Invasion of Ureaplasma diversum in Hep-2 cells.

BACKGROUND: Understanding 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. RESULTS: The 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. CONCLUSIONS: The results presented herein will help better understand U. diversum infections, aswell as cellular attachment and virulence.

Survey of feline leukemia virus and feline coronaviruses in captive neotropical wild felids from Southern Brazil.

A total of 57 captive neotropical felids (one Leopardus geoffroyi, 14 Leopardus pardalis, 17 Leopardus wiedii, 22 Leopardus tigrinus, and three Puma yagouaroundi) from the Itaipu Binacional Wildlife Research Center (Refúgio Bela Vista, Southern Brazil) were anesthetized for blood collection. Feces samples were available for 44 animals, including one L. geoffroyi, eight L. pardalis, 14 L. wiedii, 20 L. tigrinus, and one P. yagouaroundi. Total DNA and RNA were extracted from blood and feces, respectively, using commercial kits. Blood DNA samples were evaluated by polymerase chain reaction (PCR) for feline leukemia virus (FeLV) proviral DNA, whereas reverse transcriptase-PCR was run on fecal samples for detection of coronavirus RNA. None of the samples were positive for coronaviruses. A male L. pardalis and a female L. tigrinus were positive for FeLV proviral DNA, and identities of PCR products were confirmed by sequencing. This is the first evidence of FeLV proviral DNA in these species in Southern Brazil.

Genotyping of Ureaplasma diversum isolates using pulsed-field electrophoresis.

Ureaplasma diversum has been associated with reproductive disorders in cattle and in the present study genotypic variations among U. diversum isolates obtained from the vaginal mucus of healthy cattle and sick animals were analyzed by enzymatic digestion and pulsed-field gel electrophoresis (PFGE). The influence of time and broth volume was important in obtaining sufficient cell sediment and DNA for PFGE. The method presented a high discriminatory power and satisfactory reproducibility for the analysis of detected variations among U. diversum isolates and strains. Different band profiles and wide genotypic heterogeneity were detected but no association between DNA polymorphism and sick or healthy animals could be established.

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.

Validation of FoodChek™ - Salmonella for Rapid Detection of Salmonella in Eggs, Derivative Products, and the Environment.

The FoodChek™ - Salmonella assay is an immunomagnetic lateral flow assay for the rapid detection (shorter than 24 h) of the most frequently isolated Salmonella (groups B-E) in eggs, egg-derivative products, and environmental surfaces. The FoodChek - Salmonella assay correctly identified 99.6% (239/240) of the samples tested in the matrix studied, and the statistical analysis of the method comparison study results demonstrated that it performs as well as U.S. culture-based reference methods. Ninety-nine percent of the 103 Salmonella strains tested belonging to serogroups B-E were detected during the inclusivity study. Concerning the exclusivity, 31 nontarget strains were tested. No cross-reactivity was observed in FoodChek - Salmonella assay enrichment conditions. In addition, the assay shows strong robustness, good stability, and consistency among lots. The present study proves that the assay is an effective tool for the rapid detection of Salmonella spp. in whole liquid eggs, liquid egg white (liquid egg albumen), shell eggs, dried whole eggs, dried egg yolks, and environmental surfaces as stainless steel, plastic, rubber, ceramic tiles, and sealed concrete.

Ureaplasma diversum Genome Provides New Insights about the Interaction of the Surface Molecules of This Bacterium with the Host.

Whole genome sequencing and analyses of Ureaplasma diversum ATCC 49782 was undertaken as a step towards understanding U. diversum biology and pathogenicity. The complete genome showed 973,501 bp in a single circular chromosome, with 28.2% of G+C content. A total of 782 coding DNA sequences (CDSs), and 6 rRNA and 32 tRNA genes were predicted and annotated. The metabolic pathways are identical to other human ureaplasmas, including the production of ATP via hydrolysis of the urea. Genes related to pathogenicity, such as urease, phospholipase, hemolysin, and a Mycoplasma Ig binding protein (MIB)-Mycoplasma Ig protease (MIP) system were identified. More interestingly, a large number of genes (n = 40) encoding surface molecules were annotated in the genome (lipoproteins, multiple-banded antigen like protein, membrane nuclease lipoprotein and variable surface antigens lipoprotein). In addition, a gene encoding glycosyltransferase was also found. This enzyme has been associated with the production of capsule in mycoplasmas and ureaplasma. We then sought to detect the presence of a capsule in this organism. A polysaccharide capsule from 11 to 17 nm of U. diversum was observed trough electron microscopy and using specific dyes. This structure contained arabinose, xylose, mannose, galactose and glucose. In order to understand the inflammatory response against these surface molecules, we evaluated the response of murine macrophages J774 against viable and non-viable U. diversum. As with viable bacteria, non-viable bacteria were capable of promoting a significant inflammatory response by activation of Toll like receptor 2 (TLR2), indicating that surface molecules are important for the activation of inflammatory response. Furthermore, a cascade of genes related to the inflammasome pathway of macrophages was also up-regulated during infection with viable organisms when compared to non-infected cells. In conclusion, U. diversum has a typical ureaplasma genome and metabolism, and its surface molecules, including the identified capsular material, represent major components of the organism immunopathogenesis.

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

Abstract 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.

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.

Invasion of Ureaplasma diversum in bovine spermatozoids.

BACKGROUND: Ureaplasma 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. FINDINGS: U. 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. CONCLUSIONS: The 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.

Evaluation of a PCR multiplex for detection and differentiation of Mycoplasma synoviae, M. gallisepticum, and M. gallisepticum strain F-vaccine / Avaliação de uma PCR multiplex para detecção e diferenciação de Mycoplasma synoviae, Mycoplasma gallisepticum e Mycoplasma gallisepticum cepa F vacinal

Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are the mycoplasma infections of most concern for commercial poultry industry. MG infection is commonly designated as chronic respiratory disease (CRD) of chickens and infections sinusitis of turkeys. MS causes sub clinical upper respiratory infection and tenosynovitis or bursitis in chickens and turkeys. The multiplex PCR was standardized to detect simultaneously the MS, MG field strains and MG F-vaccine strain specific. The generic PCR for detection of any species of Mollicutes Class was performed and compared to the multiplex PCR and to PCR using species-specific primers. A total of 129 avian tracheal swabs were collected from broiler-breeders, layer hens and broilers in seven different farms and were examined by multiplex PCR methods. The system (multiplex PCR) demonstrated to be very rapid, sensitive, and specific. Therefore, the results showed a high prevalence of MS in the flocks examined (27.9%), and indicate that the MS is a recurrent pathogen in Brazilian commercial poultry flocks...

Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) são micoplasmas que causam infecção de maior preocupação para a indústria avícola. MG é a bactéria responsável pela infecção, comumente designada, como doença crônica respiratória (DCR) de galinhas e sinusite infecciosa de perus. MS é responsável por infecções subclínicas do trato respiratório superior e tenosinovite ou bursite em galinha e perus. A reação da PCR multiplex foi padronizada para detectar simultaneamente MS, MG cepa de campo e MG-F cepa vacinal. A PCR genérica para detecção de qualquer espécie de Mycoplasma foi realizada e comparada a PCR multiplex e a PCR com primers específicos. O total de 129 amostras de suabes de traqueia foi coletado de reprodutoras pesadas, poedeiras e frangos em sete diferentes empresas avícolas e então foram examinados por PCR multiplex. O sistema da PCR multiplex demonstrou ser muito rápido, sensível e específico. Então, os resultados mostraram uma alta prevalência de MS nos lotes examinados ( 27,9%), e indica que MS é um patógeno recorrente nos lotes de aves comerciais brasileiro...

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 / Mollicutes isolation and PCR on ovine vaginal mucous and its association with reproductive problems in Piedade, SP, Brazil

Pesquisou-se Mycoplasma spp, Ureaplasma spp e Acholeplasma laidlawiii em amostras de muco vaginal de 60 ovinos, criados na região de Piedade no Estado de São Paulo, Brasil, que apresentavam ou não vulvovaginite no exame específico do sistema genital. A caracterização desses microrganismos baseou-se no cultivo e detecção do respectivo DNA pela Reação da Polimerase em Cadeia (PCR) com os primers para classe Mollicutes (GPO e MGSO), para o gênero Ureaplasma (UGPF e UGPS) e a espécie Acholeplasma laidlawii (UNI e ACH3). A presença de micoplasmas não foi associada com distúrbios do trato reprodutivo dos animais, entretanto todos os isolados obtidos de Ureaplasma spp foram provenientes de animais com distúrbios reprodutivos, sugerindo o possível envolvimento desse agente nas enfermidades da reprodução. A PCR para a espécie Acholeplasma laidlawii detectou somente uma amostra positiva.

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.

Isolates of Ureplasma diversum genotyped by single-enzyme amplified length polymorphism

Isolates of Ureaplasma diversum recovered from bovines with reproductive disorders and healthy ones of four premises were compared by SE-AFLP. Twenty-eight SE-AFLP profiles without monomorphic fragments were obtained. The ureaplasma studied were divided in clusters A and B. Cluster A was divided in subclusters A1 and A2, while A1 was divided in subclusters A1a and A1b. Cluster B grouped only the reference strains. The clusters obtained were not associated with the reproductive disorders. The dendrogram obtained showed high heterogeneity among the studied ureaplasmas and indicated a low genomic stability as detected in other species of microorganisms of class Mollicutes.

Cepas de referência e 30 estirpes de Ureaplasma diversum isoladas do muco vaginal de bovinos apresentando ou não distúrbios reprodutivos, de quatro diferentes propriedades, foram comparadas por meio da metodologia da SE-AFLP (single-enzyme amplified fragment length polymorphism). Foram obtidos 28 perfis, com ausência de fragmentos monomórficos. No dendrograma, as amostras foram divididas em grupos A e B. O grupo A foi subdividido em A1 e A2 e o A1 dividiu-se em A1a e A1b. As amostras de referência formaram o grupo B. Não houve diferenciação entre as estirpes isoladas de animais doentes ou sadios. Evidenciou-se grande heterogeneidade entre os ureaplasmas estudados indicando baixa estabilidade genômica, como detectado em outras espécies dos microrganismos da classe Mollicutes.

Mastite bovina por Mycoplasma bovis em rebanhos leiteiros / Mastitis caused by Mycoplasma bovis in dairy cattle

Foram examinadas 713 vacas de três rebanhos leiteiros localizados na regiäo norte do Estado do Paraná e sudoeste do Estado de Säo Paulo, das quais 137 apresentaram mastite. Nas três propriedades foram detectados oito animais (1,12 por cento) com mastite clínica por Mycoplasma bovis. Destes animais, quatro tratados com oxitetraciclina e tilosina e três com enrofloxacina, näo responderam ao tratamento e foram descartados no decorrer da lactaçäo. Uma vaca medicada com enrofloxacina recuperou quase que totalmente a secreçäo láctea mas a eliminaçäo de M. bovis persistiu por toda lactaçäo. Esta vaca apresentou cura bacteriológica na lactaçäo seguinte. O descarte dos animais positivos, monitoramento bacteriológico e a aplicaçäo correta das medidas de prevençäo para as mastites contagiosas controlaram a disseminaçäo de M. bovis nos rebanhos

Etiologia das infecções intramamárias em vacas primíparas no período pós-parto / Etiology of intramammary infections in primiparous cows at postparturition

Foram analisadas 664 amostras de leite de 83 vacas primíparas da raça Holandesa. As amostras foram colhidas no primeiro e no sétimo dia após o parto. Das 664 amostras analisadas, 488 (73,50%) foram bacteriologicamente negativas e 176 (26,50%), positivas para microrganismos aeróbios. Foi observado um alto índice de mastite clínica (20,48%). Os agentes isolados com maior freqüência foram os Staphylococcus spp coagulase negativo (64,20%), Staphylococcus spp coagulase positivo (8,52%), Streptococcus spp (7,96%), Actinomyces pyogenes (4,55%), Mycoplasma bovigenitalium (3,40%) e Escherichia coli (2,84%). Foi observado um maior índice de isolamento de patógenos no primeiro dia (17,62%) em relação ao sétimo (8,88%)

Etiologia das infecções intramamárias em vacas primíparas no período pós-parto

A total of 664 foremilk samples from 83 Holstein cows were cultured. Quarter samples were collected at parturition and 7 days post the first parturition. From 664 milk samples examined, 488 (73.50%) were bacteriologically negative and 176 (26.50%) were positive. A high incidence (20.48%) of clinical mastitis was observed. The most frequently encountered isolates were coagulase negative staphylococci (64.20%), coagulase positive staphylococci (8.52%), streptococci (7.96%), Actinomyces pyogenes (4.55%), Mycoplasma bovigenitalium (3.40%) and Escherichia coli (2.84%). The frequency of mastitis pathogen isolation was highest at parturition (17.62%) and decreased markedly during the first week (8.88%).

Foram analisadas 664 amostras de leite de 83 vacas primíparas da raça Holandesa. As amostras foram colhidas no primeiro e no sétimo dia após o parto. Das 664 amostras analisadas, 488 (73,50%) foram bacteriologicamente negativas e 176 (26,50%), positivas para microrganismos aeróbios. Foi observado um alto índice de mastite clínica (20,48%). Os agentes isolados com maior freqüência foram os Staphylococcus spp coagulase negativo (64,20%), Staphylococcus spp coagulase positivo (8,52%), Streptococcus spp (7,96%), Actinomyces pyogenes (4,55%), Mycoplasma bovigenitalium (3,40%) e Escherichia coli (2,84%). Foi observado um maior índice de isolamento de patógenos no primeiro dia (17,62%) em relação ao sétimo (8,88%).