Hayflick Medium Research Articles

Insights into Clinical Features and Antibiotic Response in MCCP Infection among Small Ruminants

This study documents the clinical manifestations observed during sample collection from diseased small ruminants. The samples and clinical data were collected from CCPP suspected sheep and goat flocks. The culturing of Mycoplasma was performed using Modified Hay Flick medium. Mccp species were identified by biochemical profile. Antibiogram of the isolated strains was performed according to CLSI using various antibiotics including ampicillin, ceftiofur sodium, gentamicin, cloxacillin, tylosin, metronidazole, oxytetracycline, enrofloxacin and ciprofloxacin. Among the 385 culture-positive samples, fever (91.1%) and nasal discharge (88.3%) were the most prevalent symptoms, followed by cough (82.3%), dyspnea (63.6%), anorexia (61.6%), and weakness (56.8%). Contingency coefficient analysis revealed a strong association between Contagious Caprine Pleuropneumonia (CCPP) and clinical manifestations; temperature, nasal discharge, moderate association with cough and dyspnea, and weak association with anorexia and weakness. Additionally, the study assessed the in-vitro susceptibility of Mccp isolates to various antibiotics. Enrofloxacin demonstrated the highest effectiveness, followed by ciprofloxacin and gentamicin. Tylosin, metronidazole, and oxytetracycline showed moderate effectiveness, while ceftiofur sodium and cloxacillin were completely ineffective. In summary, clinical severity such as elevated temperature, nasal excretions, and respiratory distress was notable during the infection produced by prevailing field Mccp strains. The promising antibiotics response was determined for enrofloxacin, gentamicin and enrofloxacin against the isolated Mccp species from various regions of province KPK, Pakistan.

Mycoplasma bradburyae sp. nov. isolated from the trachea of sea birds

In the search for mollicutes in wild birds, six Mycoplasma strains were isolated from tracheal swabs taken from four different species of seabirds. Four strains originated from three Yellow-legged gulls (Larus michahellis) and a Cory’s shearwater (Calonectris borealis) from Spain, one from a South African Kelp gull (Larus dominicanus), and one from an Italian Black-headed gull (Chroicocephalus ridibundus). These Mycoplasma strains presented 99 % 16S rRNA gene sequence similarity values with Mycoplasma (M.) gallisepticum. Phylogenetic analyses of marker genes (16S rRNA gene and rpoB) confirmed the close relationship of the strains to M. gallisepticum and M. tullyi. The seabirds’ strains grew well in modified Hayflick medium, and colonies showed typical fried egg morphology. They produced acid from glucose and mannose but did not hydrolyze arginine or urea. Transmission electron microscopy revealed a cell morphology characteristic of mycoplasmas, presenting spherical to flask-shaped cells with an attachment organelle. Gliding motility was also observed. Furthermore, serological tests, MALDI-ToF mass spectrometry and genomic studies demonstrated that the strains were different to any known Mycoplasma species, for which the name Mycoplasma bradburyae sp. nov. is proposed; the type strain is T158T (DSM 110708 = NCTC 14398).

Prevalence, molecular characterization and antibiogram of Mycoplasma bovis isolated from milk in Pakistan.

Mycoplasma bovis is a highly contagious pathogen that causes clinical or subclinical mastitis. The present study was aimed for the isolation, molecular characterization and antibiogram determination of M. bovis from raw milk samples. Milk samples were collected randomly from lactating cows and buffaloes from different tehsils of district Faisalabad, Pakistan. Samples were inoculated on modified Hayflick medium and biochemical tests were performed for further confirmation of isolated M. bovis. Out of total 400 milk samples, 184 (46%) samples were found positive for culture method. The 16S-rRNA gene polymerase chain reaction was performed for molecular characterization of isolated M. bovis strains. Out of total 400 milk samples, 240 (60%) positive for M. bovis through PCR method were examined. The 16S-rRNA gene PCR positive isolated M. bovis strains were sequenced and results were compared using Maximum-likelihood method and sequenced strains of M. bovis were aligned and analyzed by Clustal W software. Antibiogram of isolated M. bovis strains was analyzed by disc diffusion assay against eight commonly used antibiotics. Tylosin (30μg) and Tilmicosin (15ug) showed inhibition zones of 32.34 ± 1.10 mm and 17.12 ± 0.93 mm respectively against isolated M. bovis which were found sensitive. Isolated M. bovis was found resistant to other commonly used antibiotics. Statistical analysis revealed that p-value was < 0.05 and the odds ratio was >1.0 at 95% CI. This study complemented the lack of epidemiological knowledge of molecular characterization, comparative effectiveness and resistance trends of isolated M. bovis strains against commonly used antibiotics.

Microbiological and molecular detection of Mycoplasma bovis in milk samples from bovine clinical mastitis

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

Staphylococcal intramammary infection dynamics and the relationship with milk quality parameters in dairy goats over the dry period

The objectives of this study were (1) to report the rates of new intramammary infection (IMI) and spontaneous IMI cure over the dry period in 3 dairy goat herds; (2) to evaluate the factors predicting infection dynamics over the dry period; and (3) to define milk quality parameter thresholds that predict infection dynamics over the dry period. Two consecutive udder-half milk samples were collected 10 to 14 d apart before dry-off from 288 goats in 3 herds, and 2 consecutive udder-half samples were collected 7 to 14 d apart in the following lactation, with the first sample being collected ≤10 d in milk, from 200 of the same goats. In 2 of the herds, udder-half milk samples were also collected at the same time points (n = 312 halves; 157 goats) for measurement of milk quality parameters. Standard aerobic culture of milk samples was performed for the detection of mastitis pathogens. To rule out the presence of Mycoplasma spp. IMI, milk samples were also cultured on modified Hayflick medium. Non-Mycoplasma isolates were speciated using MALDI-TOF mass spectrometry. Staphylococcal isolates, when not identified by MALDI-TOF, were speciated using partial gene sequence analysis of rpoB or tuf. When >1 sample from an udder half yielded the same species, available isolates from the first and last positive samples for that species were strain-typed using pulsed-field gel electrophoresis. Incidence of new IMI and cure rate were computed. Generalized linear mixed regression models were built to evaluate the associations between new IMI and pre-dry somatic cell score (SCS), between IMI persistence and half-level SCS, and between IMI persistence and pre-dry IMI species. Thresholds for pre-dry SCS and lactose concentration were computed to predict IMI persistence. Overall, 12.6% (48/380) of halves had a persistent IMI. Cumulative incidence of new IMI over the dry period was 13.2%, and cure rate was 52.0%. Pre-dry SCS was not associated with odds of new IMI or IMI persistence. Pre-dry IMI species was not associated with odds of persistence. Lactose concentration was not associated with odds of persistence. Regardless of culture data, the optimal pre-dry SCS threshold to detect IMI that would persist into the next lactation was 8.7, with sensitivity and specificity of 50 and 73.8%, respectively. Further studies on the effect of control measures on species-specific incidence and cure rates during the dry period are warranted.

Johne’s disease, Mycoplasma and BVD in Utah-Bulk Tank Milk Testing and Comparison to Previous Regional Prevalence and Individual Herd Results over Time

Dairy herd-level prevalence of Mycobacterium avium subsp. paratuberculosis (MAP), causative agent of Johne’s disease (JD), Mycoplasma spp., and Bovine Viral Diarrhea (BVD) virus were estimated in Utah and surrounding states and compared to previous surveillance results. Milk was collected at 3-4 day intervals on 5 dates (duplicate samples) from each bulk tank on participating farms, samples analyzed separately. One frozen sample was shipped to a laboratory for JD/MAP testing with ELISA and real-time PCR, the other paired sample was shipped to another laboratory for mycoplasma and BVD testing. Mycoplasma was cultured on modified Hayflick medium, standard methods; BVD testing was real-time, RT-PCR. 151/209 (72%) eligible dairy farms participated. Farms detected positive (some had multiple diseases) were: 58 JD (38%); 4 mycoplasma (3%); 14 BVD (9%). Sensitivity of testing was: 284/528 milks = 54% for JD, 17/61 = 28% for mycoplasma (lower than previous reports), 41/117 = 35% for BVD. Of 67 herds positive for JD in 2009, 28 (42%) remained positive, 14 (21%) became test-negative, and 25 (37%) were lost to follow up. Of 16 herds positive for mycoplasma in 2007, one (6%) remained positive, 8 (50%) became testnegative, and 7 (44%) were lost to follow up. Bulk milk remains a practical way to screen dairy herds for presence of JD, mycoplasma and BVD, provided that repeated sampling is used. Mycoplasma-positive herds were more likely to become test-negative in bulk milk in subsequent years than were JD-positive herds. Prevalence of BVD was similar to but slightly lower than previously reported.

Interação do Mycoplasma bovigenitalium com células do cumulus in vitro após o período de maturação oocitária bovina

O Mycoplasma é considerado cosmopolita, podendo ser disseminado através do comércio internacional de animais, sêmen industrializado e de produtos de transferência de embriões. A expansão de células do cumulus é utilizada como parâmetro de avaliação de oócitos bovinos cultivados in vitro e suas alterações morfológicas são representativas. O objetivo deste trabalho foi avaliar a interação do Mycoplasma bovigenitalium, exposto experimentalmente à cultura primária de célula do cumulus, após o período de maturação. Complexos oócitos cumulus (COCs) obtidos através de punção folicular de ovários bovinos, provenientes de abatedouro, foram divididos em dois grupos para serem maturados durante 24h em meio de maturação (TCM199 + hormônios) em estufa a 38º C, 5% de CO2, 95% de umidade. Posteriormente, os oócitos foram retirados das placas, permanecendo somente com as células do cumulus aderidas. Com o monoestrato celular formado, um grupo foi infectado com 30 mL de M. bovigenitalium, replicado em meio Hayflick modificado a 37º C em estufa de microaerofilia, enquanto o outro foi mantido como controle. Os resultados mostraram que, com 24h de exposição ao patógeno, as culturas apresentaram um pequeno número de células arredondadas e granulosas, quando comparadas as dos controles. Esse efeito persistiu até o sétimo dia, onde se iniciou um processo de descolamento das células. Pode-se concluir que uma contaminação por micoplasma pode ser imperceptível às manipulações da FIV, pois células infectadas por esse grupo de bactérias não apresentam turvações no meio de cultura e, quando não lisam a célula hospedeira, tornam mais suscetível ao ambiente e outros agentes infecciosos.

Detection of Mycoplasma ovipneumoniae and M. arginini in Bighorn Sheep Using Enrichment Culture Coupled with Genus- and Species-Specific Polymerase Chain Reaction

Mycoplasma species are of interest as possible primary pathogens in the pneumonia complex of bighorn sheep (Ovis canadensis). Previous investigations have not commonly detected low frequencies of Mycoplasma spp. from free-ranging bighorn sheep, possibly due to the fastidious and slow growth of these organisms. We developed a culture protocol that employed an average initial 3-day enrichment culture in liquid Hayflick broth in a CO(2)-enhanced atmosphere. The broth was plated to solid Hayflick medium and the cultures observed for growth for up to 30 days. Polymerase chain reaction (PCR) was performed on DNA isolated from the enrichment broth and on isolates obtained from culture using Mycoplasma genus-specific PCR assays and species-specific PCR assays for M. arginini and M. ovipneumoniae. Some cultures that grew on Hayflick plates were picked as single colonies but were mixed because two organisms may grow together and appear as a single colony. Culture and PCR tests produced similar results for M. arginini, but for M. ovipneumoniae, culture alone was less accurate than PCR. Use of genus-specific primers also may allow detection of other species in samples negative for M. arginini and M. ovipneumoniae. Two methods of transport from field to laboratory (Port-a-Cul™ tubes, cryoprotectant in liquid N(2) and Fisher Transport System) gave similar results under our study conditions.

ConfirmedMycoplasma pneumoniaeEndocarditis

Confirmed<i>Mycoplasma pneumoniae</i>Endocarditis

Étude prospective de la colonisation et de l'infection néonatales à mycoplasmes génitaux

Genital mycoplasmas have been implicated in different neonatal diseases as pneumonia, sepsis and meningitis. This prospective study was conducted to specify their role in these diseases. Population and methods .- A pharyngeal or tracheal swab specimen for mycoplasmas culture was obtained from 10G infants admitted consecutively to the Neonatal Care Unit (NCU) during the first 24 hours of life. Mycoplasma culture of blood and cerebrospinal fluid was also performed. Pharyngeal and/or tracheal specimens were collected again on days 5, 15 and 28 if the child was still in the NCU. Mycoplasma hominis (Mh) and Ureaplasma urealyticum (Uu) were identified by culture in a modified Hayflick's medium. Results .- Three-hundred and ten pharyngeal or tracheal swabs were obtained (100 on day 0, 89 on day 5, 72 on day 15 and 49 on day 28). Twenty-one infants had one or more positive swabs in the first five days of life (20 on day 0 and one on day 5); those forming the “Myco +” group and the others forming the “Myco - ” group. Uu was isolated alone from 20 infants, associated with Mh from one. Both groups were similar for gestational age, birth weight, maternal fever during labor, prolonged rupture of the fetal membranes or chorioamnionitis and for the incidence of acute respiratory distress. There was a statistically significant difference for the route of delivery ( χ 2 < 0.02). One blood culture (from 92 performed) was positive for Uu and another positive for Uu and Mh. Both children were cured without any specific mycoplasmacidal therapy. Three children had probable Uu infection and were also cured without specific therapy. Conclusions .- A pharyngeal colonization with genital mycoplasmas common in the first days of life (21%) but our data do not allow us to conclude that they are accountable for newborn infections.

Mycoplasma and Epidemic Group A Meningococcal Meningitis-Reply

In Reply. — As Drs Kenny and Foy point out, mycoplasma contamination of cell cultures is a common and insidious problem. We are confident that the M hominis isolated in our study came from the specimens and not the cell cultures due to the precautions taken to avoid and detect contamination. Ten uninoculated control cell cultures of each cell type were passaged in parallel with each batch of inoculated cultures; none of these controls became positive. As part of our quality assurance policies, each cell line is also tested monthly for mycoplasma at the Centers for Disease Control using an adenosine phosphorylase test (Mycotect, Gibco) and at the American Type Culture Collection (Rockville, Md) using both direct culture on Hayflick's medium and a DNA fluorescent staining test. 1,2 All three tests were consistently negative throughout the study period. Furthermore, when study specimens were positive for mycoplasma, all four cell types

Mycoplasma mobile sp. nov., a New Species from Fish

Mycoplasma strain 163K was isolated from the gills of a tench (Tinca tinca L.) with red disease. The cells are elongated, ovoid or flask shaped, consisting of a thicker body and a more slender part ending in a hemispherical terminal structure, that is apparently stabilized by a cytoskeleton. They are able to attach to inert surfaces and living cells. The most exciting property of the organisms is their fast gliding motion, which occurs on glass and plastic surfaces and which is not interrupted by resting periods. Growth occurs between 17 and 30°C, with an optimum at 25°C, in modified Hayflick medium with horse or bovine serum and in medium with swine serum. Acid is produced from several carbohydrates, but arginine and urea are not catabolized. The organism reduces tetrazolium chloride, produces film and spots and hydrogen peroxide, and is able to hemolyze and to adsorb to erythrocytes of several animal species. Indirect immunofluorescence test, growth inhibition, and metabolism inhibition tests indicate that the mycoplasma represents a new species for which the name Mycoplasma mobile is proposed. Strain 163K (ATCC 43663) is the type strain.

M. genitalium; studies on biological characteristics and pathogenicity in the experimental animal respiratory tract

A new mycoplasma species M. genitalium (G37), isolated from urethral specimens of patients with non-gonococcal urethritis, was studied.The serologic and bacteriologic characteristics, pathogenicity and possible role in human respiratory disease was discussed. The results were as follows:1. Biological properties. M. genitalium grew slowly in classical Hayflick medium and SP-4 medium. Cholesterol was required for growth. Colonies on agarose were variable in size and adherent to glass and plastic surfaces.2. Morphology and ultrastructure. Electron microscopy of M. genitalium cells was similar to that of M. pneumoniae, but flask-shaped cells with terminal rod-like structures were seen in some M. genitalium.3. Biochemical properties. The results of various biochemical tests were as follows: fermentation of glucose-positive; hydrolysis of arginine-negative; hemadsorption to human type-0 erythrocytespositive.4. Serological properties. Cross-reactivity between M. genitalium and M. pneumoniae was shown by complement fixation test, indirect hemagglutinin test, direct immunofluorescence and double immune diffusion methods. Cross-reactivity was not demonstrated by metabolic inhibition test and growth inhibition test.5. In hymsters intratracheal infection with M. genitalium produced peribronchitis, pneumonia and perivasculitis. These pathological findings were very similar to those seen in M. pneumoniae infection. It was suggested that M. genitalium may be pathogenic in animal respiratory tracts.6. M. genitalium inhibited ciliary activity of hamster tracheal rings.These observations indicate that the characteristics of M. genitalium are very similar to those of M. pneumoniae. M. genitalium may have a pathogenic role in human respiratory disease.

Isolation of a Motile Mycoplasma from Fish

For the first time a mycoplasma has been isolated from fish. The organism, designated strain 163K, was isolated on modified Hayflick medium under aerobic conditions at 25 degrees C from the gills of a tench (Tinca tinca L.). It showed the characteristic features of mycoplasmas. In addition it was flask-shaped with a distinct head-like structure and was able to attach to inert surfaces and living cells. The most interesting property of the organism was its ability to show fast gliding motion. Movement was only in the direction of the head-like structure and was not interrupted by resting periods.

Isolation of Acholeplasma laidlawii from commercial, serum-free tissue culture medium and studies on its survival and detection.

This report documents the first isolation of Acholeplasma laidlawii from a commercial lot of serum-free Dulbecco basal medium. Experimental studies demonstrated survival of the organism for at least 1 year depending on the concentration of the contaminating organism as well as pH and temperature of storage of the serum-free medium. A comparison of isolation methods showed that concentration by filtration through 220-nm membrane filters and testing the filters for mycoplasma recovery, especially when using phenol red-diphasic Hayflick medium, was both sensitive and practical for the average laboratory.

Isolation of Acholeplasma laidlawii from Commercial, Serum-Free Tissue Culture Medium and Studies on Its Survival and Detection

This report documents the first isolation of Acholeplasma laidlawii from a commercial lot of serum-free Dulbecco basal medium. Experimental studies demonstrated survival of the organism for at least 1 year depending on the concentration of the contaminating organism as well as pH and temperature of storage of the serum-free medium. A comparison of isolation methods showed that concentration by filtration through 220-nm membrane filters and testing the filters for mycoplasma recovery, especially when using phenol red-diphasic Hayflick medium, was both sensitive and practical for the average laboratory.

Pathophysiology of Mycoplasma pneumoniae Infection in Human Fetal Tracheal Organ Culture

Nycoplasma pneumoniae-host cell interactions have been difficult to analyze: natural disease is limited to man, and low mortality provides little pathologic material. Data from experimental models suggest that the ciliated respiratory epithelium is the target cell of M. pneumoniae. Evaluation was made of fetal tracheal organ culture as a means of providing organized differentiated human epithelial cells for studies in vitro. Tracheas were removed from 15–20-week fetuses, obtained aseptically by hysterotomy for psychiatric indications; transverse sections were maintained in Hayflick's medium with Hepes buffer at 36°C in 5% CO2. The effects of M. pneumoniae were studied by observations of ciliary function, light microscopy and immunofluorescence. Ciliary motion (which could be quantitated stroboscopically) slowed, became disorganized and ceased by 96 h. Microscopic changes included epithelial cytoplasmic vacuolization and nuclear swelling, followed by loss of cilia. Immunofluorescence identified organisms among the cilia, between cells, and in surface microcolonies. No comparable changes were produced by 4 other human mycoplasma species which were tested. These findings suggest the patho-physiology of M. pneumoniae disease by revealing both functional and structural changes in parasitized human respiratory epithelium. The nature of this interaction may explain many general features of M. pneumoniae disease, particulary the frequency of tracheo-bronchitis with protracted paroxysmal cough which commonly occurs in childhood infections.

Vital staining of Mycoplasma and L-forms with Chlorazol Black E.

Vital staining of Mycoplasma colonies was attempted because other dye visualization techniques kill the organisms and preclude reisolation for further studies. The lipophilic amphoteric dye Chlorazol Black E (CBE) was the most successful of 14 vital dyes tested on Mycoplasma hominis, M. pharyngis, M. fermentans, M. arthritidis, M. salivarium, M. pneumoniae, and L-forms of Staphylococcus aureus when used in 1:1,000 (w/v) saline dilution as the sterile suspension medium for inoculation of Hayflick's medium under both aerobic and microaerophilic (Fortner method) conditions. Colonies of all species stain homogeneously in the periphery and center portion, the latter being more refractive under positive phase contrast. All stained colonies were successfully subcultured. The most striking and promising result of the use of CBE as a tool for physiological study of Mycoplasma was a very significant increase in diameter of all colonies except those of M. pneumoniae grown with CBE: 1.5 x for M. hominis and 5 x for L-form S. aureus. This size increase in M. hominis is proportional to the concentration down to a 1:50,000 dilution only under microaerophilic conditions. Whether this increase in colony size is due to an increased number of cells, to larger cells, or to the adsorption of CBE on the lipid membrane is unknown at present.