Mycoplasma Agalactiae Research Articles

Comprehensive RNA-Seq Profiling to Evaluate the Sheep Mammary Gland Transcriptome in Response to Experimental Mycoplasma agalactiae Infection.

Mycoplasma agalactiae is a worldwide serious pathogen of small ruminants that usually spreads through the mammary route causing acute to subacute mastitis progressing to chronic persistent disease that is hard to eradicate. Knowledge of mechanisms of its pathogenesis and persistence in the mammary gland are still insufficient, especially the host-pathogen interplay that enables it to reside in a chronic subclinical state. This study reports transcriptome profiling of mammary tissue from udders of sheep experimentally infected with M. agalactiae type strain PG2 in comparison with uninfected control animals using Illumina RNA-sequencing (RNA-Seq). Several differentially expressed genes (DEGs) were observed in the infected udders and RT-qPCR analyses of selected DEGs showed their expression profiles to be in agreement with results from RNA-Seq. Gene Ontology (GO) analysis revealed majority of the DEGs to be associated with mycoplasma defense responses that are directly or indirectly involved in host innate and adaptive immune responses. Similar RNA-Seq analyses were also performed with spleen cells of the same sheep to know the specific systemic transcriptome responses. Spleen cells exhibited a comparatively lower number of DEGs suggesting a less prominent host response in this organ. To our knowledge this is the first study that describes host transcriptomics of M. agalactiae infection and the related immune-inflammatory responses. The data provides useful information to further dissect the molecular genetic mechanisms underlying mycoplasma mastitis, which is a prerequisite for designing effective intervention strategies.

Comparative Therapeutic Efficacy of Different Drugs Against Caprine Contagious Agalactia

The present work was undertaken with the aim to compare the therapeutic efficacy of different drugs against contagious agalactia in lactating goats. A total of 24 lactating goats having clinical mastitis and positive for Mycoplasma agalactiae were placed randomly into four groups i.e. T1-T4, each group comprised of six mastitic goats and subjected to different therapeutic regimens. Six clinically healthy goats were kept as healthy control. Mycoplasma agalactiae was detected and confirmed as causative agent of contagious agalactia by using polymerase chain reactions which showed a specific 176 bp bands obtained from the DNA amplification of Mycoplasma agalactiae using primers ma-mp 1F and ma-mp 1R. The response of therapeutic study was evaluated on the basis of clinical score card, clinical parameters and polymerase chain reactions on day 0 pretreatment and on days 7 and 14 post treatment. At the time of sampling, the clinical signs in sick animals were of clinical mastitis i.e. swelling of udder, abnormal milk secretion, pain on palpation of the udder and raised clinical parameters. The response of therapeutic study revealed significant improvement in temperature, pulse and respiration in all the treatment groups. On the basis of score card and polymerase chain reactions results on days 7 and 14 post-treatment, goats of the group T4 that was subjected to the treatment with combination of tylosin, tocopherol and sodium selenite showed earliest recovery.

Detection and Isolation of Mycoplasma capricolum Subspecies Capricolum from East Azerbaijan Sheep Flocks.

Mycoplasma capricolum subspecies capricolum (Mcc) is one of the causative agents of contagious agalactia (CA), which is an important disease in sheep and goats in the Mediterranean and Middle East countries. Mycoplasma agalactiae is the classic agent of CA in sheep and goats. Mycoplasma mycoides subspecies Capri (Mmc), Mycoplasma capricolum subspecies capricolum (Mcc), and Mycoplasma putrefaciens (Mp) produce a clinically similar disease, more often in goats. The aim of the present study was to detect Mcc in sheep flocks in East Azerbaijan Province of Iran. Milk, ear canal, and eye swab samples were collected from 49 sheep flocks with clinical signs of CA or a history of a disease. All the samples were examined using both culture and molecular methods. In the molecular method,positive samples for the Mycoplasma genus were tested for M. mycoides cluster and Mcc. From 272 samples, 67, 87, and 62 samples were shown to be positive using the culture method, polymerase chain reaction (PCR) method, and both culture and PCR methods, respectively. Mcc was detected in all the four M. mycoides cluster positive samples, including milk, ear canal, and eye swab samples. This is the first report of Mcc detection from East Azerbaijan. Our results showed that eye, milk, and ear canal samples could be suitable sources for Mcc detection in sheep flocks.

Detection of Mycoplasma agalactiae in Small Ruminants of Southeast Iran.

Agalactia is an infectious and contagious disease of small ruminants caused by Mycoplasma agalactiae (M. agalactiae). Although different microorganism strains contribute to this disease, M. agalactiae is known as the most prominent causative agent. Therefore, this study aimed to investigate the rate of M. agalactiae involvement in contagious agalactia in the southeast region of Iran. Sampling was performed from milk, conjunctiva, ear lesions, and joints exudate of suspicious sheep and goat flocks according to the reports of Iran Veterinary Organization. The presence of Mycoplasma and its species, namely M. agalactiae, was evaluated through microbial culture and polymerase chain reaction (PCR) techniques. The detected microorganisms were confirmed to be Mycoplasma and M. agalactiae by the PCR amplification of 16S rRNA and lipoprotein target genes. According to the findings of present study, 14.8% and 36.0% of the samples were diagnosed as positive for Mycoplasma by culture and PCR, respectively. Moreover, the incidence of M. agalactiae was determined as 6.1% using the specific PCR method. Therefore, it is recommended to identify the other species of Mycoplasma in small ruminant samples involved with contagious agalactiae disease.

Genetic loci of Mycoplasma agalactiae involved in systemic spreading during experimental intramammary infection of sheep

Mycoplasmas are amongst the most successful pathogens of both humans and animals yet the molecular basis of mycoplasma pathogenesis is poorly understood. This is partly due to the lack of classical virulence factors and little similarity to common bacterial pathogenic determinants. Using Mycoplasma agalactiae as a model we initiated research in this direction by screening a transposon mutant library in the natural sheep host using a negative selection method. Having successfully identified putative factors involved in the colonization of local infection and lymphogenic sites, the current study assessed mutants unable to spread systemically in sheep after experimental intramammary infection. Analysis of distant body sites for complete absence of mutants via SSM PCR revealed that additional set of genes, such as pdhB, oppC, oppB, gtsB, MAG1890, MAG5520 and MAG3650 are required for systemic spreading apart from those that were necessary for initial colonization. Additional in vitro studies with the mutants absent at these systemic sites confirmed the potential role of some of the respective gene products concerning their interaction with host cells. Mutants of pdhB, oppC and MAG4460 exhibited significantly slower growth in the presence of HeLa cells in MEM medium. This first attempt to identify genes exclusively required for systemic spreading provides a basis for further in-depth research to understand the exact mechanism of chronicity and persistence of M. agalactiae.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-016-0387-0) contains supplementary material, which is available to authorized users.

Severe otitis and pneumonia in adult cattle with mixed infection of Mycoplasma bovis and Mycoplasma agalactiae

Respiratory disease and otitis were seen on a northern Italian farm in weaned bulls obtained from multiple sources. Mycoplasma bovis was consistently identified in nasal swabs, tonsil, ears, eyes and...

Mycoplasma agalactiae Induces Cytopathic Effects in Infected Cells Cultured In Vitro.

Mycoplasma agalactiae is the etiological agent of the contagious agalactia syndrome in sheep and goats and causes significant economic losses worldwide. Yet the mechanism of pathogenesis is largely unknown. Even whole-genome sequence analysis of its pathogenic type strain did not lead to any conclusions regarding its virulence or pathogenicity factors. Although inflammation and tissue destruction at the local site of M. agalactiae infection are largely considered as effects of the host immune response, the direct effect of the agent on host cells is not completely understood. The aim of this study was to investigate the effect of M. agalactiae infection on the quality and viability of host cells in vitro. Changes in cell morphology including cell elongation, cytoplasm shrinkage and membrane blebbing were observed in infected HeLa cells. Chromatin condensation and increased caspase-3 cleavage in infected HeLa cells 48 h after infection suggests an apoptosis-like phenomenon in M. agalactiae-infected cells. In compliance with these results, decreased viability and cell lysis of M. agalactiae-infected HeLa cells was also observed. Measurement of the amount of LDH released after M. agalactiae infection revealed a time- and dose-dependent increase in HeLa cell lysis. A significant decrease in LDH released after gentamicin treatment of infected cells confirmed the major role of cytadherent M. agalactiae in inducing host cell lysis. This is the first study illustrating M. agalactiae’s induction of cytopathic effects in infected HeLa cells. Further detailed investigation of infected host tissue for apoptotic markers might demonstrate the association between M. agalactiae-induced host cell lysis and the tissue destruction observed during M. agalactiae natural infection.

Detecting asymptomatic rams infected with Mycoplasma agalactiae in ovine artificial insemination centers

Mycoplasma agalactiae (Ma) is the main causative agent of ovine contagious agalactia, which is a serious disease of small ruminants. In endemic areas, its most common clinical situation consists of chronically infected herds, and asymptomatic infected individuals represent an epidemiological risk regarding the transmission of this disease. The aim of this work was to detect the presence of asymptomatic rams infected with Ma in different artificial insemination centers, and to determine the most effective way to identify these individuals so as to implement adequate surveillance protocols. For this purpose, 215 rams and 14 teaser sheep were sampled taking auricular, nasal, and vaginal swabs and serum samples. In addition, ejaculates from 147 rams were analyzed. These samples were subjected to specific culture and molecular techniques to isolate and identify mycoplasmas, and to a serological test to detect antibodies against Ma. Mycoplasma agalactiae was detected in 47 (4.4%) of the 1077 samples analyzed, and also one individual resulted seropositive. Thus, 37 (17.2%) of the 215 studied rams were infected with Ma. The specimens which proportionally yielded the greatest number of positive results for this pathogen were semen samples (13.6%), followed by nasal swabs (5.8%). In contrast, the sampling of the external auricular canal and the serological analyses resulted insufficient to effectively detect infected individuals. Asymptomatic rams infected with Ma were detected in all the analyzed artificial insemination centers, highlighting the need to implement adequate surveillance protocols to prevent the presence of these individuals in these centers, reducing the risk of transmitting contagious agalactia.

Synthesis of Recombinant P48 of Mycoplasma agalactiae by Site Directed Mutagenesis and its Immunological Characterization

ABSTRACTContagious agalactia caused by Mycoplasma agalactiae is an economically important disease of sheep and goats and has been prevalent worldwide including India. The present study was undertaken to evaluate the membrane protein P48 of M. agalactiae for specific diagnosis of disease. For this, p48 gene of the organism was amplified by PCR and subjected to site directed mutagenesis to convert three TGA codons to TGG’s and, subsequently, cloned into prokaryotic expression vector pPRO EX HTb. Purified recombinant P48 protein reacted to anti-P48 serum in western blotting, which confirmed its immunogenic nature. Furthermore, the immune-blotting of the cell lysates from various Indian isolates of M. agalactiae against anti-P48 serum resulted in a single band at ∼ 48 kDa among all isolates, indicating the conserved nature of P48 antigen in M. agalactiae. Also, the cross reactivity of P48 antigen among various Mycoplasma spp. was checked by western blotting which revealed reactivity only with M. agalactiae and M. bovis. Hence, this antigen could be exploited to differentiate M. agalactiae from other pathogenic Mycoplasma species except M. bovis. However, the inability of P48 to distinguish M. agalactiae from M. bovis does not downgrade the significance of P48 as the two species are usually host specific.

Immunoproteomic identification of MbovP579, a promising diagnostic biomarker for serological detection of Mycoplasma bovis infection.

A lack of knowledge regarding the antigenic properties of Mycoplasma bovis proteins prevents the effective control of bovine infections using immunological approaches. In this study, we detected and characterized a specific and sensitive M. bovis diagnostic biomarker. After M. bovis total proteins and membrane fractions were separated with two dimensional gel electrophoresis, proteins reacting with antiserawere detected using MALDI-TOF MS. Thirty-nine proteins were identified, 32 of which were previously unreported. Among them, immunoinformatics predicted eight antigens, encoded by Mbov_0106, 0116, 0126, 0212, 0275, 0579, 0739, and 0789, to have high immunological value. These genes were expressed in E. coli after mutagenesis of UGA to UGG using overlap extension PCR. A lipoprotein, MbovP579, encoded by a functionally unknown gene, was a sensitive and specific antigen for detection of antibodies in sera from both M. bovis-infected and vaccinated cattle. The specificity of MbovP579 was confirmed by its lack of cross-reactivity with other mycoplasmas, including Mycoplasma agalactiae. An iELISA based on rMbovP579 detected seroconversion 7 days post-infection (dpi). The ELISA had sensitivity of 90.2% (95% CI: 83.7%, 94.3%) and a specificity of 97.8% (95% CI: 88.7%, 99.6%) with clinical samples. Additional comparative studies showed that both diagnostic and analytic sensitivities of the ELISA were higher than those of a commercially available kit (p<0.01). We have thus detected and characterized the novel antigen, MbovP579, and established an rMbovP579-based ELISA as a highly sensitive and specific method for the early diagnosis of M. bovis infection.

Mixed infection zones may be important in the epidemiology of contagious agalactia

Abstract Introduction: The current study was designed to detect Mycoplasma agalactiae (Ma), Mycoplasma mycoides subsp. capri (Mmc), Mycoplasma capricolum subsp. capricolum (Mcc) and Mycoplasma putrefaciens (Mp) in sheep and goats with clinical signs consistent with contagious agalactia. Material and Methods: A total of 299 samples were collected from 55 mixed herds in Azarbaijan-e-Sharghi province, Iran. Samples were examined using PCR and culture methods. Results: The findings showed that in 40 herds at least one sample was positive by PCR or culture method. Moreover, out of 274 sheep samples, 101 were proved to be positive using the PCR technique and 76 were found positive using the culture method. Out of 25 goat samples, 10 were found positive using PCR and 9 were positive through the culture method. Less than 20% of isolated mycoplasmas were Ma. Ma was detected from almost all studied regions in the province while Mmc, Mcc, and Mp were detected only in a very limited area that was deemed by the research group the mixed infection zone. Conclusion: In vaccination or eradication projects, it would be more economical to focus on mixed infection zones. Further investigation on mixed infection zones could facilitate better understanding of contagious agalactia epidemiology.

Mycoplasma lipoproteins are major determinants of neutrophil extracellular trap formation.

Neutrophil granulocytes are paramount to innate responses as major effectors of acute inflammation. Among the various strategies enacted by neutrophils to eliminate microbes NETosis is a novel distinct antimicrobial activity in which an interlacement of chromatin fibres rich in granule-derived antimicrobial peptides and enzymes is extruded (NETs, neutrophils extracellular traps ). NETs contribute to the pathogenesis of acute and chronic inflammatory disorders. The interactions of mycoplasmas and innate immune cells, in particular neutrophil granulocytes, are poorly defined. Here, we describe NET formation in vivo in the mammary gland and milk of sheep naturally infected by Mycoplasma agalactiae. Also, we assess the contribution of liposoluble proteins, the most abundant component of the Mycoplasma membrane, in inducing NETosis. We demonstrate that Mycoplasma liposoluble proteins induce NET release at levels comparable to what observed with other stimuli, such as lipopolysaccharides and phorbol 12-myristate 13-acetate. Stimulation of neutrophils with synthetic diacylated lipopeptides based on the M. agalactiae P48, P80, and MAG_1000 proteins, combined in a mix or used individually, suggests that NETosis might not be dependent on a specific lipopeptide sequence. Also, NETosis is partially abolished when TLR2 is blocked with specific antibodies. The results presented in this work provide evidences for the mechanisms underlying NET activation in mycoplasma infections, and on their contribution to pathogenesis of mycoplasmosis.

Mycoplasma agalactiae Secretion of β-(1→6)-Glucan, a Rare Polysaccharide in Prokaryotes, Is Governed by High-Frequency Phase Variation.

Mycoplasmas are minimal, wall-less bacteria but have retained the ability to secrete complex carbohydrate polymers that constitute a glycocalyx. In members of the Mycoplasma mycoides cluster, which are important ruminant pathogens, the glycocalyx includes both cell-attached and cell-free polysaccharides. This report explores the potential secretion of polysaccharides by M. agalactiae, another ruminant pathogen that belongs to a distant phylogenetic group. Comparative genomic analyses showed that M. agalactiae possesses all the genes required for polysaccharide secretion. Notably, a putative synthase gene (gsmA) was identified, by in silico reconstruction of the biosynthetic pathway, that could be involved in both polymerization and export of the carbohydrate polymers. M. agalactiae polysaccharides were then purified in vitro and found to be mainly cell attached, with a linear β-(1→6)-glucopyranose structure [β-(1→6)-glucan]. Secretion of β-(1→6)-glucan was further shown to rely on the presence of a functional gsmA gene, whose expression is subjected to high-frequency phase variation. This event is governed by the spontaneous intraclonal variation in length of a poly(G) tract located in the gsmA coding sequence and was shown to occur in most of the M. agalactiae clinical isolates tested in this study. M. agalactiae susceptibility to serum-killing activity appeared to be dictated by ON/OFF switching of β-(1→6)-glucan secretion, suggesting a role of this phenomenon in survival of the pathogen when it invades the host bloodstream. Finally, β-(1→6)-glucan secretion was not restricted to M. agalactiae but was detected also in M. mycoides subsp. capri PG3(T), another pathogen of small ruminants. Many if not all bacteria are able to secrete polysaccharides, either attached to the cell surface or exported unbound into the extracellular environment. Both types of polysaccharides can play a role in bacterium-host interactions. Mycoplasmas are no exception despite their poor overall metabolic capacity. We showed here that M. agalactiae secretes a capsular β-(1→6)-glucopyranose thanks to a specific glycosyltransferase with synthase activity. This secretion is governed by high-frequency ON/OFF phase variation that might be crucial in mycoplasma host dissemination, as cell-attached β-(1→6)-glucopyranose increases serum-killing susceptibility. Our results provide functional genetic data about mycoplasmal glycosyltransferases with dual functions, i.e., assembly and export of the sugar polymers across the cell membrane. Furthermore, we demonstrated that nonprotein epitopes can be subjected to surface antigenic variation in mycoplasmas. Finally, the present report contributes to unravel the role of secreted polysaccharides in the virulence and pathogenicity of these peculiar bacteria.

Diversity and variation in antimicrobial susceptibility patterns over time in Mycoplasma agalactiae isolates collected from sheep and goats in France.

Mycoplasma agalactiae is responsible for Contagious Agalactia, a severe syndrome affecting small ruminants worldwide and resulting in significant economic losses in countries with an important dairy industry. The aim of this study was to examine the antimicrobial susceptibility patterns of M. agalactiae isolates in France, their evolution over the last 25 years and their relationships with the genetic diversity of isolates and their origin (geographical and animal host). Susceptibility patterns were determined by measuring minimal inhibitory concentrations (MICs) of several antimicrobials used against mycoplasmas. Caprine M. agalactiae strains showed increased MICs over time for most of the antimicrobials tested, except fluoroquinolones. This susceptibility loss was homogeneous despite the considerable genetic and geographical heterogeneity of the isolates. In contrast, all the ovine isolates originating from a single clone and the same region showed increased MICs only to some macrolides. MICs have evolved differently depending on the origin of the isolates but the overall loss in susceptibility has remained far more moderate than that of Mycoplasma bovis, a cattle pathogen closely related to M. agalactiae. Several hypotheses are proposed to explain the differences in susceptibility patterns, such as local, specific, nonmycoplasma-targeting antibiotic treatments and the genetic background of isolates in connection with their animal host.

Presence of Mycoplasma agalactiae in semen of naturally infected asymptomatic rams

The purpose of the present study was to assess the presence of Mycoplasma agalactiae (Ma), the main causative agent of ovine contagious agalactia (CA), in semen of naturally infected rams. Therefore, semen samples from 167 rams residing in three different artificial insemination (AI) centers of a CA–endemic area were studied by microbiological and molecular techniques. In addition, serial ejaculates from the same rams were evaluated to determine the excretion dynamics of Ma. Of the 384 samples studied, Ma was detected in 56 (14.58%) which belonged to 44 different rams (26.35%). These findings confirm the ability of Ma to be excreted in semen of asymptomatic rams. Furthermore, these results also evidence the presence of these asymptomatic carriers of Ma in ovine AI centers, representing a serious health risk regarding the spread and maintenance of CA, especially in endemic areas. Moreover, the excretion of Ma in semen also points to the risk of venereal transmission of this disease. The current results highlight the need to implement control measures to prevent the admission of infected rams in AI centers and the necessity to continuously monitor semen samples to effectively detect infected individuals.

Isolation and Detection of Mycoplasma agalactiae from Semen Samples of Goats.

Contagious agalactia (CA) is a highly infectious disease of goats and sheep, and is a form of Mycoplasmosis, which is usually enzootic. Since Mycoplasma agalactiae (M. agalactiae) is the main cause of this disease in goats, the aim of this study was to isolate and detect M. agalactiae from semen of goat bucks. Thirty-nine semen samples were collected from goat bulks, and all samples were cultured in PPLO broth medium supplemented for M. agalaciae isolation. The bacteria DNAs were extracted from clinical samples and the PCR assay was applied to detect Mycoplasma genus and M. agalactiae species using specific primers, which amplified a 163bp fragment in 16SrRNA gene and a 375bp fragment in lipoprotein gene. The PCR evaluations were performed for both the clinical samples and the cultures. Out of the 39 samples, 29 (74.3%) of the cultures were shown positive and typical Mycoplasma colonies grew on PPLO agar, which could be considered as the diagnostic method. In addition, 38 (97.4%) samples had positive PCR results for Mycoplasma genus and six (15.3%) of the samples were shown to be positive using PCR for M. agalactiae as the diagnostic method. In the present study, M. agalactiae was detected in semen of goat bulks for the first time in Iran. Therefore, it is recommended to concern semen as one of the significant sources for this pathogen and the possibility for transmission to the female goats through semen is highlighted. Moreover, presence of this microorganism in semen could be involved in infertility of goat population.

Comparison of PCR tests for the detection of Mycoplasma agalactiae in sheep and goats

The aim of this study was to compare PCR tests for the detection of Mycoplasma agalactiae in sheep and goats. Samples obtained from sheep and goats in Turkey were tested to determine the presence of M. agalactiae. Mycoplasma culture and two direct PCR methods based on the uvrC gene and polC gene were compared with PCR of the 16S rRNA gene followed by denaturing gradient gel electrophoresis (PCR/DGGE) on DNA extracted from clinical samples (following culture enrichment). A total of 234 samples were examined: 9.4% and 13.2% were positive for M. agalactiae based on the uvrC gene and polC gene PCRs, respectively, while the culture method revealed that 12.8% of the samples were mycoplasma-positive. The PCR/DGGE method identified M. agalactiae in 15.0% of the samples; it also detected and identified other Mycoplasma species in the samples. M. agalactiae was only detected in goats in this study. This is the first time 16S rRNA PCR/DGGE was performed on various samples from sheep and goats in Turkey. This study confirmed that M. agalactiae and other Mycoplasma species are present in goats, indicating a requirement for effective control strategies for contagious agalactia and Mycoplasma species in Turkey.

Sensitivity of two methods to detect Mycoplasma agalactiae in goat milk.

BackgroundLaboratory diagnostic techniques able to detect Mycoplasma agalactiae are essential in contagious agalactia in dairy goats. This study was designed: 1) to determine the detection limits of PCR and culture in goat milk samples, 2) to examine the effects of experimental conditions including the DNA extraction method, PCR technique and storage conditions (fresh versus frozen stored milk samples) on these methods and 3), to establish agreement between PCR and culture techniques using milk samples from goats with mastitis in commercial dairy herds. The study was conducted both on artificially inoculated and field samples.ResultsOur findings indicate that culture is able to detect M. agalactiae in goat milk at lower concentrations than PCR. Qualitative detection of M.agalactiae by culture and PCR was not affected by sample freezing, though the DNA extraction method used significantly affected the results of the different PCR protocols. When clinical samples were used, both techniques showed good agreement.ConclusionsThe results from this study indicate that both culture and PCR are able to detect M. agalactiae in clinical goat mastitis samples. However, in bulk tank milk samples with presumably lower M. agalactiae concentrations, culture is recommended within the first 24 h of sample collection due to its lower limit of detection. To improve the diagnostic sensitivity of PCR in milk samples, there is a need to increase the efficiency of extracting DNA from milk samples using protocols including a previous step of enzymatic digestion.

Sheep primary cells as in vitro models to investigate Mycoplasma agalactiae host cell interactions.

Appropriate infection models are imperative for the understanding of pathogens like mycoplasmas that are known for their strict host and tissue specificity, and lack of suitable cell and small animal models has hindered pathogenicity studies. This is particularly true for the economically important group of ruminant mycoplasmas whose virulence factors need to be elucidated for designing effective intervention strategies. Mycoplasma agalactiae serves as a useful role model especially because it is phylogenetically very close to M. bovis and causes similar symptoms by as yet unknown mechanisms. Here, we successfully prepared and characterized four different primary sheep cell lines, namely the epithelial and stromal cells from the mammary gland and uterus, respectively. Using immunohistochemistry, we identified vimentin and cytokeratin as specific markers to confirm the typical cell phenotypes of these primary cells. Furthermore, M. agalactiae’s consistent adhesion and invasion into these primary cells proves the reliability of these cell models. Mimicking natural infections, mammary epithelial and stromal cells showed higher invasion and adhesion rates compared to the uterine cells as also seen via double immunofluorescence staining. Altogether, we have generated promising in vitro cell models to study host–pathogen interactions of M. agalactiae and related ruminant pathogens in a more authentic manner.

Investigation of Mycoplasma agalactiae in sheep in Kurdistan province by PCR

Contagious agalactia (CA) is a serious disease syndrome of sheep and goats that is characterized by mastitis, arthritis, keratoconjunctivitis and, occasionally, abortion. Mycoplasma (M.) agalactiae is the main cause of the disease in sheep and goats. The aim of the present study was to detect M. agalactiae in conjunctival, synovial fluid, nasal, ear and milk samples in sheep herds with or without CA sign in Kurdistan province. One hundred and seventy three samples analyzed were taken from sheep herds reared in a CA endemic area. Of the samples tested, 130 were positive by PCR for Mycoplasma spp. and of these, 19 showed a positive result for M. agalactiae . M. agalactiae was detected in conjunctival (7/59), synovial fluid (1/8) and milk (11/21) samples. Ear and nasal swap samples were free of M. agalactiae . The results of 60 samples in sheep without signs of CA syndrome showed that Mycoplasma spp. and M. agalactiae were detected in 25 and 6 samples, respectively. Our findings indicate that in Kurdistan province; M. agalactiae was not the main etiological agents of the CA syndrome. Also, this species can be isolated from animals without clinical signs of disease. Our results suggested that milk secretion is suitable for PCR detection of M. agalactiae .