Mycoplasma Broth Research Articles

The prevalence of Mycoplasma hominis in Outpatients at a Tertiary Care Hospital in East India.

Introduction Mycoplasma hominis (M. hominis) is the first Mycoplasma isolated from humans in the year 1937. Though regarded as a commensal of the urogenital tract, it has been implicated in various genital and extra-genital infections namely bacterial vaginosis, cervicitis, pelvic inflammatory diseases, pyelonephritis, premature rupture of the membrane in pregnancy, infertility, sepsis in newborns, etc. The pathogenesis, prevalence, and epidemiology of genital mycoplasmas in general and M. hominis in particular in Indian women have been studied very minimally. This study aimed to study the prevalence of M. hominis carriage among symptomatic and asymptomatic sexually active women attending to the outpatient department of a tertiary care hospital in East India with or without clinically suspected genitourinary infections and to compare the detection of M. hominis by polymerase chain reaction (PCR) as compared to that of culture. Methods In this observational study, sterile Dacron swabs were used to collect two samples each from the genitourinary tract of 110 sexually active women aged 15-45 years (80 cases and 30 control). One sample was inoculated in mycoplasma broth for culture. The other was used for PCRto detect the presence of theM. hominis gene. Results Culture positivity for mycoplasma was seen in 4/80 (5%) patients clinically suspected of genitourinary infection (cases) based on their presenting signs and symptoms. In those without such suspicion (control), all cultures were negative (p=0.021). PCR was positive for M. hominis in 22 (20%) samples. Considering the PCR as the gold standard the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of culture are found to be 18.18%, 100%, 100%, and 88.25% respectively. The highest prevalence of M. hominis was in the age group 20-24 years (9/24) and 50% of all detections (11/22) were among 25-29 years. Detections were more frequent among patients with multiparity, multiple sexual partners, intrauterine contraceptive devices, lower socioeconomic status, and lower educational status. Conclusion Our study results showed that the presence of M. hominis is significantly higher in cases than in the control group. The study also indicates the need for continued research on this bacterium both in patients with genital symptoms and in asymptomatic patients.

Isolation and molecular detection of avian mycoplasmosis in selected areas of Mymensingh district in Bangladesh

Avian mycoplasmosis caused by several species of Mycoplasma including Mycoplasma gallisepticum, M synoviae, M. meleagridis and M. iowae. Among these Mycoplasma gallisepticum is the most important poultry pathogen in Bangladesh. For effective control of Mycoplasmosis, proper early diagnosis is the corner stone. The research work was designed, a total of 20 samples, lung exudates, swabs from trachea and air sacs were collected from dead birds of different poultry farms in Mymensingh district during October-December, 2007. Samples were collected in 10% buffered formalin for histopathological study. Swabs were collected in mycoplasma broth supplemented with supplement-G. Additionally Kanamycin solution was added to prevent the growth of gram–Ve bacteria and then the organisms were transferred into mycoplasma agar for isolation. Histopathological studies were conducted using routine procedure in Hematoxylin and Eosin stain. Isolated Mycoplasma were subjected to DNA extraction, Nested PCR was done using a commercial PCR kit. The histopathological study revealed the presence of mycoplasmal related tissue changes, such as severe congestion and infiltration of mononuclear cells in different organs. The extracted DNA accumulated at the upper position of DNA ladder as band without any smear formation. The DNA from avian mycoplasmas was amplified and gave amplified product of 975 bp by outer primer and 395 bp by inner primer which was much smaller than the expected size. In this study, preliminary results from field samples suggest that culture using mycoplasma agar and broth supplement with Supplement-G and Kanamycin solution could be useful for the isolation of pathogenic avian mycoplasmas. Asian J. Med. Biol. Res. 2021, 7 (2), 182-190

Pathological and Immunohistochemical Studies of Experimental Mycoplasma pneumoniae in Gerbils (Meriones unguiculatus)

Mycoplasma pneumoniae (Mp) is a leading cause of human community-acquired pneumonia. To investigate the pathogenesis of the infection, 36 gerbils were intranasally inoculated with Mp culture (30 animals) or sterile mycoplasma broth (6 animals) and euthanized from 1 to 5 weeks post infection. A morphological and immunohistochemical study was carried out in all animals to determine the cellular populations present in lung parenchyma. Polyclonal and monoclonal antibodies were used to detect antigens of Mp and CD3, CD4, CD8 and CD79 lymphocytes, as well as cells containing S100 and major histocompatibility complex class II (MHC-II) antigens. There was progressive infiltration of mononuclear cells in the lamina propria of bronchi and bronchioles, and hyperplasia of the bronchus-associated lymphoid tissue (BALT) in the infected animals. BALT contained dendritic cells immunopositive to S100 and MHC-II and numerous CD3, CD4 and CD79 lymphocytes. The immunohistochemical results showed that T lymphocytes, particularly CD4 and CD79cells, may play a role in the immune response of gerbils against Mp. This experimental model is valuable for investigation of the pathogenesis of Mp infection and may assist in the development of therapeutic strategies.

Enhancement of Apx Toxin Production in Actinobacillus pleuropneumoniae Serotypes 1, 2, and 5 by Optimizing Culture Condition.

Actinobacillus pleuropneumoniae (APP) is a causative agent of porcine pleuropneumonia. Therefore, the development of an effective vaccine for APP is necessary. Here, we optimized the culture medium and conditions to enhance the production yields of Apx toxins in APP serotype 1, 2, and 5 cultures. The use of Mycoplasma Broth Base (PPLO) medium improved both the quantity and quality of the harvested Apx toxins compared with Columbia Broth medium. Calcium chloride (CaCl2) was first demonstrated as a stimulation factor for the production of Apx toxins in APP serotype 2 cultures. Cultivation of APP serotype 2 in PPLO medium supplemented with 10 μg/ml of nicotinamide adenine dinucleotide (NAD) and 20 mM CaCl2 yielded the highest levels of Apx toxins. These findings suggest that the optimization of the culture medium and conditions increases the concentration of Apx toxins in the supernatants of APP serotype 1, 2, and 5 cultures and may be applied for the development of vaccines against APP infection.

Immune responses to vaccination and infection with Mycoplasma gallisepticum in turkeys

ABSTRACTInfection with Mycoplasma gallisepticum induces severe lymphoproliferative lesions in multiple sites along the respiratory tract in chickens and turkeys. These immunopathological responses have been well-characterized in chickens, but have not been studied closely in turkeys. The aim of the study described here was to examine the immune responses of turkeys after live vaccination and infection with M. gallisepticum. In a strain comparison study, the mean log10 antibody titre of birds exposed to an aerosol culture of M. gallisepticum strain Ap3AS was found to be significantly higher at day 14 than that of birds exposed to strain 100809/31. In a dose–response study, there was a significant difference in the mean log10 antibody titre between birds exposed to mycoplasma broth and birds exposed to the highest dose of strain Ap3AS at day 7 after exposure. Immunohistochemical analysis of the tracheal mucosa and the air sacs revealed similar patterns of distribution of CD4+ and CD8+ lymphocytes to those seen in the tracheal mucosa of chickens, implicating these cell types in the pathogenesis of respiratory mycoplasmosis in turkeys. Turkeys that had been vaccinated with M. gallisepticum GapA+ ts-11 had significantly higher antibody titres than unvaccinated birds at both 7 and 14 days after challenge with strain Ap3AS. Vaccination with GapA+ ts-11 protected against the lymphoproliferative response to infection with virulent M. gallisepticum in both the tracheal mucosa and the air sacs, suggesting that this strain may be a useful vaccine candidate for use in turkeys.

DIAGNOSIS OF MYCOPLASMOSIS IN BROILER BREEDERS AND ITS TREATMENT

Two hundred broiler breeders of 13 month old were examined from flock of 52000 hens . These breeders showing clinical signs in the form of rales , depression , low egg production and low hatchability . The rate of the embryos death reached 20% and dead embryos showing dwarfing and generalized odema. Mycoplasma gallisepticum (M.G.) was isolated from breeders and their dead chicken embryos using mycoplasma broth and Mycoplasma plate agar . The antibodies against M.G. were elevated in the affected breeders . The hyperplasia of their lining epithelial ,parabronchial lymphoid aggregation , in addition to pneumonia and thickened air sacs by leukocytes and lymphocytes could be seen . After treatment with tylosin for one-month failure of isolation of M.G. was noticed . Clinical signs , lesions , egg production and hatchability were improved . The antibodies were declined after 5 months of treatment . The tylosin was effective in the treatment and control of M.G. infection .

Pathogenicity of local isolates of Mycoplasma ovipneumoniae and Mycoplasma arginini in experimental West African Dwarf goats

Objective: This study was carried out to assess the pathogenicity of local isolates of Mycoplasma ovipneumoniae and M . arginini in West African dwarf goats (kids) in Nigeria. Materials and methods: A total of 22 goats aged less than 1-year were purchased from markets. The goats were divided into six groups comprising of four experimental groups (EG; 4 in each) and two control groups (CG; 3 in each). The goats were fed ad libitum with standard diets and safe water. Groups EG1 and EG2 were infected with M . ovipneumoniae through trans-tracheal (TT) and intravenous (IV) routes, respectively, while those in groups EG3 and EG4 were infected with M . arginini through the same routes. Goats in groups CG1 and CG2 were inoculated with sterile Mycoplasma broth through TT and IV routes, respectively. In all cases, the amount of bacteria inoculated was 1.5x10 8 cells/mL. After the onset of the disease in goats, re-isolation of Mycoplasma was performed by culturing on mycoplasma agar supplemented with mycoplasma supplement G. The goats were monitored for 14 days post-infection (PI) to observe respiratory signs and mortality. Post-mortem (PM) examination was performed on each animal that died, while one surviving goat from each of the groups was sacrificed at 14 days PI for PM. After PM, histopathology was performed to observe the changes in tissues. Results: Cough and nasal discharges were observed in all the experimentally infected goats seven days PI. Mortalities were recorded in goats in EG1 (two goats), EG2 (one goats), EG3 (two goats) and EG4 (one goat). At PM, pneumonic lesions were observed in the lungs of all the experimentally infected goats. Conclusion: This study provides evidence that the local isolates of M . ovipneumoniae and M . arginini strains are pathogenic for goats in Nigeria. http://doi.org/10.5455/javar.2016.c161

Characterization and optimization of the haemozoin-like crystal (HLC) assay to determine Hz inhibiting effects of anti-malarial compounds.

BackgroundThe haem-haemozoin biocrystallization pathway is an attractive target where several efficacious and safe anti-malarial drugs act. Consequently, in vitro haemozoin (Hz) inhibition assays have been developed to identify novel compounds. However, results may differ between assays and often require complex methods or sophisticated infrastructure. The recently reported growth of haemozoin-like crystals (HLC) appears to be a simple alternative although the endproduct is structurally different to Hz. This study set out to characterize this assay in depth, optimize it, and assess its performance.MethodsThe HLC assay was used as previously described but a range of different growth conditions were examined. Obtained HLCs were investigated and compared to synthetic (sHz) and natural haemozoin (nHz) using scanning electron microscopy, powder X-ray diffraction (PXRD), Fourier Transform Infrared spectroscopy (FTIR) and Raman spectroscopy (RS). Interactions of HLC with quinolines was analysed using RS. Inhibitory effects of currently used anti-malarial drugs under four final growth conditions were established.ResultsHLC growth requires Mycoplasma Broth Base, Tween 80, pancreatin, and lysed blood or haemin. HLCs are similar to nHz and sHz in terms of solubility, macroscopic and microscopic appearance although PXRD, FTIR and RS confirm that the haem aggregates of HLCs are structurally different. RS reveals that CQ seems to interact with HLCs in similar ways as with Hz. Inhibition of quinoline drugs ranged from 62.5 µM (chloroquine, amodiaquine, piperaquine) to 500 µM in mefloquine.ConclusionsThe HLC assay provides data on inhibiting properties of compounds. Even if the end-product is not structurally identical to Hz, the inhibitory effects appear consistent with those obtained with sHz assays, as illustrated by the results obtained for quinolines. The assay is simple, inexpensive, robust, reproducible and can be performed under basic laboratory conditions with a simple visual positive/negative read-out.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0913-y) contains supplementary material, which is available to authorized users.

English

Aim: The present study was undertaken to determine the seroprevalence of mycoplasma infection and possible isolation from local chickens in Niger State, Nigeria. We have looked into this problem using a combined MG/MS ELISA kit and cultural isolation. Methods: A total of 552 blood samples were randomly collected from exanguinated chickens for serology. Tracheal swabs were collected into screwed cap bijou bottles containing 2ml of mycoplasma broth medium. Results: The sero prevalence of indigenous chickens from Niger State was 91.83% by MG/MS Elisa. A total of 126 swabs yielded to the growth of avian mycoplasma on mycoplasma agar. Key words: Isolation, Mycoplasma species, Avian, indigenous chickens

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%).

A simplified PCR assay for fast and easy mycoplasma mastitis screening in dairy cattle

A simplified polymerase chain reaction (PCR) assay was developed for fast and easy screening of mycoplasma mastitis in dairy cattle. Species of major mycoplasma strains [Mycoplasma (M.) bovis, M. arginini, M. bovigenitalium, M. californicum, M. bovirhinis, M. alkalescens and M. canadense] in cultured milk samples were detected by this simplified PCR-based method as well as a standard PCR technique. The minimum concentration limit for detecting mycoplasma by the simplified PCR was estimated to be about 2.5 × 103 cfu/mL and was similar to that of the standard PCR. We compared the specificity and sensitivity of the simplified PCR to those of a culture method. Out of 1,685 milk samples cultured in mycoplasma broth, the simplified PCR detected Mycoplasma DNA in 152 that were also positive according to the culture assay. The sensitivity and specificity of the simplified PCR were 98.7% and 99.7%, respectively, for detecting mycoplasma in those cultures. The results obtained by the simplified PCR were consistent with ones from standard PCR. This newly developed simplified PCR, which does not require DNA purification, can analyze about 300 cultured samples within 3 h. The results from our study suggest that the simplified PCR can be used for mycoplasma mastitis screening in large-scale dairy farms.

Nutritional effects of culture media on mycoplasma cell size and removal by filtration

Careful media filtration prior to use is an important part of a mycoplasma contamination prevention program. This study was conducted to increase our knowledge of factors that influence efficient filtration of mycoplasma. The cell size of Acholeplasma laidlawii was measured after culture in various nutritional conditions using scanning electron microscopy. The maximum cell size changed, but the minimum cell size remained virtually unchanged and all tested nutritional conditions resulted in a population of cells smaller than 0.2 microm. Culture in Tryptic Soy Broth (TSB) resulted in an apparent increase in the percentage of very small cells which was not reflected in increased penetration of non-retentive 0.2 microm rated filters. A. laidlawii cultured in selected media formulations was used to challenge 0.2 microm rated filters using mycoplasma broth base as the carrier fluid. We used 0.2 microm rated filters as an analytical tool because A. laidlawii is known to penetrate 0.2 microm filters and the degrees of penetration can be compared. Culture of A. laidlawii in TSB resulted in cells that did not penetrate 0.2 microm rated filters to the same degree as cells cultured in other media such as mycoplasma broth or in TSB supplemented with 10% horse serum.

Survey of bulk tank milk in New Zealand for Mycoplasma bovis, using species-specific nested PCR and culture

AIM: To survey the dairy cattle population in New Zealand for the presence or absence of Mycoplasma bovis. METHODS: A random cross-sectional survey of bulk tank milk from dairy herds in New Zealand based on regionally proportioned sampling, weighted towards herds with a high bulk tank milk somatic cell count (SCC) was used to detect M. bovis at a between-herd prevalence of 2%, with 99% confidence. Bulk tank milk samples collected on-farm were tested using a nested M. bovis PCR, and bacteriological culture employing enrichment in mycoplasma broth and direct plating onto mycoplasma agar. RESULTS: Mycoplasma bovis was not detected in any of the 244 bulk tank milk samples by either PCR or culture. CONCLUSIONS: This survey provides further evidence that M. bovis is not present in the dairy cattle population in New Zealand.

Effects of cyclosporin A on the immune responses and pathogenesis of a virulent strain of Mycoplasma gallisepticum in chickens

Immune responses to the virulent S6 strain of Mycoplasma gallisepticum in immunocompetent and cyclosporin A (CsA)-treated specific pathogen free chickens were investigated, and pathogenesis of the M. gallisepticum strain was also examined. Ten-day-old specific pathogen free chickens were inoculated by eye-drop with M. gallisepticum, and a control uninfected group was inoculated with mycoplasma broth. Blood was collected weekly for 4 weeks from five birds in each group and whole blood lymphocyte transformation assayed against concanavalin A and lipopolysaccharide. Blood samples were also collected at intervals for serological assays. Live body weight, clinical signs and lesions were monitored, and recovery of M. gallisepticum was attempted from choanal cleft of live birds and also from various sites at necropsy. In parallel to the aforementioned groups, another set of two groups of chicks treated with CsA was infected with M. gallisepticum S6 or mycoplasma broth. These groups were subjected to the same experimental procedures. In the immunocompetent chickens, M. gallisepticum caused temporary T-cell suppression at 2 weeks post-infection. Comparison of the clinical signs and macroscopic lesions produced in immunocompetent and CsA-treated chickens indicated that T cells may not play an active role in disease development. The percentage of birds with mycoplasma isolation and the load of mycoplasmas suggested that T cells may have some role in resisting mycoplasma colonization or in the elimination of the infection.

Poor systemic antibody response after vaccination of commercial broiler breeders with Mycoplasma gallisepticum vaccine ts-11 not associated with susceptibility to challenge.

A live attenuated Mycoplasma gallisepticum vaccine, ts-11, has been used for control of M gallisepticum in several countries. The rapid serum agglutination test is usually used as an indicator of flock response to vaccination; however, in some flocks, the detected response may be weak or absent. We investigated whether the low level, or lack, of systemic antibodies in ts-11-vaccinated flocks is correlated with susceptibility to infection after challenge with a virulent M. gallisepticum strain. Birds from 2 separate ts-11-vaccinated commercial flocks with no, or weak, rapid serum agglutination responses (at 11 or 14 wk postvaccination) were randomly selected and subjected to aerosol challenge with either M gallisepticum strain Ap3AS or sterile mycoplasma broth. A group of nonvaccinated specific-pathogen-free chickens at similar age were also exposed to aerosolization with M. gallisepticum strain Ap3AS and used as positive controls. Postmortem examination of the birds, performed 2 wk after challenge, revealed no significant difference in microscopic tracheal lesions or mucosal thicknesses between the ts-11-vaccinated field birds irrespective of their aerosolization treatment. However, both microscopic tracheal lesions and tracheal mucosal thicknesses of nonvaccinated challenged birds were significantly greater than those of ts-11 vaccinates. Hence, broiler breeders vaccinated in the field showed significant protection against virulent M. gallisepticum challenge even when no serum antibody was detected by rapid serum agglutination test. These results reveal that seroconversion detected by rapid serum agglutination test after ts-11 vaccination is not a reliable predictor of protection against M. gallisepticum infection. The possible significance of local antibody response and cell-mediated immunity against M. gallisepticum infection is discussed.

Immunohistochemical Characterization of Lung Lesions Induced Experimentally by Mycoplasma agalactiae andMycoplasma bovis in Goats

Goats aged 3 months were inoculated with a recent isolate of Mycoplasma agalactiae (five animals) or Mycoplasma bovis (five animals) by a combined (intratracheal+intranasal) route. Two control goats were inoculated by the same route with sterile mycoplasma broth. Animals were killed 14 or 21 days after infection. At necropsy, tracheal and lung tissue was taken for pathological and immunohistochemical examination to determine changes in the lymphocyte subpopulations in the bronchus-associated lymphoid tissue (BALT). Consolidation of the lungs was not observed in any animal. M. agalactiae or M. bovis was recovered from the respiratory tract and lung of all but two infected animals. BothMycoplasma spp. induced a moderate bronchointerstitial pneumonia, characterized by lymphoid hyperplasia of the BALT and infiltration of mononuclear cells into the alveolar walls. The predominant phagocytic cell in the pulmonary parenchyma and the airways was the macrophage. The main cellular type in the BALT was the CD3+T lymphocyte, and the ratio of CD4+: CD8+cells was >1. It is likely that cellular immune mechanisms, through the activation of CD4+T lymphocytes, plays a prominent role in the acute and subacute phase of these infections.

Field Investigation of Mycoplasma gallisepticum Infections in House Finch (Carpodacus mexicanus) Eggs and Nestlings

We conducted a field study to investigate the occurrence of Mycoplasma gallisepticum (MG) in eggs and nestlings from nests of house finches (Carpodacus mexicanus). Forty-three nests were located between the months of April and August 1998 and were followed with one to three sampling efforts. Vitelline membrane of fresh eggs, whole embryos, or swabs from the choanal cleft or conjunctiva of nestlings were inoculated into mycoplasma broth for MG isolation and polymerase chain reaction (PCR) testing. No isolation of MG was made from 39 eggs or 110 nestlings sampled during the study. Pooled choanal and conjunctival swab samples from two broods of nestlings, however, tested positive for MG by PCR. None of the nestlings examined showed clinical signs of conjunctivitis, and no nestling mortality could be linked to MG infection. Serologic tests from 37 older nestlings were negative for antibodies to MG. The results suggest transmission of MG is occurring between breeding adults and their dependent offspring (pseudovertical transmission). Evidence supporting transovarian transmission of MG was not found in these house finches.

The development of lameness and bone deformity in the broiler following experimental infection with Mycoplasma gallisepticum or Mycoplasma synoviae

Summary Groups of eight 7‐day‐old Mycoplasma‐free broiler chicks were inoculated with one of two strains of M. gallisepticum (MG/S6, MG/B31/85) or one of two strains of M. synoviae (MS/B31/88 and MS/B94/91) into the left hock joint. Controls were similarly inoculated with sterile mycoplasma broth. The birds were assessed at 2, 3, 4 and 5 weeks of age (1, 2, 3 and 4 weeks post‐injection) for lameness and joint and limb lesions. At 5 weeks of age they were killed and examined for gross skeletal abnormalities and microscopic lesions of both hock joints. The in‐contact controls showed no joint swelling or lameness. The mycoplasma did not apparently spread to cause gross pathology in the joints of in‐contact control birds, or to other joints in a bird injected with mycoplasma. Each mycoplasma strain caused specific pathologies manifest as varying degrees of lameness, left hock joint swelling, bone deformity and differences in the severity of articular cartilage and chondrodystrophic growth plate pathology. Shortening and bowing of the right tibiotarsus in the birds injected with mycoplasma indicated unilateral increased limb loading contributing to bone deformity. Chondrodystrophy seen with three mycoplasmal strains may have contributed to bone deformity in these birds. Lameness appeared to be a consequence of either joint swelling and/or bone deformity resulting in mechanical dysfunction. The role of joint pain in the lameness was not determined.

The use of sodium pyruvate and sodium hydroxide to increase the number of colonies and coloured antigen yield of Mycoplasma gallisepticum

A trial to increase the number of colonies and coloured antigen yield of Mycoplasma gallisepticum in mycoplasma broth medium was conducted by comparing the use of normal medium, medium with sodium pyruvate and medium with sodium hydroxide. The result showed that medium with §odium pyruvate had increased the number of colonies and antigen yield and was highly significant different (P< 0.01) compared to both normal medium and medium with sodium hydroxide. Medium with sodium hydroxide produced the number of colonies and antigen yield lower than medium with sodium pyruvate, but higher and was highly significant different (P< 0.01) compared to the normalmedium. Key words: Sodium pyruvate, sodium hydroxide, Mycoplasma gallisepticum antigen

Detection of Mycoplasma mycoides subspecies mycoides by monoclonal antibody-based sandwich ELISA

Monoclonal antibodies (MAbs) were produced from a mouse immunised with Mycoplasma mycoides subsp. mycoides small colony ( MmmSC) antigen and their use to detect and differentiate strains within the Mycoplasma mycoides cluster investigated in an antigen capture ELISA format. The MAbs produced could not distinguish between MmmSC and M. mycoides subsp. mycoides large colony ( MmmLC) strains. However, the sandwich ELISAs developed were able to specifically distinguish these two biotypes from the other four members of the M. mycoides cluster, and from all other mycoplasma or bacteria species examined. The most sensitive application of the test was a combination of enrichment and capture by overnight or 48-h incubations of samples inoculated into mycoplasma broth in antibody-coated microtiter wells.