Mycoplasma Agalactiae Research Articles

Isolation and identification of mycoplasma strains in the inner ear of cattle and small ruminants in Mali

Mycoplasmas are microorganisms characterized by the absence of a cell wall and affecting animals and humans. In domestic ruminants, the role of inner ear swabbing was determined in the isolation of mycoplasmas in cattle and small ruminants in Mali. From 250 asymptomatic carriers, inner ear swabs were taken from 126 cattle and 124 small ruminants in the regions of Kayes, Koulikoro, Sikasso, Ségou and the District of Bamako. Microbiological analysis by isolation on Hyflick medium revealed 38 strains of mycoplasma including 23 in cattle (6 strains from Kayes, 3 strains from Sikasso, 1 strain from Ségou, 13 strain from Bamako) and 15 strains from small ruminants (1 strain from Sikasso, 2 from Ségou, 12 from Bamako). Growth inhibition and PCR tests (FusA, Polc) made it possible to identify in cattle 8 strains of Mycoplasma bovis, 13 strains of Mycoplasma yeatsii and 2 strains of Mycoplasma alkalescens and in small ruminants, 5 strains of Mycoplasma mycoides capri, 3 strains of Mycoplasma yeatsii and 7 strains of Mycoplasma agalactiae. Overall, the prevalence rate of mycoplasmas from inner ear swabs was 42.11% (16/38) of Mycoplasma yeatsii, 21.05% (8/38) of Mycoplasma bovis, 18.42% (7 /38) of Mycoplasma agalactiae, 13.16% (5/38) of Mycoplasma mycoides capri and 5.26% (2/38) of Mycoplasma alkalescens. The results of this study confirmed that the ear canal of cattle and small ruminants could be suitable sources for the detection of pathogenic strains such as Mycoplasma bovis, Mycoplasma agalactiae, Mycoplasma mycoides subsp. capri in asymptomatic herds of cattle and small ruminants.

First Description of Mycoplasma agalactiae Anatomical Localization in Naturally Infected Hard Ticks (Rhipicephalus bursa).

Mycoplasma agalactiae (Ma) is considered the primary causative agent of contagious agalactia (CA) in sheep and goats, which causes severe losses to the small ruminant dairy industry. As early as 1816, it was thought that environmental factors played a role in pathogen maintenance in endemic areas. Specifically, recent studies hypothesized a vector role for arthropods in the epidemiology of disease. The aim of this study was to investigate the presence and anatomical localization of Ma in naturally infected Riphicephalus bursa ticks to better evaluate tick-pathogen interactions. Salivary glands and ovaries of confirmed Ma-positive R. bursa were analyzed to look for the Ma antigen using immunohistochemistry (IHC). IHC showed strong positivity to Ma in the cytoplasm of salivary cells as well as in cells from the ovary. Our work demonstrated for the first time the crossing of the tick midgut barrier by Ma and the subsequent infection of organs capable of spreading the infection, and this result represents an absolute novelty in disease-related knowledge. Our preliminary results provide conclusive evidence of the potential vector role represented by hard ticks in the epidemiology of CA. Further field and laboratory investigations are necessary to confirm the tick role in the transmission of clinical CA.

Diagnosis of Mycoplasma agalactiae from various specimens of goats

The aim of this study was to determine the presence of Mycoplasma agalactiae (M. agalactiae) by bacteriological methods and to reveal the prevalence of the disease in a goat farm with pneumonia, mastitis and arthritis symptoms and deaths. This study was carried out in a Saanen goat farm in Isparta province between January 2015 and January 2017. Samples (milk, intra-articular fluid, internal organs of deceased animals) were brought to Burdur Mehmet Akif Ersoy University, Faculty of Veterinary Medicine Microbiology Laboratory and examined by bacteriological methods. Blood samples were collected from 813 goats in aged 1-6 years in the goat farm. Blood sera were tested for Mycoplasma agalactiae with a commercial ELISA kit (IDEXX, France) according to the kit instructions. Mycoplasma colonies were isolated from all samples. As a result of serological examination, 83 (10.2%) animals were positive, 9 (1.1%) animals were suspicious, and the rest were negative by ELISA. The seropositivity of M. agalactiae was ranged from 5.1 % to 28.7% according to age, and it was found quite high in three-year-old animals. With this study, it was determined that the seroprevalance of M. agalactiae is quite high in goats aged three years and above, it can be detected of M. agalactiae in animals that do not show clinical symptoms using ELISA and can be used in eradication of the disease from flocks.

A Preliminary Study Evaluating Some Biochemical Parameters In Saanen Goats Naturally Infected With Mycoplasma agalactiae

Mycoplasma agalactiae in sheep and goats causes significant economic losses worldwide. The disease can cause mastitis, arthritis, ophthalmitis and less commonly abortion. This study aimed to reveal what kind of changes the disease causes in routine biochemical parameters and PCT and CRP levels in naturally infected goats. The material of the study consisted of 15 naturally infected with Mycoplasma agalactiae and 9 healthy (control group) female goats in a private commercial enterprise. M. agalactiae was diagnosed by growth inhibition test in milk and blood serum. Serum biochemical analysis was performed by autoanalyzer. In the statistical study conducted between the control and infected groups, a significant decrease was found in albumin levels and A/G ratio in the infected group, and a significant increase in globulin, chlorine and potassium levels. No significance could be determined in levels of ALT, AST, D.BIL, T.BIL, BUN, Creatinine, Urea, Glucose, Na, P, CRP and PCT. As a result, it was revealed that PCT is not an important biomarker in goats with Mycoplasma agalactiae.

In Vitro Interaction between Mycoplasma agalactiae and Small Ruminants' Endogenous Bacterial Strains of Enterococcus spp. and Coagulase-Negative Staphylococcus.

Recently, an antimicrobial effect on Mycoplasma agalactiae (Ma), the main etiological agent of contagious agalactia (CA), was reported in vitro with strains of Enterococcus spp. from ovine and caprine milk. The aim of this work was to evaluate the interaction of Ma with the same Enterococcus spp. isolated from other anatomical locations (vagina) and other bacterial populations present in milk, such as coagulase-negative staphylococci (CNS). The vaginal Enterococcus strains and the raw milk CNS were isolated from sheep and goats. Experimental in vitro conditions were prepared to assess the growth of Ma with and without the presence of these strains. The selected vaginal strains were identified as Enterococcus (E.) hirae and E. mundtii, and the strains of CNS were identified as Staphylococcus petrasii. Different interactions of Ma with ovine and caprine wild vaginal strains of Enterococcus and dairy strains of CNS are described for the first time: Ma can grow exponentially during 15 h with the selected strains, although with certain strains, its optimal growth can be negatively affected (p < 0.05). The colonization and/or excretion of Ma could, therefore, be influenced by certain endogenous bacterial strains. Our results increase the knowledge about possible bacterial ecology dynamics surrounding CA.

Mycoplasma agalactiae Vaccines: Current Status, Hurdles, and Opportunities Due to Advances in Pathogenicity Studies.

Contagious agalactia (CA) is a serious multietiological disease whose classic etiological agent is Mycoplasma agalactiae and which causes high morbidity and mortality rates in infected herds. CA is classified as a notifiable disease by the World Organization for Animal Health due to its significant worldwide economic impact on livestock, primarily involving goat and sheep farms. The emergence of atypical symptoms and strains of M. agalactiae in wildlife ungulates reestablishes its highly plastic genome and is also of great epidemiological significance. Antimicrobial therapy is the main form of control, although several factors, such as intrinsic antibiotic resistance and the selection of resistant strains, must be considered. Available vaccines are few and mostly inefficient. The virulence and pathogenicity mechanisms of M. agalactiae mainly rely on surface molecules that have direct contact with the host. Because of this, they are essential for the development of vaccines. This review highlights the currently available vaccines and their limitations and the development of new vaccine possibilities, especially considering the challenge of antigenic variation and dynamic genome in this microorganism.

Antibacterial potential of commercial and wild lactic acid bacteria strains isolated from ovine and caprine raw milk against Mycoplasma agalactiae.

The complexity of fighting contagious agalactia (CA) has raised the necessity of alternative antimicrobial therapies, such as probiotics. Lactic acid bacteria (LAB) are present in the mammary gland of small ruminants and their antimicrobial effect have been previously described against species like Mycoplasma bovis but never against Mycoplasma agalactiae (Ma). This in vitro study aims to evaluate the antimicrobial activity against Ma of ovine and caprine LAB strains and a human commercial probiotic (L2) of Lactobacillus spp. A total of 63 possible LAB strains were isolated from nine ovine and caprine farms in Spain, three isolates (33B, 248D, and 120B) from the 63 strains were selected, based on their capacity to grow in a specific medium in vitro, for an in vitro experiment to assess their antimicrobial activity against Ma in Ultra High Temperature (UHT) processed goat milk (GM). A women commercial vaginal probiotic was also included in the study. The inoculum of L2 was prepared at a concentration of 3.24 × 108 CFU/mL and the average concentration of the inoculum of the wild LAB varied from 7.9 × 107 to 8.4 × 108 CFU/mL. The commercial probiotic L2 significantly reduced the concentration of Ma to 0.000 log CFU/mL (p < 0.001), strain 33B reduced it from 7.185 to 1.279 log CFU/mL (p < 0.001), and 120B from 6.825 to 6.466 log CFU/mL (p < 0.05). Strain 248D presented a bacteriostatic effect in GM. Moreover, the three wild strains and the commercial probiotic produced a significative reduction of the pH (p < 0.001). This is the first in vivo report of the antimicrobial potential of LAB strains against Ma and its interaction. Our results support possible future alternative strategies to antibiotic therapy, previously not contemplated, to fight CA in small ruminants. Further studies are necessary to elucidate the action mechanisms through which these LAB are able to inhibit Ma and to assess the safety of using these strains in possible in vivo studies.

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.

Designing a P48-40 Chimeric Protein of Mycoplasma agalactiae and Highly Expression in E. coli, Applicable for Indirect ELISA

Designing a P48-40 Chimeric Protein of Mycoplasma agalactiae and Highly Expression in E. coli, Applicable for Indirect ELISA

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

BackgroundThe 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 methodsThe 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.ResultsWestern 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.ConclusionWe proposed a novel designed chimeric protein of Mycoplasma agalactiae as a potential marker for serodiagnostic assays but still further field research is required.

Preliminary Study on the Host Response to Bivalent and Monovalent Autogenous Vaccines against Mycoplasma agalactiae in Dairy Sheep.

In Italy, dairy sheep farming represents a vital agro-industry sector, but it is still challenged by contagious agalactia (CA), which is endemic there, and vaccination is the most economical and sustainable tool for control. This study aimed to evaluate the combined Mycoplasma agalactiae (Ma)-Staphylococcus aureus (Sa) vaccine (Ma-Sa) against the Ma monovalent vaccine in ewes. Twelve primiparous Ma-free ewes were randomly grouped into three equal groups: first, the control group injected with placebo, second, the group vaccinated with the Ma monovalent vaccine, and third, the group vaccinated with Ma-Sa combined vaccine, with two S/C doses at 45-day intervals. The animals were examined for serological, hematological, and somatic cell count (SCC) changes for 17 successive weeks. A significant increase in anti-Ma antibody mean titers, leukocytes, and platelets was observed in the vaccinated animals, with the highest values in those who received the combined vaccine. Neutrophils were high only in the animals who received the combined vaccine. SCC was lower in the vaccinated animals during the first six weeks. This study concludes that the combined Ma-Sa vaccines enhance immune response and potentiate its efficacy against Ma. This improvement might be attributed to the sensitization/activation effect of S. aureus on platelets, which are recoded to act as a key regulator for the coordination of all components of the innate immune system. Even though this study included a small number of animals, its findings about the potentialities of this inactivated vaccine in the control of CA are strongly encouraging. Further confirmation might be needed through additional replicates and a challenge study is needed before proceeding with widespread use.

Susceptibility of caprine mastitis pathogens to tildipirosin, gamithromycin, oxytetracycline, and danofloxacin: effect of serum on the in vitro potency of current macrolides

Mastitis is a significant disease in dairy ruminants, causing economic losses to the livestock industry and severe risks to public health. Antibiotic therapy is one of the most crucial practices to treat mastitis, although the susceptibility of caprine mastitis pathogens to current antibiotics has not been tested under standard or modified incubation conditions. This work evaluated the in vitro activity of tildipirosin, gamithromycin, oxytetracycline, and danofloxacin against caprine mastitis pathogens incubated following standard conditions of Clinical and Laboratory Standards Institute (CLSI) and deviation method by 25% supplementation with goat serum. Mycoplasma agalactiae, Escherichia coli, Staphylococcus aureus, Streptococcus spp., and coagulase-negative Staphylococci (CNS) were isolated from dairy goats with mastitis in Spain. Minimum inhibitory concentrations (MICs) were determined using the broth microdilution technique. The lowest MIC90 under standard conditions was obtained with danofloxacin for mastitis-causing pathogens. An exception was M. agalactiae, where danofloxacin and oxytetracycline obtained low values. However, after adding serum, gamithromycin showed the lowest MIC50 for S. aureus, Streptococcus spp., and CNS. The lowest MIC50 was obtained with all the antibiotics tested (< 0.125 µg/ml) against M. agalactiae. Supplementing with serum resulted in a significant variation in tildipirosin and gamithromycin MIC values for CNS, S. aureus, M. agalagtiae, and E. coli. In brief, the MIC for antibiotics used against mastitis should be determined under conditions closely resembling intramammary infections to obtain representative susceptibility patterns against mastitis pathogens. Caprine mastitis pathogens were broadly susceptible to danofloxacin under standard conditions. The potency of macrolides against caprine mastitis pathogens increases when serum is present in culture media.

Serum Resistance of Mycoplasma agalactiae Strains and Mutants Bearing Different Lipoprotein Profiles.

In order to spread systemically, resistance against complement and other factors present in serum is an important trait in pathogenic bacteria. The variable proteins of Mycoplasma agalactiae (Vpmas) have been shown to affect differential adhesion, invasion and immune evasion, and undergo high-frequency phase-variation in expression. However, nothing is known about their involvement in M. agalactiae’s serum susceptibility. To evaluate this, the PG2 strain, the GM139 strain and the six Vpma phase-locked mutants (PLMs, PLMU to PLMZ) were tested for their ability to survive in the presence of non-sensitized and sensitized sheep serum, as well as guinea pig complement. Additionally, the reactivity of the sensitized sheep serum was analysed on the strains via western blotting. Overall data demonstrate PG2 strain to be more susceptible to sheep serum compared to the GM139 strain bearing a different Vpma profile. Significant differences were also observed between the different PLMs, with PLMU and PLMX showing the highest serum susceptibility in serum, while the other PLMs expressing longer Vpma proteins were more resistant. The results are in good correlation with previous studies where shorter lipoprotein variants contributed to a higher susceptibility to complement. Since none of the tested strains and PLMs were susceptible to non-sensitized sheep serum, antibodies seem to play an important role in serum killing.

Influence of Natural and Climatic Conditions on the Distribution and Forms of Contagious Agalactia in Sheep in Bessarabia, Ukraine.

Contagious agalactia of ruminants is an endemic disease caused by Mycoplasma agalactiae in flicting significant losses on farms in deaths and forced slaughter of sick animals, abortions, births of sick young animals, and reduced milk and wool production. The aim of the study was to determine the influence of hydrometeorological conditions on the distribution and forms of contagious agalactia in sheep in Bessarabia, Ukraine. The epizootic situation regarding contagious agalactia was studied during 2011-2021 on sheep farms in the south of the Odesa region in Bessarabia. Over two million blood samples from sheep aged 1-6 years were serologically investigated and the prevalence of agalactia was correlated with Selyaninov's hydrothermal coefficient for each sampling year. High rates of infection of sheep with contagious agalactia (from 13.1% to 14.4%) were registered in 2012, 2016 and 2021, years which according to the hydrothermal coefficient of 1.0 were sufficiently moist. The lowest incidence rates, from 6.5% to 7.4%, were registered in the very dry 2013, 2014 and 2019 with hydrothermal coefficients of 0.5‒0.6. In sufficiently moist years, contagious agalactia of sheep manifested itself in the mastitic form, while in the dry period the mastitic form was half as prevalent, and the mixed, articular and ocular forms of the disease proliferated. The results indicate the circulation of Mycoplasma agalactiae among small ruminants in Bessarabia, and that the prevalence and the course of the associated disease depend on the humidity of the climate, i.e. the value of the hydrothermal coefficient.

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

BackgroundContagious 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.ObjectivesDtermination 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.Methods20 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.ResultsAfter 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).ConclusionsThis 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.

Sheep Infection Trials with 'Phase-Locked' Vpma Expression Variants of Mycoplasma agalactiae-Towards Elucidating the Role of a Multigene Family Encoding Variable Surface Lipoproteins in Infection and Disease.

The significance of large multigene families causing high-frequency surface variations in mycoplasmas is not well-understood. Previously, VpmaY and VpmaU clonal variants of the Vpma family of lipoproteins of M. agalactiae were compared via experimental sheep infections using the two corresponding ‘Phase-Locked Mutants’. However, nothing is known about the infectivity of the remaining four Vpma expression variants VpmaX, VpmaW, VpmaZ and VpmaV as they were never evaluated in vivo. Here, in vivo infection and disease progression of all six Vpma expressers constituting the Vpma family of type strain PG2 were compared using the corresponding xer1-disrupted PLMs expressing single well-characterized Vpmas. Each of the six PLMs were separately evaluated using the intramammary sheep infection model along with the control phase-variable wildtype strain PG2. Thorough bacteriological, pathological and clinical examinations were performed, including assessment of milk quality, quantity and somatic cell counts. Altogether, the results indicated that the inability to vary the Vpma expression phase does not hamper the initiation of infection leading to mastitis for all six PLMs, except for PLMU, which showed a defect in host colonization and multiplication for the first 24 h p.i. and pathological/bacteriological analysis indicated a higher potential for systemic spread for PLMV and PLMX. This is the first study in which all isogenic expression variants of a large mycoplasma multigene family are tested in the natural host.

Host cell interactions of novel antigenic membrane proteins of Mycoplasma agalactiae

BackgroundMycoplasma 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.ResultsAdhesion 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.ConclusionsTo 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.

Comparison of Commercial Enzyme-linked Immunosorbent Assays for Diagnosis of Contagious Agalactia Caused By Mycoplasma Agalactiae.

IntroductionContagious agalactia (CA) is a disease affecting small ruminants with worldwide distribution and caused by several mycoplasmas, especially M. agalactiae. The main option for systematic diagnosis under monitoring control programmes is the enzyme-linked immunosorbent assay (ELISA) test.Material and MethodsThis study was designed to appraise the performance of two commercial indirect ELISA tests using M. agalactiae p48 protein and one using total protein, for antibody detection in small ruminants after natural infection with different M. agalactiae strains. We carried out the test evaluation using sera of confirmed M. agalactiae-positive goats with clinical signs. In addition, test agreement was assessed by kappa between the three commercial ELISA tests.ResultsAll three ELISA tests showed high validity scores (Youden’s J: 72.9–84%). The sensitivity values for the P48 protein-based tests were 76.9% and 84.6%, and was 79% for the total protein-based test. The specificity of all tests was 100%. In addition, between the total protein-based ELISA test and the other two ELISA tests based on the P48 protein, the agreement was substantial (kappa: 0.762–0.763) and the agreement between the latter two tests was almost perfect (kappa: 0.93).ConclusionThe validity parameters for all tests allowed their application for diagnostic purposes in lactating goats excreting M. agalactiae in milk and presenting clinical signs. The agreements show that any of these ELISA tests could be equally well used for diagnosis in programmes against CA.

Report on first confirmed outbreaks of contagious agalactia in small ruminants from Maharashtra State of India

Mycoplasma agalactiae causes contagious agalactia, a serious, economically important and OIE listed diseasesyndrome, in sheep and goats. There are no reports of this disease from Maharashtra state of India. The aim ofthe present investigation was to record epidemiological information and to detect M. agalactiae in clinical samplesfrom natural outbreaks of contagious agalactia. Clinically, anorexia, reluctance to follow the herd, lethargy and feverwere the initial clinical signs noted. Later, mastitis, arthritis and keratoconjunctivitis were noted as important clinicalsigns in both sheep and goats. However, all signs were not present in every case. DNA was extracted from mastiticmilk and articular exudate collected from suspected cases and PCR was performed using primers specific for 16S rRNA gene of Mycoplasma spp. The agarose gel electrophoresis of PCR product revealed the presence of bands of expected size (278 bp) indicating positive amplification. The nucleotide sequence analysis of PCR amplicons showed 100% identity with 16S rRNA gene of Mycoplasma agalactiae isolates JF4428 (Accession No. LT578418.1). The outbreaks of contagious agalactia were confirmed for the first time in Maharashtra state of India and carries epidemiological significance.