral stocks for the presence of mycoplasma contaminants. This PCR-based mycoplasma detection test has proven capable of quickly clarifying the status of viral stocks that occasionally yield indeterminate results when tested for mycoplasma by the Hoechst-staining method.

Influence of mycoplasma infection on the incorporation of different precursors into RNA components of tissue culture cells

Seven different tissue culture cells have been cultured with and without mycoplasma ( M. hyorhinis) in the presence of various precursors of RNA. Total cellular RNA was isolated and analysed by electrophoresis on polyacrylamide gels. The results obtained with mycoplasma-infected cells can be summarized as follows: 1. 1. When cells are labelled with [8- 3H]guanosine or [5- 3H]uridine there is some incorporation into host cell 28S and 18S rRNA, but it is less than into mycoplasma 23S and 16S rRNA. [8- 3H]guanosine or [5- 3H]uridine are also incorporated into host cell and mycoplasma tRNA and mycoplasma 4.7S RNA, but the incorporation into host cell 5S rRNA and low molecular weight RNA components (LMW RNA) is reduced. 2. 2. [5- 3H]uracil is not incorporated into host cell RNA but into mycoplasma tRNA, 4.7S RNA, a mycoplasma low molecular weight RNA component M 1 and 23S and 16S rRNA. 3. 3. [ 3H]methyl groups are incorporated into mycoplasma tRNA, 23S and 16S rRNA, but not into host cell 28S, 18S, 5S rRNA nor into mycoplasma 4.7S RNA. 4. 4. With [ 32P]orthophosphate or [ 3H]adenosine as precursors, the labelling is primarily in the host RNA. Mycoplasma infection influences the labelling of RNA primarily by an effect on the utilization of the exogenously added radioactive RNA precursors, since the generation time of mycoplasma infected cells is about the same as that of uninfected cells. Mycoplasma infection may completely prevent the identification of LMW RNA components.