Population pharmacokinetics and pharmacokinetic/pharmacodynamic evaluation of marbofloxacin against Coagulase-negative staphylococci, Staphylococcus aureus and Mycoplasma agalactiae pathogens in goats.

Marbofloxacin is a broad-spectrum fluoroquinolone, and an extra-label use has been reported in horse, sheep and goat. However, extrapolation of dosage regimens from cattle to horse and small ruminants could lead to incorrect dosing due to pharmacokinetic differences among species, increasing the risk of antimicrobial resistance or toxicity. Pharmacokinetic properties of marbofloxacin, including PK/PD analysis, have been studied by intravenous, intramuscular and subcutaneous administration in lactating and non-lactating goats. A population pharmacokinetic model of marbofloxacin in goats was built using 10 pharmacokinetic studies after intravenous, intramuscular, and subcutaneous administration at a dose of 2, 5 and 10 mg/kg. Serum or plasma and milk concentration-time profiles were simultaneously fitted with a non-linear mixed effect model with Monolix software. Level of milk production (lactating and non-lactating) and health status (healthy and un-healthy) were retained as covariates on volume of distribution and clearance. Marbofloxacin concentrations were well described in plasma/serum and milk by the population model. Simulated dose regimens of marbofloxacin administered at 2, 5 and 10 mg/kg by intramuscular route for five days were evaluated (n = 5000 per group). Steady-state fAUCs for each dose regimen were obtained. Probability of target attainment of fAUC/MIC ratios were determined and PK/PDco values (highest MIC for which 90% of individuals can achieve a prior numerical value of the fAUC/MIC index) were established using Monte Carlo simulations (n = 50,000). MIC values for wild type isolates of Staphylococcus aureus, coagulase negative staphylococci, and Mycoplasma agalactiae were determined and tentative epidemiological cutoff (TECOFF) were obtained at 1.0, 0.5 and 0.5 mg/L, respectively. The PK/PDco for the dose regimen of 2 mg/kg/24 h and 5 mg/kg/24 h (0.125 and 0.25 mg/L) were lower than TECOFF (0.5 and 1 mg/L). The dosage regimen of 10 mg/kg/24 h was adequate for intermediate MIC values of 0.125-0.50 mg/L and could be effective for a population with a target fAUC/MIC ratio ˂ 48 for Coagulase negative staphylococci and Mycoplasma agalactiae, but not for Staphylococcus aureus. Results obtained in this study could be taken as a starting point by committees that set the clinical breakpoints and justifies expert rules to optimize marbofloxacin dose regimens.

A novel fusion protein candidate for the serodiagnosis of Mycoplasma agalactiae infection.

BACKGROUND: The aim of current study was to construct, express, purify and immunogenicity evaluate of a novel recombinant fusion protein including Pyruvate dehydrogenase beta subunit (PDHB) and high antigenic region of lipoprotein P80 of Mycoplasma agalactiae. Using bioinformatics tools, antigenicity and physiochemical properties of fused protein were assessed. MATERIAL AND METHODS: The recombinant fusion protein of GST-PDHB-P80 were expressed in pGEX4T-1 and purified then verified by Western blot assay. The purified protein was successfully used for immunization of mice. 30 female BALB/c mice were divided into three groups (10 mice per each group) injected with GST-PDHB-P80, inactivated bacteria vaccine and PBS as negative control, separately. RESULTS: Western blot analysis confirmed the interaction between the immunized mice serum and the blotted recombinant protein GST-PDHB-P80, demonstrating the immunogenicity of this protein. Moreover, the sera of vaccinated mice with inactivated bacteria vaccine, containing whole cell proteins, detected the recombinant protein GST-PDHB-P80 confirming the antigenicity of PDHB-P80. Negative control displayed no reactivity with GST-PDHB-P80. CONCLUSION: We proposed a novel designed chimeric protein of Mycoplasma agalactiae as a potential marker for serodiagnostic assays but still further field research is required.

Assessment of the duration of maternal-derived antibodies specific to the Mycoplasma agalactiae vaccine in goat kids.

BACKGROUND: Contagious agalactia (CA) is one of the most important diseases in the small ruminant industry in Iran. The historical aetiology of this disease is Mycoplasma agalactiae (Ma). The main way to control this disease, in addition to management measures, is vaccination. In ruminant newborns, determining the age of first vaccination against Ma is a challenge due to the interference between colostrum-derived maternal immunity and vaccination-induced immunity. The aim of this study was to evaluate the consistency of maternal-derived antibodies specific to the Ma in goat kids blood serum born from the vaccinated does. OBJECTIVES: Dtermination of level of antibody against Ma in goat kids born from vaccinated dams against Ma. Assessment of duration of protective level of maternal derived antibody in goat kids serum, after receiving colostrum from vaccinted mother with Ma vaccine. Determination the best time vaccination against Ma in goat kids receiving colostrum from vaccinated dams. METHODS: 20 Saanen goat kids were studied in two groups of 10 animals including control (receiving colostrum from unvaccinated does) and treatment (receiving colostrum from vaccinated does). Indirect Elisa was used to evaluate serum specific antibodies to Ma in goat kids (control and treatment groups) from birth to 100 days of age. RESULTS: After receiving a sufficient amount of colostrum, the goat kids in the treatment group had a significantly higher S/P% than the control group until 56 days after birth (p < 0.05) and at 70-100 days after birth, there was no significant difference between the treatment and control groups (p > 0.05). CONCLUSIONS: This study showed that 56-70 days of age could be a good age to give the first dose of CA vaccine in goat kids, but more studies are needed on the effectiveness of this vaccine at this age.

Impacts of Mycoplasma agalactiae restriction-modification systems on pan-epigenome dynamics and genome plasticity.

DNA methylations play an important role in the biology of bacteria. Often associated with restriction modification (RM) systems, they are important drivers of bacterial evolution interfering in horizontal gene transfer events by providing a defence against foreign DNA invasion or by favouring genetic transfer through production of recombinogenic DNA ends. Little is known regarding the methylome of the Mycoplasma genus, which encompasses several pathogenic species with small genomes. Here, genome-wide detection of DNA methylations was conducted using single molecule real-time (SMRT) and bisulphite sequencing in several strains of Mycoplasma agalactiae, an important ruminant pathogen and a model organism. Combined with whole-genome analysis, this allowed the identification of 19 methylated motifs associated with three orphan methyltransferases (MTases) and eight RM systems. All systems had a homolog in at least one phylogenetically distinct Mycoplasma spp. Our study also revealed that several superimposed genetic events may participate in the M. agalactiae dynamic epigenomic landscape. These included (i) DNA shuffling and frameshift mutations that affect the MTase and restriction endonuclease content of a clonal population and (ii) gene duplication, erosion, and horizontal transfer that modulate MTase and RM repertoires of the species. Some of these systems were experimentally shown to play a major role in mycoplasma conjugative, horizontal DNA transfer. While the versatility of DNA methylation may contribute to regulating essential biological functions at cell and population levels, RM systems may be key in mycoplasma genome evolution and adaptation by controlling horizontal gene transfers.

Host cell interactions of novel antigenic membrane proteins of Mycoplasma agalactiae.

BACKGROUND: Mycoplasma agalactiae is the main etiological agent of Contagious Agalactia syndrome of small ruminants notifiable to the World Organization for Animal Health. Despite serious economic losses, successful vaccines are unavailable, largely because its colonization and invasion factors are not well understood. This study evaluates the role of two recently identified antigenic proteins (MAG_1560, MAG_6130) and the cytadhesin P40 in pathogenicity related phenotypes. RESULTS: Adhesion to HeLa and sheep primary mammary stromal cells (MSC) was evaluated using ELISA, as well as in vitro adhesion assays on monolayer cell cultures. The results demonstrated MAG_6130 as a novel adhesin of M. agalactiae whose capacity to adhere to eukaryotic cells was significantly reduced by specific antiserum. Additionally, these proteins exhibited significant binding to plasminogen and extracellular matrix (ECM) proteins like lactoferrin, fibrinogen and fibronectin, a feature that could potentially support the pathogen in host colonization, tissue migration and immune evasion. Furthermore, these proteins played a detrimental role on the host cell proliferation and viability and were observed to activate pro-apoptotic genes indicating their involvement in cell death when eukaryotic cells were infected with M. agalactiae. CONCLUSIONS: To summarize, the hypothetical protein corresponding to MAG_6130 has not only been assigned novel adhesion functions but together with P40 it is demonstrated for the first time to bind to lactoferrin and ECM proteins thereby playing important roles in host colonization and pathogenicity.

Mycoplasma agalactiae ST35: a new sequence type with a minimal accessory genome primarily affecting goats.

BACKGROUND: Mycoplasma agalactiae, causing agent of contagious agalactia, infects domestic small ruminants such as sheep and goats but also wild Caprinae. M. agalactiae is highly contagious and transmitted through oral, respiratory, and mammary routes spreading rapidly in an infected herd. RESULTS: In an outbreak of contagious agalactia in a mixed herd of sheep and goats, 80% of the goats were affected displaying swollen udders and loss of milk production but no other symptom such as kerato-conjunctivitis, arthritis or pulmonary distress commonly associated to contagious agalactia. Surprisingly, none of the sheep grazing on a common pasture and belonging to the same farm as the goats were affected. Whole genome sequencing and analysis of M. agalactiae strain GrTh01 isolated from the outbreak, revealed a previously unknown sequence type, ST35, and a particularly small, genome size of 841'635 bp when compared to others available in public databases. Overall, GrTh01 displayed a reduced accessory genome, with repertoires of gene families encoding variable surface proteins involved in host-adhesion and variable antigenicity being scaled down. GrTh01 was also deprived of Integrative Conjugative Element or prophage, and had a single IS element, suggesting that GrTh01 has a limited capacity to adapt and evolve. CONCLUSIONS: The lack of most of the variable antigens and the Integrative Conjugative Element, both major virulence- and host specificity factors of a M. agalactiae strain isolated from an outbreak affecting particularly goats, indicates the implication of these factors in host specificity. Whole genome sequencing and full assembly of bacterial pathogens provides a most valuable tool for epidemiological and virulence studies of M. agalactiae without experimental infections.

Sonographic and histological udder parenchyma changes after intramammary infection of sheep with Mycoplasma agalactiae.

INTRODUCTION: The sonographic findings of the udder parenchyma and udder lymph nodes in 30 lactating sheep after experimental infection with Mycoplasma agalactiae are described. The objective of the study was to describe infection related changes in the udder parenchyma and udder lymph nodes using physical, sonographic, and histological examination and to detect associations between sonographic and histological changes of the tissues. Animals were intramammarily infected with different mutant cocktails and the wild type PG2. One group served as a negative control. A 15 MHz linear transducer (Esaote MyLab 30 CV, Esaote, Florence, Italy) was used for sonographic examinations. Compared with the uninfected control group with homogeneously granular parenchyma, the udder lymph nodes were larger and the udder parenchyma was more inhomogeneous and partially hyperechoic. The corresponding histological findings in infected mammary glands comprised proliferation of interstitial connective tissue, non-purulent interstitial mastitis, and purulent galactophoritis. The infected udder lymph nodes showed reactive hyperplasia. The findings obtained in this study may improve the diagnosis of Mycoplasma mastitis in sheep.

INTRODUCTION: Les constatations échographiques sur le parenchyme de la mamelle et des ganglions lymphatiques de la mamelle chez 30 brebis en lactation après une infection expérimentale avec Mycoplasma agalactiae sont décrits. L'objectif de l'étude était de décrire les modifications liées à l'infection dans le parenchyme mammaire et les ganglions lymphatiques de la mamelle à l'aide d'un examen physique, échographique et histologique et de détecter les associations entre les altérations échographiques et histologiques des tissus. Les animaux ont été infectés par voie intramammaire avec différents cocktails de mutants et le type sauvage PG2. Un groupe a servi de contrôle négatif. Une sonde linéaire de 15 MHz (Esaote MyLab 30 CV, Esaote, Florence, Italie) a été utilisé pour les examens échographiques. Comparativement au groupe témoin non infecté avec un parenchyme granulaire homogène, les ganglions lymphatiques de la mamelle étaient plus gros et le parenchyme de la mamelle était plus inhomogène et partiellement hyperéchogène. Les résultats histologiques correspondants dans les glandes mammaires infectées comprenaient une prolifération de tissu conjonctif interstitiel, une mammite interstitielle non purulente et une galactophorite purulente. Les ganglions lymphatiques de la mamelle infectée présentaient une hyperplasie réactive. Les résultats obtenus dans cette étude peuvent améliorer le diagnostic de la mammite à Mycoplasma chez le mouton.

Detecting Mollicutes by PCR in goats in southwestern Bahia, Brazil.

Brazil has a herd of over 9 million goats, and the northeast of Brazil is home to over 93% of this herd. Caprine mycoplasmosis are widely disseminated worldwide, being highly contagious with high rates of morbidity and mortality, causing considerable economic loss to goat herders. In addition, there has been a lack of research using molecular testing to monitor the health and detect Mollicutes in this herd in Brazil. Therefore, the aim of this study is to associate animal management with the presence of the caprine origin Mollicutes in goats, in the southwest region of the state of Bahia, Brazil. A cross-sectional study was conducted on twelve farms, and statistical analyses were performed to identify associations between the presence of Mollicutes and the management of goats. Molecular testing identified Mollicutes class, Mycoplasma agalactiae (Ma) and M. conjunctivae (Mc), in the samples analyzed. Statistical associations were observed between animals from intensive livestock facilities and the presence of Mollicutes in nasal samples and dairy ranch animals and the presence of Mollicutes in ocular samples and animals from extensive ranching sites and positive results of Mollicutes in genital samples. We conclude that mycoplasmas are present in goat herds in the southwestern region of Bahia, which supports the need for more focused studies of mycoplasmas throughout the country. Our research also demonstrated the presence of two important opportunistic bacteria, Mc and Ma, and, to the best of our knowledge, this is the first time that M. conjunctivae was detected in Brazilian goats by molecular testing.

Molecular Identification of Mycoplasma agalactiae in Iran Based on P30 Gene.

Mycoplasma agalactiae (M. agalactiae) is known as the main etiological agent of contagious agalactia (CA). The CA is a disease affecting dairy sheep and goats, the main characteristics of which include keratoconjunctivitis, arthritis, and mastitis. This pathogen results in milk production reduction and suppression, thereby leading to serious economic loss. In the present study, 125 sheep and goat samples were collected from 15 provinces of Iran. Cultural and molecular methods were used for sample characterization. After extracting genomic DNAs using the phenol/chloroform method, the PCR technique was employed to detect Mycoplasma genus in 163bp fragment of 16S rRNA gene (M-PCR) and M. agalactiae in 800bp fragment of conserve and specific P30 lipoprotein gene (P30-PCR) in cultural and clinical samples. Finally, to validate the experimental approach, a 375 bp amplicon of P80 lipoprotein was amplified using the MA-PCR. Out of 125 samples under investigation, 43 cases were positive, and Mycoplasma colonies were observed in the pleuropneumonia-like organisms agar culture. Based on the results of the M-PCR method, 61 specimens (out of 125 samples) were scored positive for Mycoplasma presence. Furthermore, 20 samples were positive according to the P30-PCR data. It should be mentioned that the MA-PCR was performed based on the P80 gene on 125 total samples to furtherverify the results for M.agalactiae detection. Based on the obtained data, P30 and P80 genes were presented and amplified in all Iranian M. agalactiae isolates (n=20). Our results indicated that the P30 gene was conserved and specific to all Iranian M. agalactiae isolates and this new P30-PCR method (as an MA-PCR technique) might be useful in the detection of this pathogen.

Identification of conserved Mycoplasma agalactiae surface antigens by immunoproteomics.

Contagious agalactia represents one of the most relevant infectious diseases of dairy sheep, with Mycoplasma agalactiae being the primary etiological agent. The early, sensitive, and specific identification of infected animals, as well as the development of efficient prophylactic tools, remain challenging. Here, we present a comprehensive characterization of M. agalactiae antigens focusing on those shared among different isolates. Leveraging on previous proteomic data obtained on individual strains, we adopted a strategy entailing sample pooling to optimize the identification of conserved proteins that induce an immune response. The liposoluble proteins from previously characterized field isolates and the type strain PG2T were enriched by Triton X-114 fractionation, pooled, analysed by one-dimensional (1D) and two-dimensional (2D) electrophoresis, and subjected to western immunoblotting against sheep sera collected during natural infection with M. agalactiae. Immunodominant antigens were identified by Matrix-Assisted Laser Desorption-Time-Of-Flight-Mass Spectrometry (MALDI-TOF-MS). This combined immunoproteomic approach confirmed the role of several known immunogens, including P80, P48, and P40, and most variable surface proteins (Vpmas), and unveiled novel immunodominant, conserved antigens, including MAG_1000, MAG_2220, MAG_1980, phnD, MAG_4740, and MAG_2430. Genomic context, functional prediction, subcellular localization, and invariable expression of these proteins in all isolates suggest their possible involvement in bacterial pathogenicity and metabolism. Moreover, most of the identified antigens elicit a host humoral response since the early stages of infection, persisting for at least 270 days. The immunodominant, conserved antigen panel identified in this work supports the development of effective vaccines and diagnostic tools with higher sensitivity and specificity in all the natural infection stages.

Ultrasensitive Electrochemical Impedance Detection of Mycoplasma agalactiae DNA by Low-Cost and Disposable Au-Decorated NiO Nanowall Electrodes.

Nanostructured electrodes detecting bacteria or viruses through DNA hybridization represent a promising method, which may be useful in on-field applications where PCR-based methods are very expensive, time-consuming, and require trained personnel. Indeed, electrochemical sensors combine disposability, fast response, high sensitivity, and portability. Here, a low-cost and high-surface-area electrode, based on Au-decorated NiO nanowalls, demonstrates a highly sensitive PCR-free detection of a real sample of Mycoplasma agalactiae (Ma) DNA. NiO nanowalls, synthesized by aqueous methods, thermal annealing, and Au decoration, by electroless deposition, ensure a high-surface-area platform for successful immobilization of Ma thiolated probe DNA. The morphological, chemical, and electrochemical properties of the electrode were characterized, and a reproducible detection of synthetic Ma DNA was observed and investigated by impedance measurements. Electrochemical impedance spectroscopy (EIS) ascribed the origin of impedance signal to the Ma DNA hybridization with its probe immobilized onto the electrode. The electrode successfully discriminates between DNA extracted from healthy and infected sheep milk, showing the ability to detect Ma DNA in concentrations as low as 53 ± 2 copy number µL-1. The Au-decorated NiO nanowall electrode represents a promising route toward PCR-free, disposable, rapid, and molecular detection.

Novel antigenic proteins of Mycoplasma agalactiae as potential vaccine and serodiagnostic candidates.

Contagious agalactia (CA) is a serious disease notifiable to the World Organisation for Animal Health (OIE) causing severe economic losses to sheep and goat producers worldwide. Mycoplasma agalactiae, considered as its main etiological agent, inflicts a variety of symptoms in infected animals, including keratoconjunctivitis, mastitis, arthritis, ankylosis, abortions, stillbirths and granular vulvovaginitis. Despite its significance, developing a successful vaccine remains elusive, mostly due to the lack of knowledge about M. agalactiae's pathogenicity factors and pathogenic mechanisms, including its "core" antigens. The aim of this study was to identify, characterize and express antigenic proteins of M. agalactiae as potential vaccine candidates. Predicted proteins of type strain PG2 were analyzed using bioinformatic algorithms to assess their cellular localization and to identify their linear and conformational epitopes for B cells. Out of a total of 156 predicted membrane proteins, three were shortlisted as potential antigenic surface proteins, namely [MAG_1560 (WP_011949336.1), MAG_6130 (WP_011949770.1) and P40 (WP_011949418.1)]. These proteins were expressed in recombinant Escherichia coli strains. Purified proteins were evaluated for their antigenicity using Western blot and ELISA using sera of M. agalactiae-naturally infected and non-infected sheep and goats. All 3 proteins were specifically recognized by the tested sera of M. agalactiae-infected animals. Also, specific rabbit antisera raised against each of these 3 proteins confirm their membrane localization using TritonX-114 phase partioning, Western and colony immunoblotting. In conclusion, our study successfully identified P40 (as proof of concept and validation) and two novel antigenic M. agalactiae proteins as potential candidates for developing effective CA vaccines.

Inferring Active Metabolic Pathways from Proteomics and Essentiality Data.

Here, we propose an approach to identify active metabolic pathways by integrating gene essentiality analysis and protein abundance. We use two bacterial species (Mycoplasma pneumoniae and Mycoplasma agalactiae) that share a high gene content similarity yet show significant metabolic differences. First, we build detailed metabolic maps of their carbon metabolism, the most striking difference being the absence of two key enzymes for glucose metabolism in M. agalactiae. We then determine carbon sources that allow growth in M. agalactiae, and we introduce glucose-dependent growth to show the functionality of its remaining glycolytic enzymes. By analyzing gene essentiality and performing quantitative proteomics, we can predict the active metabolic pathways connected to carbon metabolism and show significant differences in use and direction of key pathways despite sharing the large majority of genes. Gene essentiality combined with quantitative proteomics and metabolic maps can be used to determine activity and directionality of metabolic pathways.

Seroprevalence and associated risk factors of Mycoplasma agalactiae and investigation of coinfection with the caprine lentivirus in Rio Grande do Norte, Brazil.

Contagious agalactia is a disease caused by Mycoplasma agalactiae that leads to a reduction or complete stop of milk production. Caprine arthritis encephalitis (CAE) is an infectious disease caused by a lentivirus of the Retroviridae family, member of the small ruminant lentivirus (SRLV) group. Although these diseases are caused by distinct pathogens, the clinical presentation is similar. Hence, this study aimed to perform a serological investigation, as well as to assess correlation between both diseases and risk factors associated in two mesoregions of Rio Grande do Norte, Brazil. Enzyme-linked immunosorbent assay (ELISA) was used for contagious agalactia and western blot for CAE. A total of 538 serum samples were used in this study that were collected from goats and sorted from a blood bank of the Brazilian Agricultural Research Corporation. Seroprevalence of M. agalactiae in flocks from Rio Grande do Norte was 7.8% (42/538). In both regions that were investigated, 25.9% (14/54) of farms had positive animals. CAE results revealed that 3.9% (21/538) of animals and 42.6% (23/54) of farms had this disease. Concerning risk factors, only sex and animal category presented significant relevance (P < 0.05) for contagious agalactia, in which females presented higher frequency of seropositive individuals (10.1%; 39/387). In the animal category, 4.3% (14/326) and 11.1% (36/323) of female breeders were positive for CAE and contagious agalactia, respectively, and significance was identified only in the latter (P < 0.05). In conclusion, there was no correlation between the investigated diseases, considering that no animal demonstrated antibodies for both pathogens.

Microbiological and molecular detection of Mycoplasma bovis in milk samples from bovine clinical mastitis / Detecção microbiológica e molecular de Mycoplasma bovis em amostras de leite oriundas de mastite clínica bovina

The genus Mycoplasma includes more than 200 bacterial species that cause disease in animals. It is responsible for causing mastitis in bovines and may be related to other manifestations, such as arthritis and pneumonia in calves and heifers. The present study aimed to detect Mycoplasma bovis isolated from milk samples of bovine clinical mastitis, and to compare the isolation rates in two culture media: Hayflick and SP4. An initial screening was performed in order to detect the presence of the class Mollicutes in 1166 milk samples from clinical mastitis by the conventional Polymerase Chain Reaction (PCR) technique. According to the 1166 milk samples evaluated, 8.6% (100/1166) were positive to class Mollicutes. Regarding molecular analyses, 1.1% (13/1166) of conventional PCR for positive M. bovis was obtained and 0.9% (11/1166) in real-time PCR. The results of the microbiological culture of the 100 samples previously screened demonstrated that 6% (6/100) of colony growth have been developed when using the Hayflick medium, and 11% (11/100) when using the SP4 medium (including the positive on Hayflick medium). Concerning the 11 isolates obtained in the microbiological culture, conventional PCR confirmed M. bovis in nine of them, and two cultures were negative. In the phylogenetic analysis of the isolates, all of them were grouped in M. bovis and M. agalactiae clusters. The results confirmed the importance of the presence of M. bovis in the etiology of bovine clinical mastitis and reinforced the need for further studies to elucidate other Mycoplasma species that may be involved in bovine clinical mastitis in Brazil.(AU)

O gênero Mycoplasma inclui mais de 200 espécies que causam doenças nos animais. É responsável por quadros de mastite em bovinos, podendo também estar relacionado à outras manifestações como artrite e pneumonia em bezerros e novilhas. O presente estudo objetivou a detecção de Mycoplasma bovis isolados a partir de amostras de leite de mastite clínica bovina, bem como, a comparação da taxa de isolamento em dois meios de cultura: Hayflick e SP4. Para efeito de triagem amostral, foram avaliadas quanto à presença da classe Mollicutes 1166 amostras de leite de casos de mastite clínica pela técnica de PCR convencional. Das 1166 amostras de leite avaliadas, 8,6% (100/1166) foram positivas à classe. Nas análises moleculares, obteve-se 1,1% (13/1166) de positividade para Mycoplasma bovis na PCR convencional e 0,9% (11/1166) na PCR em tempo real. Os resultados do cultivo microbiológico das 100 amostras triadas previamente demonstraram 6% (6/100) de crescimento de colônias ao se utilizar o meio Hayflick e 11% (11/100) ao se utilizar o meio SP4 (incluindo as positivas ao primeiro). A partir dos 11 isolados obtidos no cultivo microbiológico, a PCR convencional confirmou Mycoplasma bovis em nove deles, e dois foram negativos para o agente. Na análise filogenética dos isolados, todos agruparam no cluster Mycoplasma bovis e Mycoplasma agalactiae. Frente aos resultados, ressalta-se a importância da presença de Mycoplasma bovis na etiologia da mastite clínica bovina e reforça a necessidade de estudos mais aprofundados para a elucidação de outras espécies de micoplasmas que possam estar envolvidas na mastite bovina.(AU)

A Study on Mycoplasma agalactiae and Chlamydophila abortus in Aborted Ovine Fetuses in Sistan and Baluchestan region, Iran.

Abortion is one of the most important economic issues in sheep flocks. Chlamydophila abortus is an agent of enzootic abortions in sheep. Mycoplasma agalactiae is the main etiological agent of contagious agalactia, which can cause abortion in sheep. The aim of this study was to investigate the prevalence of M. agalactiae and C. abortus among aborted ovine fetuses in Sistan and Baluchestan, Iran. Sheep owners were asked to transfer their aborted fetuses to a nearby veterinary clinic; furthermore, they were taught biosecurity principles. A total of 78 aborted sheep fetuses were collected from all over Sistan region in the autumn of 2015 and winter of 2016. The samples were then transferred in ice to the Anatomy Laboratory of the Veterinary Faculty of Zabol University, Zabol, Iran. The spleen and abomasum contents of the fetuses were sampled under sterile and safe conditions. Polymerase chain reaction was used to detect M. agalactiae and C. abortus. The results showed that 24 (30.8%) cases were infected with M. agalactiae. However, infection with C. abortus was not detected in any fetuses. There was no statistically significant relationship between such independent variables as the location of livestock, history of abortion, fetal gender and age, age and parity of ewe, and fetal infection with M. agalactiae. The high incidence of Mycoplasma contamination in this study may be due to inappropriate biosecurity measures and lack of vaccination against agalactia in sheep herds in Sistan region.

Metabolic Engineering of Saccharomyces cerevisiae to Improve Glucan Biosynthesis.

ß-Glucan is a chief structural polymer in the cell wall of yeast. ß-Glucan has attracted intensive attention because of its wide applications in health protection and cosmetic areas. In the present study, the ß-glucan biosynthesis pathway in S. Cerevisiae was engineered to enhance ß-glucan accumulation. A newly identified bacterial ß-1, 6-glucan synthase GsmA from Mycoplasma agalactiae was expressed, and increased ß-glucan content by 43%. In addition, other pathway enzymes were investigated to direct more metabolic flux towards the building of ß-glucan chains. We found that overexpression of Pgm2 (phosphoglucomutase) and Rho1 (a GTPase for activating glucan synthesis) significantly increased ß-glucan accumulation. After further optimization of culture conditions, the ß-glucan content was increased by 53.1%. This study provides a new approach to enhance ß-glucan biosynthesis in Saccharomyces cerevisiae.

Contagious agalactia monitoring in caprine herds through regular bulk tank milk sampling.

Surveillance and control of Mycoplasma spp. responsible for contagious agalactia (CA) in caprine herds are important challenges in countries with a large small-ruminant dairy industry. In the absence of any clinical signs, being able to determine the potential circulation of mycoplasmas within a herd could help to prevent biosecurity issues during animal exchanges between farms and improve health management practices. The objective of this study was to determine whether regular sampling of bulk tank milk was suitable for such surveillance. Twenty farms were sampled once a month for 2 yr and CA-responsible mycoplasmas were detected by real-time PCR on DNA extracted from milk, using 3 different DNA extraction methods. The pattern of mycoplasma excretion in bulk tank milk was assessed over time and several herd characteristics were recorded together with any event occurring within the herds. In general, the results obtained with the different detection methods were comparable and mainly agreed with the culture results. Several patterns of excretion were observed but were not related to herd characteristics (size, breed, and so on). Recurrence of the same (sub)species and same pulsed-field gel electrophoresis subtype during the 2-yr period is indicative of the considerable persistence of mycoplasmas. This persistence was associated with intermittent excretion. In conclusion, bulk tank milk sampling could be valuable for controlling CA in caprine herds provided it is repeated several times, yet to be defined, per year and analyzed using an appropriate methodology and the right cut-off for interpretation.

A European interlaboratory trial to evaluate the performance of different PCR methods for Mycoplasma bovis diagnosis.

BACKGROUND: Several species-specific PCR assays, based on a variety of target genes are currently used in the diagnosis of Mycoplasma bovis infections in cattle herds with respiratory diseases and/or mastitis. With this diversity of methods, and the development of new methods and formats, regular performance comparisons are required to ascertain diagnostic quality. The present study compares PCR methods that are currently used in six national veterinary institutes across Europe. Three different sample panels were compiled and analysed to assess the analytical specificity, analytical sensitivity and comparability of the different PCR methods. The results were also compared, when appropriate, to those obtained through isolation by culture. The sensitivity and comparability panels were composed of samples from bronchoalveolar fluids of veal calves, artificially contaminated or naturally infected, and hence the comparison of the different methods included the whole workflow from DNA extraction to PCR analysis. RESULTS: The participating laboratories used i) five different DNA extraction methods, ii) seven different real-time and/or end-point PCRs targeting four different genes and iii) six different real-time PCR platforms. Only one commercial kit was assessed; all other PCR assays were in-house tests adapted from published methods. The analytical specificity of the different PCR methods was comparable except for one laboratory where Mycoplasma agalactiae was tested positive. Frequently, weak-positive results with Ct values between 37 and 40 were obtained for non-target Mycoplasma strains. The limit of detection (LOD) varied from 10 to 103 CFU/ml to 103 and 106 CFU/ml for the real-time and end-point assays, respectively. Cultures were also shown to detect concentrations down to 102 CFU/ml. Although Ct values showed considerable variation with naturally infected samples, both between laboratories and tests, the final result interpretation of the samples (positive versus negative) was essentially the same between the different laboratories. CONCLUSION: With a few exceptions, all methods used routinely in the participating laboratories showed comparable performance, which assures the quality of diagnosis, despite the multiplicity of the methods.

Ocurrence and risk factors associated with Mycoplasma agalactiae infection in dairy goat herds of Paraíba State, Brazil / Ocorrência e fatores de risco associados à infecção por Mycoplasma agalactiae em rebanhos caprinos leiteiros do estado da Paraíba, Brasil

Mycoplasmosis is a disease that may cause severe economical losses in goat and sheep herds, and it is associated with mastitis, polyarthritis, agalactia, conjunctivitis, pneumonia and reproductive failure. The objective of this study was to determine the occurrence of Mycoplasma agalactiae in milk samples and investigate the main risk factors associated with infection in goats from farms of the state of Paraíba, Brazil. For Mycoplasma agalactiae diagnosis, 251 milk samples were submitted to DNA extraction using a commercially available kit, following the manufacturer's instructions and Polymerase Chain Reaction (PCR) was performed. In addition, questionnaires were applied to identify the main risk factors associated with contagious agalactia. Out of the two hundred fifty-one samples analyzed, 50 (19.9%, I.C. 15.1-25.4%) were PCR positive for M. agalactiae. In the risk factors analysis, some associations were observed for the following variables: size of the herd (P<0.001, OR=7.1, I.C. 2.4-20.6), replacement of farm animals (P<0.001, OR=4.7, I.C. 1.8-12.2) and participation of animals in fairs and exhibitions (P=0.029, OR=2.0, I.C.1.0-3.9). The results allowed confirming the occurrence of Mycoplasma agalactiae in milk samples of goats from Paraíba. Therefore, it is strictly necessary to monitor dairy goat flocks and to raise the awareness of farmers about the economic importance of the disease, since it causes severe economic losses for producers of the state. Identification of risk factors is essential for adoption of control measures and for the correction of the management factors in farms where there are animals with positive diagnosis, avoiding, so, pathogen dissemination.(AU)

As micoplasmoses ocasionam prejuízos econômicos nas criações de ovinos e caprinos, e estão associados com quadros de mastite, poliartrite, agalaxia, conjuntivite, pneumonia e falhas reprodutivas. Objetivou-se neste estudo determinar a ocorrência de Mycoplasma agalactiae em amostras de leite e investigar os principais fatores de risco associados à infecção em caprinos provenientes de propriedades rurais do estado da Paraíba, Brasil. Para o diagnóstico de Mycoplasma agalactiae, foram analisadas 251 amostras de leite, que foram submetidas à extração do DNA genômico usando um kit comercial, seguindo as recomendações do fabricante. Para diagnóstico da infecção utilizou-se a Reação em Cadeia da Polimerase (PCR). Além disso, foram aplicados questionários para identificar os principais fatores de risco associados infecção à agalaxia contagiosa. Das 251 amostras analisadas, 50 (19,9%; I.C. 15,1-25,4%) foram positivas na PCR para M. agalactiae. Observaram-se na análise dos fatores de risco, algumas associações para as seguintes variáveis: tamanho do rebanho (P<0,001; OR 7,1), reposição de animais da propriedade (P<0,001; OR 4,7) e participação dos animais em feiras e exposições (P= 0,029; OR 2,0). Os resultados permitiram confirmar a ocorrência do Mycoplasma agalactiae em amostras de leite de caprinos da Paraíba. Portanto, é necessário o monitoramento dos rebanhos caprinos leiteiros e a conscientização dos produtores rurais para a importância econômica da doença, visto que a mesma acarreta severos prejuízos econômicos para os produtores do estado. A identificação dos fatores de risco são imprescindíveis para a adoção de medidas de controle e para a correção dos fatores de manejo em propriedades que tenham animais com diagnóstico positivo, evitando assim, a disseminação do patógeno.(AU)