Association of spontaneous abortion and Ureaplasma parvum detected in placental tissue.

Spontaneous abortion is considered a public health problem having several causes, including infections. Among the infectious agents, bacteria of the vaginal microbiota and Ureaplasma parvum have been associated with abortion, but their participation needs to be further elucidated. This study aims to evaluate the influence of Mollicutes on the development of spontaneous abortion. Women who underwent spontaneous abortion and those with normal birth (control) were studied. Samples of cervical mucus (CM) and placental tissue were collected to identify Mollicutes using the quantitative polymerase chain reaction methodology. Eighty-nine women who had a miscarriage and 20 women with normal pregnancies were studied. The presence of Mollicutes in placental tissue increased the chance of developing miscarriage sevenfold. The prevalence of U. parvum in women who experienced spontaneous abortion was 66.3% in placental tissue. A positive association was observed between the detection of U. parvum in samples of placental tissue and abortion. There was a significant increase in microbial load in placental tissue for M. hominis, U. urealyticum and U. parvum compared to the control group. Detection of U. parvum in CM in pregnant women can ascend to the region of the placental tissue and trigger a spontaneous abortion.

Ureaplasma urealyticum and U. parvum in sexually active women attending public health clinics in Brazil.

Ureaplasma urealyticum and U. parvum have been associated with genital infections. The purpose of this study was to detect the presence of ureaplasmas and other sexually transmitted infections in sexually active women from Brazil and relate these data to demographic and sexual health, and cytokines IL-6 and IL-1ß. Samples of cervical swab of 302 women were examined at the Family Health Units in Vitória da Conquista. The frequency of detection by conventional PCR was 76·2% for Mollicutes. In qPCR, the frequency found was 16·6% for U. urealyticum and 60·6% U. parvum and the bacterial load of these microorganisms was not significantly associated with signs and symptoms of genital infection. The frequency found for Trichomonas vaginalis, Neisseria gonorrhoeae, Gardnerella vaginalis and Chlamydia trachomatis was 3·0%, 21·5%, 42·4% and 1·7%, respectively. Higher levels of IL-1ß were associated with control women colonized by U. urealyticum and U. parvum. Increased levels of IL-6 were associated with women who exhibited U. parvum. Sexually active women, with more than one sexual partner in the last 3 months, living in a rural area were associated with increased odds of certain U. parvum serovar infection.

Evaluation of Mollicutes Microorganisms in Respiratory Disease of Cattle and Their Relationship to Clinical Signs.

BACKGROUND: Bovine respiratory disease (BRD) is an important problem in cattle production that is responsible for economic losses in dairy herds. Mycoplasma spp. are described as an important etiological agent of BRD. HYPOTHESIS: To evaluate the occurrence of the most important mycoplasmas in the lower respiratory tract of healthy and BRD cattle in relationship to clinical signs of BRD. ANIMALS: Sixty young dairy cattle were classified as healthy (n = 32) or cattle showing clinical signs of BRD (n = 28). METHODS: Tracheal lavage samples were collected and added to tubes containing Hayflick media. Mycoplasma spp. were identified by the presence of "fried egg" like colonies, biochemical tests and polymerase chain reaction (PCR). Occurrence of Mollicutes, M. bovis, M. mycoides subsp. mycoides SC and M. dispar was evaluated. The association between clinical signs of BRD and the presence of Mycoplasma spp. also was evaluated. RESULTS: Colonies were obtained from a 1-year-old BRD calf only. However, species identification was not possible. Mollicutes (P = .035) and M. dispar (P = .036) were more common in BRD cattle. The relationship between Mollicutes and crackle (P = .057) was not significant. M. dispar was associated to tachypnea (P = .045) and mixed dyspnea (P = .003). Relationships to heart rate (P = .062) and crackle (P = .062) were not significant. CONCLUSIONS AND CLINICAL IMPORTANCE: The results confirmed the importance of mycoplasma as an etiologic agent of BRD and suggested M. dispar as part of the respiratory microbiota and its possible role in the development of BRD.

Qualitative analysis of the vaginal microbiota of healthy cattle and cattle with genital-tract disease.

The microbial community of the reproductive appara-tus, when known, can provide information about the health of the host. Metagenomics has been used to characterize and obtain genetic infor-mation about microbial communities in various environments and can relate certain diseases with changes in this community composition. In this study, samples of vaginal surface mucosal secretions were col-lected from five healthy cows and five cows that showed symptoms of reproductive disorders. Following high-throughput sequencing of the isolated microbial DNA, data were processed using the Mothur soft-ware to remove low-quality sequences and chimeras, and released to the Ribosomal Database Project for classification of operational taxo-nomic units (OTUs). Local BLASTn was performed and results were loaded into the MEGAN program for viewing profiles and taxonomic microbial attributes. The control profile comprised a total of 15 taxa, with Bacteroides, Enterobacteriaceae, and Victivallis comprising the highest representation of OTUs; the reproductive disorder-positive profile comprised 68 taxa, with Bacteroides, Enterobacteriaceae, His-tophilus, Victivallis, Alistipes, and Coriobacteriaceae being the taxa with the most OTU representation. A change was observed in both the community composition as well as in the microbial attributes of the profiles, suggesting that a relationship might exist between the patho-gen and representative taxa, reflecting the production of metabolites to disease progression.

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.

A quantitative TaqMan PCR assay for the detection of Mycoplasma suis.

AIM: To develop a TaqMan probe-based, highly sensitive and specific quantitative PCR (qPCR) assay for the detection and quantification of Mycoplasma suis in the blood of pigs. METHODS AND RESULTS: Primers and probes specific to Myc. suis 16S rRNA gene were designed. The qPCR assay's specificity, detection limit, intra- and inter-assay variability were evaluated and its performance was compared with a Myc. suis conventional PCR assay (cPCR). Blood of two experimentally infected pigs, 40 Indiana pigs, 40 Brazilian sows and 28 peccaries were tested. The assay detected as few as ten copies of Myc. suis plasmids and was 100-fold more sensitive than the cPCR. No cross-reactivity with nontarget pig mycoplasmas was observed. An average of 1·62 × 10(11) and 2·75 × 10(8) target copies ml(-1) of blood were detected in the acutely and chronically infected pigs, respectively. Three (7·5%) pigs and 32 (80·0%) sows were positive while all peccaries were negative for Myc. suis. CONCLUSION: The developed qPCR assay is highly sensitive and specific for Myc. suis detection and quantification. SIGNIFICANCE AND IMPACT OF THE STUDY: TaqMan qPCR is an accurate and quick test for detection of Myc. suis infected pigs, which can be used on varied instrumentation platforms.

Detection of Bartonella spp. in neotropical felids and evaluation of risk factors and hematological abnormalities associated with infection.

Although antibodies to Bartonella henselae have been described in all neotropical felid species, DNA has been detected in only one species, Leopardus wiedii. The aim of this study was to determine whether DNA of Bartonella spp. could be detected in blood of other captive neotropical felids and evaluate risk factors and hematological findings associated with infection. Blood samples were collected from 57 small felids, including 1 Leopardus geoffroyi, 17 L. wiedii, 22 Leopardus tigrinus, 14 Leopardus pardalis, and 3 Puma yagouaroundi; 10 blood samples from Panthera onca were retrieved from blood banks. Complete blood counts were performed on blood samples from small felids, while all samples were evaluated by PCR. DNA extraction was confirmed by amplification of the cat GAPDH gene. Bartonella spp. were assessed by amplifying a fragment of their 16S-23S rRNA intergenic spacer region; PCR products were purified and sequenced. For the small neotropical felids, risk factors [origin (wild-caught or zoo-born), gender, felid species, and flea exposure] were evaluated using exact multiple logistic regression. Hematological findings (anemia, polycythemia/hyperproteinemia, leukocytosis and leukopenia) were tested for association with infection using Fisher's exact test. The 635bp product amplified from 10 samples (10/67=14.92%) was identified as B. henselae by sequencing. Small neotropical felid males were more likely to be positive than females (95% CI=0.00-0.451, p=0.0028), however other analyzed variables were not considered risk factors (p>0.05). Hematological abnormalities were not associated with infection (p>0.05). This is the first report documenting B. henselae detection by PCR in several species of neotropical felids.

Detection of Mycoplasma pulmonis in laboratory rats and technicians.

Five species of mycoplasma are associated with several rat diseases. Mycoplasma pulmonis is the most important and most studied, possibly causing disease in rats and undermining the validity of laboratory experiments. M. pulmonis was isolated in 144/240 laboratory rats and identified by PCR in 155/240. This species was also detected in 12 human individuals (technicians of a laboratory animal house hold) in contact with these rats. The results were confirmed by sequencing of DNA products. Mycoplasma species are host specific; however, M. pulmonis was identified in humans, suggesting a case of unspecific colonization. Statistical analysis shows a greater risk for M. pulmonis colonizing individuals who are exposed to infected rats in animal facilities than individuals who do not. The detection of M. pulmonis in humans indicates a new status for this mollicute mycoplasmas in animal-holding facilities.

Detecção de Mycoplasma spp. e Ureaplasma diversum em vacas com distúrbios reprodutivos / Detection of Mycoplasma spp. and Ureaplasma diversum in cown with reproductive disorders

Foram utilizadas 112 amostras de muco vulvovaginal, coletados de vacas com distúrbios reprodutivos, para pesquisa de Mycoplasma e Ureaplasma. Para isolamentos, foram usados meios específicos para micoplasmas (SP-4) e para ureaplasmas. PCR genérica, PCR específica para Mycoplasma bovis e nested-PCR em tubo único para Ureaplasma diversum foram realizados com os DNAs extraídos das amostras. Mycoplasma spp. e U. diversum foram detectados em 12,5 e 25,0 por cento, respectivamente. A PCR genérica resultou em reações positivas em 63,4 por cento das amostras transportadas em SP-4 e em 69,6 por cento das transportadas em meio de ureaplasma. M. bovis foi detectado, na PCR específica, em 9,8 por cento das amostras e U. diversum, na nested-PCR, em 37,5 por cento. Houve maior sensibilidade na metodologia da PCR quando comparada à técnica de cultivo para Mycoplasma e Ureaplasma

In the study, 112 samples of vulvovaginal mucus of cows bearing reproductive disturbance were investigated for Mycoplasma and Ureaplasma. Specific media for the culture of mycoplasmas (SP-4) and ureaplasmas were used. PCR with general primers, PCR specific for Mycoplasma bovis, and nested-PCR in a single tube for Ureaplasma diversum were performed to detect DNA of the sample. Mycoplasma spp. and U. diversum were isolated in 12.5 and 25.0 percent, respectively. With generic PCR, positive reaction was obtained in 63.4 percent of the samples transported in SP-4 and 69.6 percent in ureaplasma medium. M. bovis was detected in 9.8 percent of samples and nested-PCR in a single tube for U. diversum resulted in 35.0 percent of positive reaction. Results demonstrated increased sensitivity of PCR methodology compared with culture technique applied to the search of microorganisms of Mycoplasma and Ureasplasma genera

Use of a polymerase chain reaction for detection of Mycoplasma dispar in the nasal mucus of calves.

A polymerase chain reaction (PCR) assay was developed for the detection of Mycoplasma dispar in nasal mucus samples collected from calves. The target DNA sequence was the 16S rRNA gene, and the fragment was selected within a region of high polymorphism. The specificity and detection limit of the method were determined. This method was then used for the detection of M. dispar in nasal swabs collected from 301 calves, including 155 clinical samples from animals showing signs of respiratory disease and 146 samples from healthy animals. PCR with generic primers was applied to the detection of Mollicutes, followed by the detection of M. dispar. Mollicutes were detected in 52.05% of clinical samples from healthy animals and in 90.96% of samples from sick animals. Mycoplasma dispar was detected in 6.16% of healthy animals and in 34.84% of sick animals. The PCR assay was useful in verifying the presence of M. dispar in calves and may be a useful tool in monitoring this mycoplasma in cattle herds.

Molecular characterization of MG isolates using RAPD and PFGE isolated from chickens in Brazil.

In the present study, 27 primers were screened under different cycles by random amplified polymorphic DNA (RAPD) method. Mathematical models were used for analysis of the genetic relationships among strains, including vaccinal, reference strains and nine field isolates previously characterized as Mycoplasma gallisepticum (MG)F by RAPD and pulsed field gel electrophoresis (PFGE). The PFGE was considered as laborious, expensive and time-consuming than RAPD method. These methods improved the typeability for epidemiological studies of MG with regard to differentiation from vaccinal and field strains.

Detection of multiple mycoplasma infection in cell cultures by PCR.

A total of 301 cell cultures from 15 laboratories were monitored for mycoplasma (Mollicutes) using PCR and culture methodology. The infection was detected in the cell culture collection of 12 laboratories. PCR for Mollicutes detected these bacteria in 93 (30.9%) samples. Although the infection was confirmed by culture for 69 (22.9%) samples, PCR with generic primers did not detect the infection in five (5.4%). Mycoplasma species were identified with specific primers in 91 (30.2%) of the 98 samples (32.6%) considered to be infected. Mycoplasma hyorhinis was detected in 63.3% of the infected samples, M. arginini in 59.2%, Acholeplasma laidlawii in 20.4%, M. fermentans in 14.3%, M. orale in 11.2%, and M. salivarium in 8.2%. Sixty (61.2%) samples were co-infected with more than one mycoplasma species. M. hyorhinis and M. arginini were the microorganisms most frequently found in combination, having been detected in 30 (30.6%) samples and other associations including up to four species were detected in 30 other samples. Failure of the treatments used to eliminate mycoplasmas from cell cultures might be explained by the occurrence of these multiple infections. The present results indicate that the sharing of non-certified cells among laboratories may disseminate mycoplasma in cell cultures.

Detection of multiple mycoplasma infection in cell cultures by PCR

A total of 301 cell cultures from 15 laboratories were monitored for mycoplasma (Mollicutes) using PCR and culture methodology. The infection was detected in the cell culture collection of 12 laboratories. PCR for Mollicutes detected these bacteria in 93 (30.9 percent) samples. Although the infection was confirmed by culture for 69 (22.9 percent) samples, PCR with generic primers did not detect the infection in five (5.4 percent). Mycoplasma species were identified with specific primers in 91 (30.2 percent) of the 98 samples (32.6 percent) considered to be infected. Mycoplasma hyorhinis was detected in 63.3 percent of the infected samples, M. arginini in 59.2 percent, Acholeplasma laidlawii in 20.4 percent, M. fermentans in 14.3 percent, M. orale in 11.2 percent, and M. salivarium in 8.2 percent. Sixty (61.2 percent) samples were co-infected with more than one mycoplasma species. M. hyorhinis and M. arginini were the microorganisms most frequently found in combination, having been detected in 30 (30.6 percent) samples and other associations including up to four species were detected in 30 other samples. Failure of the treatments used to eliminate mycoplasmas from cell cultures might be explained by the occurrence of these multiple infections. The present results indicate that the sharing of non-certified cells among laboratories may disseminate mycoplasma in cell cultures.

[Evaluation of bacterial contamination in disinfectants for domestic use]. / Avaliação da contaminação bacteriana em desinfetantes de uso domiciliar.

OBJECTIVE: To evaluate disinfectants for domestic use for the presence of bacteria, identify them, and determine their tolerance level to benzalkonium chloride. METHODS: Fifty-two samples of commercially available disinfectants for domestic use were acquired at random in the metropolitan area of São Paulo, Brazil, and analyzed to detect the presence of bacterial contaminants. The isolated organisms were identified and their tolerance level to benzalkonium chloride was determined by broth macrodilution method. RESULTS: Sixteen (30.77%) of fifty-two disinfectants sampled were contaminated by Gram-negative bacteria, with counts varying between 10(4) and 10(6) UFC/ml. Alcaligenes xylosoxidans, Burkholderia cepacia and Serratia marcescens were the predominant organisms found. The minimum inhibitory concentration (MIC: mg/ml) of benzalkonium chloride for these bacteria were 2.48, 1.23 and 0.30 to S. marcescens, A. xylosoxidans and B. cepacia, respectively. CONCLUSIONS: The disinfectant formulation containing quaternary ammonium compounds (QACs) may be exposed to contamination by Gram-negative bacteria. The MICs of benzalkonium chloride against the isolated bacteria were low, indicating that the bacteria grown in culture media without QACs lost their tolerance to this biocide.

Detection of Ureaplasma diversum in cattle using a newly developed PCR-based detection assay.

Ureaplasma diversum has been associated with different clinical manifestations including bovine vulvitis, endometritis, salpingitis, spontaneous abortion and infertility. Because the isolation of this ureaplasma from clinical samples is difficult, there is a need for improved detection methods. We developed a PCR assay based on amplification of a region of the gene encoding 16S rRNA. The specificity of the amplification was verified by sequence analysis. Female bovine vaginal swabs (n=168) were collected and the presence of U. diversum evaluated by both culture methods and by the PCR assay. Culture was positive for 60 samples (35.7%), and PCR-specific amplification was obtained for 89 samples (52.9%). These results indicated a high prevalence of U. diversum in the selected animals and the higher sensitivity of this PCR assay as compared to culture.

Mycoplasma arginini enhances cytotoxicity of thioglycollate-elicited murine macrophages toward YAC-1 tumor cells through production of NO.

Bacterial products stimulate macrophage tumoricidal activity through release of tumor necrosis factor (TNF) and nitric oxide (NO). We show here that thioglycollate-elicited macrophages acquire cytotoxic activity when cocultured with Mycoplasma arginini-infected YAC-1 tumor cells and release TNF and NO. Fixed mycoplasma-infected cells, supernatants from infected-cell cultures, or purified heat-killed mycoplasma obtained from cell-free cultures were all able to induce TNF and NO production. Thus, the mycoplasma per se and not a product of infected cells induce the release of these molecules. Addition of prostaglandin E2 (PGE2) to the cocultures, which reduced TNF release, or antibodies to TNF, did not affect macrophage cytotoxicity nor NO release. Inhibition of NO production by L-NAME or aminoguanidine reduced the cytotoxicity, and treatment with a NO donor was toxic to YAC-1 cells. These results indicate that M. arginini activates thioglycollate-elicited murine macrophages for NO and TNF release increasing their cytotoxic activity toward YAC-1 cells and that this activity is dependent on NO but not TNF release.

Thioglycollate-elicited murine macrophages are cytotoxic to Mycoplasma arginini-infected YAC-1 tumor cells

Macrophages are important components of natural immunity involved in inhibition of tumor growth and destruction of tumor cells. It is known that these cells can be activated for tumoricidal activity by lymphokines and bacterial products. We investigated whether YAC-1 tumor cells infected with Mycoplasma arginini stimulate nitric oxide (NO) release and macrophage cytotoxic activity. Thioglycollate-elicited macrophages from male BALB/c mice were co-cultured for 20 h with YAC-1 tumor cells infected or not with Mycoplasma arginini. The cytotoxic activity was evaluated by MTT assay and nitrite levels were determined with the Griess reagent. Thioglycollate-elicited macrophages co-cultured with noninfected YAC-1 cells showed low cytotoxic activity (34.7 ñ 8.6per cent) and low production of NO (4.7 ñ 3.1 µM NO2-). These macrophages co-cultured with mycoplasma-infected YAC-1 cells showed significantly higher cytotoxic activity (61.4 ñ 9.1 per cent; P=0.05) and higher NO production (48.5 ñ 13 µM NO2-; P=0.05). Addition of L-NAME (10 mM), an inhibitor of NO synthesis, to these co-cultures reduced the cytotoxic activity to 37.4 ñ 2per cent (P=0.05) and NO production to 3 ñ 4 µM NO2- (P=0.05). The present data show that Mycoplasma arginini is able to induce macrophage cytotoxic activity and that this activity is partially mediated by NO.

Thioglycollate-elicited murine macrophages are cytotoxic to Mycoplasma arginini-infected YAC-1 tumor cells.

Macrophages are important components of natural immunity involved in inhibition of tumor growth and destruction of tumor cells. It is known that these cells can be activated for tumoricidal activity by lymphokines and bacterial products. We investigated whether YAC-1 tumor cells infected with Mycoplasma arginini stimulate nitric oxide (NO) release and macrophage cytotoxic activity. Thioglycollate-elicited macrophages from male BALB/c mice were co-cultured for 20 h with YAC-1 tumor cells infected or not with Mycoplasma arginini. The cytotoxic activity was evaluated by MTT assay and nitrite levels were determined with the Griess reagent. Thioglycollate-elicited macrophages co-cultured with noninfected YAC-1 cells showed low cytotoxic activity (34.7 +/- 8.6%) and low production of NO (4.7 +/- 3.1 microM NO2-). These macrophages co-cultured with mycoplasma-infected YAC-1 cells showed significantly higher cytotoxic activity (61.4 +/- 9.1%; P < 0.05) and higher NO production (48.5 +/- 13 microM NO2-; P < 0.05). Addition of L-NAME (10 mM), an inhibitor of NO synthesis, to these co-cultures reduced the cytotoxic activity to 37.4 +/- 2% (P < 0.05) and NO production to 3 +/- 4 microM NO2- (P < 0.05). The present data show that Mycoplasma arginini is able to induce macrophage cytotoxic activity and that this activity is partially mediated by NO.