Isolation Of Mycoplasmas Research Articles

Isolation and Identification of Mycoplasma agalactiae by Culture and Polymerase Chain Reaction Methods in the Sheep Herds in Guilan Province, Iran.

Contagious agalactia is an infectious syndrome of sheep that is characterized by mastitis with reduction of milk production, arthritis, abortion, and keratoconjunctivitis. The disease is rapidly spread by the contact of the infected animals with the healthy ones. Domestic sheep and goats of both sexes can be infected at an equivalent frequency. Most of the researchers use culture and molecular methods for the isolation and identification of Mycoplasma. Mycoplasma agalactiae is the main cause of the disease in sheep. The aim of this study was to isolate and identify M. agalactiae by using culture and polymerase chain reaction (PCR) assay in the sheep herds in Guilan province, Iran. A total of 71 specimens were collected from seven sheep herds with clinical signs of agalactia disease. All of the seven sheep herds (100%) were positive either in PPLO agar or Mycoplasma PCR test. Out of the 71 specimens, 50 (70.4%) cases were positive; however, 21 (29.6%) samples were negative. Furthermore, 40 (80%) cases of the positive samples were detected for the presence of Mycoplasma by the PCR method; nonetheless, 34 (68%) samples were positive in culture. Additionally, out of the 40 positive samples in Mycoplasma PCR, 11 (27.5%) samples were detected in M. agalactiae-specific PCR. The samples that were positive for Mycoplasma were mostly detected in the ear/vaginal, milk, and ear swab samples, respectively, by culture and PCR methods. The most positive samples of Mycoplasma / M. agalactiae were obtained from the ear and vaginal samples. Our findings demonstrated that Mycoplasma was one of the main etiological agents of the contagious agalactia in Guilan province. In addition, PCR was found to be more successful than the culture method in the detection of Mycoplasma.

Molecular Characterization of Mycoplasma Isolated From Chicken

In order to study the Molecular characterization of mycoplasma isolated from chicken. A total number of 200 samples collected from birds showing respiratory manifestations and apparently healthy chicken of different ages(two weeks: two months) from different localities(al-ismailia & sharkeya Governorates). These samples included 110 samples from respiratory organs(trachea -lung -air sac) & 75 swabs from nasal cleft and 15 samples from fluid of swollen joints. A trial for isolation and identification of different Mycoplasma was done using conventional and recent techniques. All the results were finally confirmed by polymerase chain reaction (PCR) which showed products of the amplified16S rRNA gene and mgc2 gene of MG, detection of 16s rRNA gene of MS and 16S rRNA gene of un typed Mycoplasma in chickens using a set of primers tested and compared according to reference strain .The PCR amplification products were electrophoresed on 1% agrose gel stained with ethidium bromide. All the examined field isolates were identified as Mycoplasma (gave a characteristic common band at different levels of bp fragment). Primary isolation of the microorganism on PPLO medium, which appeared as fried egg when examined by dissecting microscope yielded118 positive samples with a total incidence rate 59%. The highest recovery rate was from respiratory organs (72.7%) followed by swabs from nasal claft (46.7%) and samples from swollen joints (20%).Sequence analysis of two Mycoplasma gallisepticum (strains for mgc2 gene from chicken revealed high similarity with the homologous reference strains on Gene Bank. and take these experessions on Gene Bank as Man-Reh.1/Mg/CK/EG016 acc# KY404986 and Man-Reh.2/Mg/CK/EG016 acc# KY404987.

In Vitro Activities of Gepotidacin (GSK2140944) and Other Antimicrobial Agents against Human Mycoplasmas and Ureaplasmas

Gepotidacin, a novel first-in-class triazaacenaphthylene topoisomerase II inhibitor, was tested against 85 type strains and clinical isolates of Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma parvum, and Ureaplasma urealyticum in comparison to levofloxacin, moxifloxacin, azithromycin or clindamycin, and tetracycline. Gepotidacin MIC90s (μg/ml) were 0.125 (M. pneumoniae), 0.032 (M. genitalium), 2 (M. hominis), and 8 (Ureaplasma species). Gepotidacin activity was not affected by resistance to fluoroquinolones, tetracyclines, or macrolides in the strains tested. Gepotidacin merits further study for treating infections caused by these organisms.

Therapeutic efficacy of azithromycin, clarithromycin, minocycline and tosufloxacin against macrolide-resistant and macrolide-sensitive Mycoplasma pneumoniae pneumonia in pediatric patients.

ObjectiveTo clarify therapeutic effects of azithromycin, clarithromycin, minocycline and tosufloxacin against macrolide-resistant Mycoplasma pneumoniae (MRMP) pneumonia and against macrolide-sensitive Mycoplasma pneumoniae (MSMP) pneumonia in pediatric patients.MethodsA prospective, multicenter observational study was conducted from July 2013 to August 2015. The therapeutic effects of azithromycin, clarithromycin, minocycline and tosufloxacin were evaluated in 59 patients with pneumonia caused by MRMP and in 50 patients with pneumonia caused by MSMP. In vitro activities of antimicrobial agents against isolates of Mycoplasma pneumoniae were also measured.ResultsMean durations of fever following commencement of treatment in patients infected with MRMP and MSMP were 5.2 and 1.9 days, respectively (log-rank test, P < 0.0001). Among patients infected with MRMP, mean durations of fever were 4.6, 5.5, 1.0 and 7.5 days for patients treated with azithromycin, clarithromycin, minocycline and tosufloxacin, respectively (log-rank test, P < 0.0001). Among patients infected with MSMP, mean durations of fever were 2.5, 1.7, 0.9 and 4.3 days for patients treated with azithromycin, clarithromycin, minocycline and tosufloxacin, respectively (log-rank test, P = 0.0162). The MIC90s of azithromycin and clarithromycin among the 27 isolates of MRMP were 64 and 256 μg/ml, respectively, and those among the 23 isolates of MSMP were <0.000125 and 0.001 μg/ml, respectively. The MIC90s of minocycline and tosufloxacin among the 27 isolates of MRMP were 1.0 and 0.25 μg/ml, respectively, and those among the 23 isolates of MSMP were 1.0 and 0.5 μg/ml, respectively.ConclusionBoth minocycline and tosufloxacin showed good in vitro activities against MRMP. Minocycline, but not tosufloxacin, shortened the duration of fever in pediatric patients infected with MRMP compared to the duration of fever in patients treated with macrolides.

A systematic review and meta-analysis of the association between Mycoplasma spp and upper and lower respiratory tract disease in cats

OBJECTIVE To critically assess available data from controlled observational studies on the pathogenic role of Mycoplasma spp in the upper respiratory tract (URT) and lower respiratory tract (LRT) of cats. DESIGN Systematic review and meta-analysis. SAMPLE 12 studies. PROCEDURES Seven electronic databases were searched for relevant publications. Risk of bias was assessed via the Newcastle-Ottawa Scale. Meta-analyses, stratified by URT versus LRT disease, were performed to estimate pooled ORs and 95% confidence intervals (CIs) for the association between Mycoplasma isolation and URT or LRT disease. Subanalyses by diagnostic method, sampling site, and environment (shelter vs nonshelter) were planned for studies on URT disease. RESULTS A significant association was found between isolation of mycoplasmal organisms and URT disease (pooled OR, 1.65; 95% CI, 1.14 to 2.40) but not LRT disease (pooled OR, 1.56; 95% CI, 0.51 to 4.76). The association with URT disease was only significant when conjunctival or pharyngeal samples from nonshelter cats were analyzed with a Mycoplasma felis-specific PCR assay. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that M felis may be a primary pathogen in cats with URT disease, warranting treatment in infected cats. The environment was important to consider when interpreting a mycoplasma-positive sample because of aclinical carriage, especially in shelter cats. Further investigations are needed to determine the role, if any, of mycoplasmal organisms in LRT disease of cats.

Contagious caprine pleuropneumonia: The first isolation and molecular characterization of Mycoplasma capricolum subsp. capripneumoniae in the Kingdom of Saudi Arabia

Classical contagious caprine pleuropneumonia is one of the most fatal contagious disease of goats listed by World Organization for Animal Health that leads to major economic losses. It is caused by infection with Mycoplasma capricolum subspecies capripneumoniae. In order to isolate the causative agents of CCPP for the first time in the Kingdom of Saudi Arabia, fifteen flocks from Eastern region (Al Ahsa, Dammam and Hafr Albaten) and ten flocks from Riyadh and Al-Kharj regions were selected for this study. A total of 700 samples (400 nasal swabs, 300 pleural fluid samples and lung samples (from necropsied animals)) were collected from goats showing typical signs of CCPP. The clinical signs of diseased cases revealed serous to mucoid nasal discharge, coughing, dyspnea, frothy salivation, and fever (40–42°C). Necropsied animals showed fibrinous pleuropneumonia and increased pleural fluid. Of 400 nasal swabs, 190 pleural fluid, and 110 lung samples, 26 (6.5%), 31 (16.3%) and 19 (17.3%) Mycoplasma isolates were recovered, respectively. Biochemically, all isolates were sensitive to digitonin and fermented glucose. Sixty seven of Mycoplasma isolates were belonged to Mycoplasma mycoides cluster based on detection of 16S rRNA. Polymerase chain reaction screening of Mycoplasma isolates using specific primer for M. capricolum subsp. capripneumoniae confirmed 55 isolates to be M. capricolum subsp. capripneumoniae.

A contagious bovine pleuropneumonia outbreak on a research farm in Ethiopia, and its dynamics over an eight-month period.

Contagious bovine pleuropneumonia (CBPP) was recognised on Bako Agricultural Research Farm, in the Oromia Region of Ethiopia, for the first time on 5 May 2011. The outbreak was investigated by combining recognition of clinical signs, post-mortem examination, mycoplasma isolation and serological testing using competitive enzymelinked immunosorbent assay (c-ELISA). The clinical cases were monitored for eight months; sick animals were treated with a range of antibiotics and isolated if necessary. The outbreak of CBPP was confirmed both bacteriologically and serologically and had spread to almost the entire herd (96.7%) within the eight-month observation period. Of the animals that recovered after antibiotic treatment, 12.3% fell sick again, showed typical signs of CBPP and were considered to be carriers. The role of treatment in the prevention of the spread of CBPP was minimal. Newly purchased animals that were not tested and quarantined before being introduced onto the farm were suspected to have been the most probable source of infection.

A Novel SimpleProbe PCR Assay for Detection of Mutations in the 23S rRNA Gene Associated with Macrolide Resistance in Mycoplasma genitalium in Clinical Samples

Macrolide-resistant strains of Mycoplasma genitalium are an increasing problem throughout the world, and the implementation of a rapid and sensitive assay for mutation detection to guide treatment is needed. Macrolide-resistant strains have been shown to contain base substitutions in positions 2058 and 2059 (Escherichia coli numbering) in region V of the 23S rRNA gene. In this study, we present a SimpleProbe PCR followed by melting curve analysis to differentiate between macrolide-resistant mutants and wild types. The assay was performed on 159 Mycoplasma genitalium-positive samples, and the results were compared with DNA sequencing. We also looked at the prevalence of macrolide-resistant strains in a Norwegian population. Of 139 samples characterized successfully by sequencing, 54 (39%) were wild types and 85 (61%) were mutants, consisting of 59 (42%) A2059G, 24 (17%) A2058G, 1 (1%) A2058T, and 1 (1%) A2059C mutation. The melting curve analysis correctly differentiated between wild-type and mutant strains in all cases, but it could not identify the different mutant types. The SimpleProbe PCR proved to be a simple, rapid, and reliable method for the detection of macrolide-resistant isolates of Mycoplasma genitalium in a clinical setting.

Diagnosis of poultry mycoplasmosis by cultural isolation and PCR

The present investigation was designed for diagnosis of poultry mycoplasmosis by cultural isolation and PCR assay. Clinical specimens (159: 47 lungs, 21 trachea, 91 choanal cleft swabs) of poultry were simultaneously subjected to cultural isolation of Mycoplasma spp., PCR for detection of mycoplasmosis and for isolation of E. coli. Isolation of Mycoplsma spp. was carried out using pleuropneumonia like organism (PPLO) medium. Identification of genus Mycoplasma and differentiation from Acholeplasma and Ureaplasma was done by conventional methods. A total of 15 isolates were identified as Mycoplasma spp. with isolation rate of 9.43%. In MG (Mycoplasma gallisepticum) and MS (Mycoplasma synoviae) species specific 16S rRNA PCR assay, all 15 isolates were confirmed as MG species. Direct detection of mycoplasmosis in 159 clinical specimens by PCR, targeting MG and MS species-specific 16S rRNA gene, revealed 108 (67.92%) positive specimens. Out of 108, 105 (66.04%) and 3 (1.86%) were positive for MG and MS respectively. E. coli was found to be major pathogen associated with poultry mycoplasmosis in 28 MG-PCR positive cases out of 42 E. coli isolates recovered. Comparative results of PCR and cultural isolation showed that 16S rRNA MG and MS species specific PCR is superior to cultural isolation for crucial, rapid, specific and sensitive detection of poultry mycoplasmosis directly in clinical specimens.

The phylogeny of Mycoplasma bovis from pneumonic goats based on16S rRNA Sequences in El-Salam Canal at northern Sinai Governorate

Mycoplasma bovis (Mb) is significantruminant respiratory pathogen of economic importance. This study determined the incidence of isolation of Mb in association with pneumonia based on respiratory manifestation in goats and examined the antigenic variability between same species of Mb in El-Salam Canal at northern Sinai Governorate. Three hundred and thirty five samples were collected from goats suffering from respiratory manifestations (274 nasal swabs and 61 lung tissue). The samples were subjected to Mycoplasma (M) and Mb isolation by culture and polymerase chain reaction (PCR) based on 16S rRNA gene specific primers. Mb was recorded in 8% and 9.8% and 11.4% and16.4% from nasal swabsand pneumonic lungs to the total no. of samples by culture and PCR respectively. Results revealed that, Mb. is one of the important pathogen causing pneumonia in goats. In additions, there was great dissimilarity of 16S rRNA gene between isolated strains and the isolates was harboring a novel genotype. Histopathological findings supported the mycoplasmic lesions. This research provides the first evidence of isolation and identification of Mb from the nasal swabs and lungs of goats with respiratory problems. The study showed for the first time the sequence analysis of 16S rRNA gene of this pathogen isolated from pneumonic goats in this area.

Mycoplasma detection and isolation from one-humped camels (Camelus dromedarius).

In scientific literature, a small amount of information is found concerning mycoplasmosis in camel species. Mycoplasma (M.) arginini, Acholeplasma (A.) laidlawii, and Acholeplasma oculi have been reported to be isolated from these host species. Serologically positive results have been reported for Mycoplasma mycoides subsp. mycoides SC type, Mycoplasma capricolum subsp. capripneumoniae, and M. mycoides subsp. capri. The aims of this study were to detect, isolate, and identify mycoplasmas from camels (Camelus dromedarius). Initially, saliva and ear smears plus conjunctival and vaginal secretions were taken from five female animals, but only conjunctival secretions in three male animals, all belonging to the same farm. An unknown mycoplasma was isolated from one of the vagina samples. Additionally, another unknown and uncultured mycoplasma was detected with molecular biology in the same sample. In the second stage, 23 vaginal secretions were taken from the same farm plus another secretion from a different one. Ten isolates of the same unknown and previously isolated mycoplasma were detected, nine of them recovered from the vagina of female camels. Some mycoplasmas have been related to reproductive disorders; however, there is no evidence that the isolated mycoplasmas are related to such disorders.

ISOLATION AND IDENTIFICATION OF MYCOPLASMA FROM CASES OF PNEUMONIA IN FEEDLOT LAMBS

The present study was performed to detect and isolate the Mycoplasma as causative agent of atypical pneumonia in feedlot lambs. Samples (180) were collected from lambs suffering clinically from pneumonia. During the period October 2013 to March 2014. These samples were included nasal swabs (60), tracheal swabs (60) and infected lungs (60). Samples were cultured in the selective media (Mycoplasma Broth Base) and incubated aerobically at 35C° for 4 – 14 days. When turbidity appeared inoculum of each broth was cultured on Mycoplasma Glucose Agar medium and incubated at 35C° for 7 -14 days in candle jar. Primary isolation was demonstrated by color changing of diphasic media, The ability of staining with Dennis stain, reduction of Tetrazolium, red blood cell lysis and fried egg appearance of Mycoplasma colonies was also studied. The results of present study also showed the total rate of Mycoplasma isolation (49.4%) from total samples, and the highest rate of isolation was from Nasal swabs (58%). While, the rate of isolation from Tracheal swabs (55.3%) and from infected lungs (35%).

Studies into the prevalence of Mycoplasma species in small ruminants in Benue State, North-central Nigeria.

The indicative prevalence of respiratory Mycoplasma species in small ruminants (SR) was determined in North-central Nigeria. Nasal swabs from 172 sheep and 336 goats from the Northeast, Northwest and South Senatorial Districts of Benue State were examined. Initial Mycoplasma isolation used Mycoplasma culture techniques followed by digitonin sensitivity testing. Species identification was done using polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE). Overall, Mycoplasma organisms were isolated from 131 (25.8%) of the 508 SR examined. Prevalence rates of 18.1 and 29.8% were recorded for sheep and goats, respectively. A total of 135 isolates of Mycoplasma belonging to three different species were identified: Mycoplasma ovipneumoniae (127), Mycoplasma arginini (7) and Mycoplasma mycoides subspecies capri (1). More than one Mycoplasma species were detected in four (3.1%) of the 131 confirmed Mycoplasma positive cultures. Mycoplasma was isolated from 16.2 and 29.1% of animals with and without respiratory signs, respectively. The high isolation rate of mycoplasmas in apparently healthy and clinically sick sheep and goats in this study indicates a carrier status in these SR which may constitute a serious problem in disease control.

Detection of Mycoplasma canadense and Mycoplasma californicum in dairy cattle from Argentina

Different species of Mycoplasma can affect bovine cattle, causing several diseases. PCR sequencing and further analysis of the 16S-23S rRNA ITS region have shown a significant interspecies variability among Mollicutes. Sixteen suspected isolates of Mycoplasma spp. obtained from milk samples from dairy herds were amplified (16S-23S rRNA ITS region). Fourteen out of those 16 suspected Mycoplasma spp. isolates were PCR-positive. To confirm the identity of Mycoplasma bovis, these 14 isolates were tested by another species-specific PCR. Seven of the isolates rendered a positive result. The products of 16S-23S rRNA ITS PCR from one isolate that was identified as M. bovis and from two other isolates, identified as non- M. bovis were randomly selected, sequenced and analyzed. The three sequences (A, B and C) showed 100% similarity with M. bovis, Mycoplasma canadense and Mycoplasma californicum respectively.

Detection, characterization, and molecular typing of human Mycoplasma spp. from major hospitals in Cairo, Egypt.

Mycoplasmas are fastidious slow growing organisms lacking a cell wall and mostly isolated from the mucosal surfaces of the respiratory and genitourinary tracts. There is a dearth of information regarding clinical Mycoplasma spp. isolates among Egyptian patients. A total of 170 samples were collected from patients and apparently healthy personnel in local public hospitals in Cairo, Egypt. Isolation of Mycoplasma spp. was carried out using appropriate culture media and further identification was carried out by biochemical tests followed by serotyping using specific antisera. Confirmation was done by PCR for detection of different Mycoplasma spp. using genus-specific primers targeting 16S ribosomal RNA gene. Characterization of the antibiotic resistance and sensitivity pattern against different antimicrobials was carried out using disc diffusion test. The results indicated the presence of six Mycoplasma spp. in 22.94% of the samples. Mycoplasmas were detected more frequently in throat swabs than sputum. Mycoplasma pneumoniae was highly sensitive to macrolides and quinolones but less sensitive to aminoglycosides and tetracyclines. Molecular techniques were found to be of more rapid, highly sensitive, able to detect nonviable organisms, and cost effective. These results shed light on difficulties of Mycoplasma detection and the superiority of molecular techniques over culture.

Presence of &amp;lt;i&amp;gt;Mycoplasma&amp;lt;/i&amp;gt; Spp. in Patients with Asthma or Allergic Rhinitis

Objective: To compare the presence of Mycoplasma spp. in pharyngeal swabs of patients with asthma, allergic rhinitis and controls, also to compare the reactivity to allergens in the three groups. Material and Methods: Throat swabs of 120 patients with asthma, 211 allergic rhinitis patients and 171 healthy persons were cultured in E media. Positive cultures were identified by PCR. Susceptibility of patients positive for isolation of Mycoplasma spp. to indoor, forest and undergrowth allergens were determined. Results: Mycoplasma spp. was isolated in 26.6% of samples from asthmatic patients, 36% in patients with allergic rhinitis and 2.9% in the group of healthy persons. Positive patients for Mycoplasma with asthma and allergic rhinitis showed susceptibility tomite, grass and thunder mainly. Conclusion: There is a higher incidence of Mycoplasma spp. in asthmatic and allergic rhinitis patients than healthy persons. Only patients with asthma or allergic rhinitis showed reactivity to environmental allergens.

Diagnosis of clinical or subclinical mastitis in ewes

Objectives of this paper are to review (i) diagnostic methods and procedures available for clinical or subclinical mastitis in ewes and (ii) applications of these procedures in the diagnosis of mastitis. Early and correct diagnosis of the disease is important for identification of affected animals. The following diagnostic procedures can be used: clinical examination, imaging techniques (ultrasonography, endoscopy), bacteriological examination of milk samples, immunological tests, identification of biomarkers (cytological examination of milk and measurement of milk electroconductivity). In most cases, diagnosis of clinical mastitis is straightforward, based on the findings of clinical examination. The differential diagnosis includes primarily (i) bacterial mastitis (usually sporadic occurrence in a flock, usually unilateral, isolation of bacteria from milk samples), (ii) mycoplasmal mastitis [usually epidemic occurrence in a flock, usually bilateral accompanied by other signs (e.g., arthritis), isolation of Mycoplasma spp. from milk samples] and (iii) infection by Small Ruminant Lentivirus [usually epidemic occurrence in a flock, usually bilateral accompanied by various signs (e.g., respiratory or neurological signs) in the same or other animals of the flock, detection of antibodies to the virus, pro-viral DNA or viral RNA in blood samples]. Subclinical mastitis should be always suspected as one of the primary causes in cases of decreased milk production in dairy flocks; it should also be considered as a possible factor in cases of suboptimal growth rate of lambs in mutton-type production flocks. Diagnosis of subclinical mastitis is based on detection of infection (i.e., isolation of microorganisms from milk samples) and/or inflammatory reaction in the mammary gland. The best method for detection of the inflammatory reaction remains the demonstration of increased cellular content in milk, although various other methods, have been proposed. For individual animals, values <0.5×106cellsmL−1 indicate a healthy mammary gland and values >1.0×106cellsmL−1 indicate a mammary gland with clinical or subclinical mastitis, with no need to perform a simultaneous bacteriological examination of milk samples to confirm the problem; values between 0.5×106 and 1.0×106cellsmL−1 indicate ‘suspected disease’, hence there is a need for performing bacteriological examination in milk. Two consecutive measurements increase accuracy of results. In bulk milk samples, counts of 0.65×106cellsmL−1 indicate approximately 15% prevalence of subclinical mastitis in the flock. In the differential diagnosis of cases of reduced milk yield in ewes, other possible causes of the problem should be taken into account (e.g., parasitic infections, chronic wasting diseases, suboptimal level of nutrition); in cases of suboptimal growth rate of lambs, other factors may be responsible (e.g., protozoan or parasitic infections, energy or micronutrient deficiency, viral disease).

Isolation of Mycoplasma spp. from broiler flocks with respiratory syndrome in Mashhad, Iran

Mycoplasmosis is one of the most important diseases in the poultry industry. Its causative agent, mycoplasma has various species, which two of them, Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are the most important species. Due to the enormous losses in the production farms of industrial poultry, achieving a rapid, accurate and definite diagnosis of mycoplasma is of great importance. An early and definite diagnosis can guarantee the farm management on keeping herd health. In many countries such as Iran, the disease and its complications have still remained as a serious problem. Given this issue, we decided to identify the mycoplasma infection from broiler poultry flocks through culture method. 150 carcasses of broiler chicken belonging to 50 broiler flocks were sampled in which the signs of air sacs involvement and secretions in the airways, trachea and bronchi were seen. Samples taken from trachea, palatine cleft, nasal passages and air sacs, were cultivated into PPLO liquid medium using membrane filters (0.45 micron). They were incubated at 37 °C and were examined for pH (color) changes for every 48 hours. During the first 24 hours after cultivation, every color change to yellow or dark visible with the bare eye was considered as bacterial contamination, therefore, the contaminated samples were removed from the incubator. The color change in the liquid media was compared with the uninoculated medium as negative control. If a color change was observed in the liquid media after 48h, subculture was done in the PPLO agar. The plates were incubated at 37 °C for 14 days. They were examined for mycoplasma colonies using a microscope with magnification of 10 in every other day. The results showed that out of 150 samples obtained from 50 broiler flocks, 16 (10.66%) were positive for mycoplasma, while in terms of contamination, 4 flocks (8%) were positive. The contamination of positive cultures was finally confirmed through PCR method with universal primer.

Isolation and Molecular Characterization of Mycoplasma gallisepticum and Mycoplasma synoviae in Chickens in Sudan.

The current study described the isolation and molecular detection of Mycoplasma gallisepticum (Mg) and Mycoplasma synoviae from tracheal swabs of diseased birds showing signs of respiratory distress in selected commercial (layer and broiler) farms and from yolk and an open air of pens of vaccinated breeder flocks in Sudan. A number of 45 Mycoplasma isolates were recovered from chickens in Khartoum, Gezira, and Equatoria states in Sudan. Of these, eight Mg and three Ms isolates were identified using growth inhibition and rapid serum agglutination (RSA) tests. The conventional PCR technique was applied to amplify 140 bp and 720 bp DNA fragments for the Mg and Ms, respectively. This research confirmed vertical and horizontal transmission of Mg from breeder farms through detection of Mg in yolk of fertile eggs and an air of pens despite previous vaccination. PCR is considered a rapid, sensitive, and cheap method and it will improve the diagnosis of Mycoplasma in chickens.

Isolation, molecular characterization and antimicrobial susceptibilities of isolates of Mycoplasma agalactiae from bulk tank milk in an endemic area of Spain

To isolate and characterize strains of Mycoplasma agalactiae from bulk tank and silo ewes' milk. Thirteen mycoplasma isolates were obtained from samples of sheep milk taken from bulk tank and large silos and identified as Myc.agalactiae by PCR-DGGE. The isolates were typed by pulsed field gel electrophoresis (PFGE), SDS-PAGE and immunoblot. The in vitro activity of 13 antimicrobials of veterinary interest was tested against these isolates. Results showed that the most effective compounds against Myc.agalactiae in vitro were clindamycin, an antibiotic not previously described as a suitable contagious agalactia (CA) treatment, with Minimum Inhibitory Concentration (MIC) values of <0·12μgml(-1) , and quinolones, with MIC values <0·12-0·5μgml(-1) , which are used as standard treatments against CA. Based on the in vitro assay, clindamycin, quinolones, tylosin and tilmicosin would be appropriate antimicrobials for CA treatment. The isolates were mostly resistant to erythromycin, indicating that it would not be a suitable choice for therapy. The isolates showed common molecular and protein profiles by PFGE and SDS-PAGE, with minor differences observed by immunoblot analysis, suggesting a clonal relationship among them. This study demonstrated the importance of the appropriate selection of antimicrobials for treatment of CA.