Maurício Yamaguti

Invasion of Ureaplasma diversum in Hep-2 cells

BackgroundUnderstanding mollicutes is challenging due to their variety and relationship with host cells. Invasion has explained issues related to their opportunistic role. Few studies have been done on the Ureaplasma diversum mollicute, which is detected in healthy or diseased bovine. The invasion in Hep-2 cells of four clinical isolates and two reference strains of their ureaplasma was studied by Confocal Laser Scanning Microscopy and gentamicin invasion assay.ResultsThe isolates and strains used were detected inside the cells after infection of one minute without difference in the arrangement for adhesion and invasion. The adhesion was scattered throughout the cells, and after three hours, the invasion of the ureaplasmas surrounded the nuclear region but were not observed inside the nuclei. The gentamicin invasion assay detected that 1% of the ATCC strains were inside the infected Hep-2 cells in contrast to 10% to the clinical isolates. A high level of phospholipase C activity was also detected in all studied ureaplasma.ConclusionsThe results presented herein will help better understand U. diversum infections, aswell as cellular attachment and virulence.

Invasion of Ureaplasma diversum in bovine spermatozoids

BackgroundUreaplasma diversum has been associated with infertility in cows. In bulls, this mollicute colonizes the prepuce and distal portion of the urethra and may infect sperm cells. The aim of this study is to analyze in vitro interaction of U. diversum isolates and ATCC strains with bovine spermatozoids. The interactions were observed by confocal microscopy and the gentamycin internalization assay.FindingsU. diversum were able to adhere to and invade spermatozoids after 30 min of infection. The gentamicin resistance assay confirmed the intracellularity and survival of U. diversum in bovine spermatozoids.ConclusionsThe intracellular nature of bovine ureaplasma identifies a new difficulty to control the reproductive of these animals.

Intraspecific variation in 16S rRNA gene of Mycoplasma synoviae determined by DNA sequencing.

Mycoplasma synoviae (MS) is an important avian pathogen may cause both respiratory disease and joint inflammation synovitis in poultry, causing economic losses to the Brazilian poultry industry. The genotypic variation in 16S rRNA gene is unknown. Partial sequences of 16S rRNA gene of 19 strains of M. synoviae were sequenced and analyzed in order to obtain molecular characterization and evaluation of the genetic variability of strains from distinct Brazilian areas of poultry production. Different polymorphic patterns were observed. The number of polymorphic alterations in the studied strains ranged from 0 to 6. The nucleotide variations, including deletion, insertion and substitutions, ranged from 3 to 5. The genotypic diversity observed in this study may be explained by spontaneous mutations that may occur when a lineage remains in the same flock for long periods. The culling and reposition in poultry flocks may be responsible for the entry of new strains in different areas.

Prevalence of mycoplasmas in the respiratory tracts of calves in Brazil

MOST Mycoplasma species found in calf respiratory tracts are considered to be opportunistic pathogens; however, several species have been described as the primary agents for respiratory diseases (Simecka and others 1992). Mycoplasma bovis and Mycoplasma dispar have been considered the most important agents of pneumonia in cattle herds in which Mycoplasma mycoides subspecies mycoides SC is absent (Rebhun and others 2000). This short communication described a study to determine the prevalence of Mycoplasma species in the respiratory tracts of calves in Brazil. A total of 301 nasal swabs were collected from calves of both sexes, up to one year of age, from 10 different farms: seven in the State of Sao Paulo, two in Minas Gerais and one in Bahia. The samples were collected from 155 animals with respiratory disease and 146 clinically healthy animals by vigorously rubbing a swab over the surface of the nasal cavity. The nasal mucus samples were transported in 3 ml of modified Friis medium (Friis 1971) at 4°C and then incubated at 37°C for four days. The DNA was extracted from the samples by the method described by Fan and others (1995). The primers GPO-3 and MGSO (Van Kuppeveld and others 1992) were used in a PCR for the detection of Mollicutes. Samples that were positive for Mollicutes were submitted to other PCR assays using primers to detect M bovis (Gonzalez and others 1995), M mycoides subspecies mycoides SC (Dedieu and others 1994), M dispar (Marques and others 2007), and Ureaplasma diversum (Cardoso and others 2000). The PCR products were analysed on a 1·5 per cent agarose gel containing 0·5 μM/ml ethidium bromide in TAE buffer (40 mM Tris-acetate, 2 mM EDTA, pH 8·0). Table 1 shows the prevalence of Mollicutes and the four species investigated. Mollicutes were detected in 76 (52·06 per cent) of the nasal mucus samples from healthy animals and in 141 (90·96 per cent) of the samples from animals with respiratory disease. Although M dispar and U diversum were detected in both sick and healthy animals, M bovis was detected only in calves with respiratory disease. M mycoides subspecies mycoides SC was not detected in any of the samples. M dispar and U diversum were detected more frequently in samples from animals with respiratory disease than healthy animals. Of the 301 nasal mucus samples, 102 (33·88 per cent) were positive for at least one of the species tested for. The frequency of Mollicute co-infections in healthy and sick animals is shown in Table 2. M mycoides subspecies mycoides SC is the classical primary agent of contagious bovine pleuropneumonia. According to the World Organisation for Animal Health (OIE), healthy cattle herds must be free of this microorganism. This species was not detected in the present study; however, the authors recommended that a wide-ranging investigation using PCR should be carried out in Brazil. M bovis has previously been isolated in healthy animals, and was originally described as a natural microorganism of the bovine respiratory system (Knudtson and others 1986, Ter Laak and others 1992b). Currently, M bovis is the second most important mycoplasma in bovine respiratory diseases (Gevaert 2006). In the present study, M bovis was detected only in sick animals, either as a single agent or in association with M dispar or U diversum. The results obtained are in agreement with other studies that have mentioned M bovis as a species associated with respiratory disease outbreaks in cattle herds (Bashiruddin and others 2001, Byrne and others 2001). M dispar was detected more frequently in sick calves than in healthy animals. Ter Laak and others (1992a) reported M dispar to be the main species isolated from lung samples from sick calves (49 per cent of the samples). In contrast, Ter Laak and others (1992b) showed a frequency of 40 per cent of M dispar detection in healthy animals. In the present study, U diversum was detected by PCR in 12 (8·22 per cent) nasal mucus samples from healthy calves, and in 49 (31·6 per cent) samples from sick animals. Ter Laak and others (1992b) also detected U diversum in healthy animals as well; however, the frequency of detection was higher in sick animals. In the present study, healthy animals that were PCRpositive for M dispar and U diversum were in close contact with sick animals infected with these species, suggesting that M dispar and U diversum may be important pathogens in the development of respiratory disease. The detection of Mollicutes in nasal mucus from both sick and healthy calves may indicate the opportunistic role of these bacteria. The host-parasite relationship of mycoplasmas is a complex subject, especially in calves, in which the disease process in the respiratory tract is not well understood. Co-infections with mycoplasmas in the respiratory tracts of calves are common (Thomas and others 2002, Levisohn and others 2004). In the present study, three mycoplasma species were detected together in four samples, but an association between U diversum and M dispar was most common. Brazil is one of the most important cattle meat producers in the world. Consequently, it is important to monitor disease occurrence among Brazilian herds, including mycoplasma infections. The PCR results for identifying mycoplasmas in TABLE 2: Frequency of co-infections with more than one Mycoplasma species in the respiratory tracts of healthy calves and calves with respiratory disease

Mycoplasma synoviae cell invasion: Elucidation of the Mycoplasma pathogenesis in chicken

Fluorochrome-labelled cells of two field isolates and Mycoplasma synoviae (Ms) were inoculated onto monolayer cultures of fluorochrome-labelled HEp-2 cells and monitored by confocal laser scanning microscopy (CLSM). Ms was detected initially adhered to and subsequently inside the host cells. Between 24 and 48 h of infection, Ms was detected in the perinuclear region, and after 72 h of infection was confirmed by gentamicin invasion assay. High and low passage Ms strains showed no differences in adherence or invasion. The morphology and the actin filaments of the infected HEp-2 cells were preserved throughout the study period. The observed invasion by Ms is consistent with the biology of Mollicutes, and could explain the difficulties in recovering field isolates of the mycoplasma and in controlling the infection in birds even after long-term antibiotic treatment.

Intraspecific sequence variation in 16S rRNA gene of Ureaplasma diversum isolates

Ureaplasma diversum infection in bulls may result in seminal vesiculitis, balanoposthitis and alterations in spermatozoids. In cows, it can cause placentitis, fetal alveolitis, abortion and the birth of weak calves. U. diversum ATCC 49782 (serogroups A), ATCC 49783 (serogroup C) and 34 field isolates were used for this study. These microorganisms were submitted to Polymerase Chain Reaction for 16S gene sequence determination using Taq High Fidelity and the products were purified and bi-directionally sequenced. Using the sequence obtained, a fragment containing four hypervariable regions was selected and nucleotide polymorphisms were identified based on their position within the 16S rRNA gene. Forty-four single nucleotide polymorphisms (SNP) were detected. The genotypic variability of the 16S rRNA gene of U. diversum isolates shows that the taxonomy classification of these organisms is likely much more complex than previously described and that 16S rRNA gene sequencing may be used to suggest an epidemiologic pattern of different origin strains.

Frequency of different human mollicutes species in the mucosa of the oropharynx, conjunctiva, and genitalia of free-ranging and captive capuchin monkeys (Cebus spp.)

This study is the first to evaluate the occurrence of several Mollicutes species in Brazilian capuchin monkeys (Cebus spp.). Mollicutes were detected by culture and polymerase chain reaction (PCR) in samples of the oropharyngeal, conjuctiva, and genital mucosae of 58 monkeys. In the oropharynx, Mollicutes in general (generic PCR to the Class), and those of the genus Ureaplasma (genus PCR), were detected in 72.4% and 43.0% of the samples, respectively. The identified species in this site included: Mycoplasma arginini (43.1%), M. salivarium (41.4%), and M. pneumoniae (19.0%). Both Ureaplasma and Mycoplasma are genera of the order Mycoplasmatales. In the preputial/vaginal mucosa, PCR detected Mollicutes in general in 27.58% of the samples, the genus Ureaplasma in 32.7%, the species M. arginini in 8.6%, and Acholeplasma laidlawii of the order Acholeplasmatales in 1.7% In the conjunctiva, Mollicutes in general were detected in 29.3% of the samples, with 1.7% being identified as A. laidlawii. Culturing was difficult due to contamination, but two isolates were successfully obtained. The Mollicutes species of this study provided new insights into these bacteria in Brazilian Cebus. Studies are lacking of the actual risk of Mollicutes infection or the frequency at which primates serve as permanent or temporary reservoirs for Mollicutes. In the present study, the samples were collected from monkeys without clinical signs of infection. The mere presence of Mollicutes, particularly those also found in humans, nevertheless signals a need for studies to evaluate the impact of these microorganisms on the health of non‐human primates (NHPs) and the possibility of cross‐species transmission between NHPs and humans. Am. J. Primatol. 75:973–978, 2013.

Isolamento e PCR para detecção de Mollicutes em muco vaginal e sua associação com problemas reprodutivos em ovinos criados na região de Piedade, São Paulo, Brasil

It was evaluated the presence of Mycoplasma spp, Ureaplasma spp and Acholeplasma laidlawiii in 60 samples of ovine vaginal mucous with the presence or absence of vulvovaginitis in the specific exam of the reproductive tract. The microorganisms were characterized based on bacteriological culture and DNA detection by Polymerase Chain Reaction (PCR) with specific primers to Mollicutes (GPO and MGSO), Ureaplasma (UGPF and UGPS) and Acholeplasma laidlawii (UNI and ACH3). The presence of mycoplasmas could not be associated with reproductive disorders in animals. The PCR to Acholeplasma laidlawii detected only one positive sample. However, all isolations of Ureaplasma spp were from animals presenting reproductive disorders, suggesting a possible involvement of this agent in reproductive diseases.

Molecular characterisation of Mycoplasma hyorhinis isolated from pigs using pulsed-field gel electrophoresis and 16S rRNA sequencing

Economic loss in pig breeding is common due to respiratory disorders, and Mycoplasma hyopneumoniae and Mycoplasma hyorhinis, namely, are the most common infectious agents. The aim of this study is to recover these mollicutes and detect their genotypic variations by pulsed-field gel electrophoresis (PFGE) and sequencing the 16 s rRNA gene. One hundred and twenty-six swabs from tonsil and nasal mucus of pigs with respiratory disorders were analysed. A total of 78 lungs were sampled, as well as two trachea and two tonsils obtained from animals with respiratory disorder. A total of 59 isolates were obtained: 1 (1.70 per cent) of M hyopneumoniae, 2 (3.40 per cent) of Mycoplasma flocculare and 56 (94.90 per cent) of M hyorhinis. The PFGE for M hyorhinis showed 10 profiles with enzyme AvaI and 9 profiles with XhoI. A low polymorphism of the 16sRNS gene was detected in M hyorhinis isolates compared with the type strain in the GenBank. M hyorhinis isolates of different herds showed a large heterogenicity with enzymes AvaI and XhoI. The sequencing of the 16S rRNA gene allowed for analysing the interspecific and intraspecific variations of isolated mycoplasmas.

Molecular characterization of ureaplasmas isolated from reproductive tract of goats and sheep from Brazil

Ovine/caprine ureaplasmas have not yet been assigned a species designation, but they have been classified into nine serotypes. Herein ureaplasmas were searched for in 120 samples of vulvo vaginal mucous from sheep and 98 samples from goats at 17 farms. In addition, semen samples were collected from 11 sheep and 23 goats. The recovered ureaplasma were from sheep and goats from animals without any reproductive disorder symptoms, but not all animals presented positive cultures. In sheep, 17 (68%) cultures of vulvovaginal mucous were positive for ureaplasma and 11 (27%) samples of semen presented positive cultures in animals with clinical signs of orchitis, balanoposthitis or low sperm motility. In goats four ureaplasma isolates were obtained from vulvovaginal mucus, but the semen samples were all negative. The isolates were submitted to Pulsed-field gel electrophoresis methodology and their 16S rRNA genes were sequenced. Fifty percent of ureaplasma recovered from sheep allowed for PFGE typing. Eleven isolates showed eight profiles genetically close to the bovine ureaplasmas. The 16S rRNA gene sequencing showed differences or similarities of isolates from sheep and goats, and the reference strains of bovine and human ureaplasma. Four clinical isolates from sheep were grouped separately. The studied ureaplasma isolates showed to be a diverse group of mollicutes.