Detection certainty of the contamination of bull sperm with mycoplasma

Tests for presence of mycoplasmas were conducted on 20 insemination bulls known as mycoplasma spreaders, with 5 sperm pellet batches of 20 pellets each being investigated for each animal. Mycoplasma contamination was positively recorded from 83 of the above 100 batches. Mycoplasma (M.) bovigenitalium, M. californicum. M. arginini, M. bovirhinis, and Acholeplasma laidlawii were typed by means of indirect immunofluorescent technique, which confirmed the presence also in the GDR of mycoplasma species described in the literature and detected in sperm samples. The solid culturing media used in the above tests, medium-B agar and cattle blood agar with staphylococcal nutrix, proved to be equally suitable for isolation of mycoplasma from sperm samples. Mycoplasma was positively identified in about one third of all pellets/batches tested. 3 pellets to one batch should be sufficient a random sample size from which to obtain information at least very close to real contamination.     

国内火鸡体内霉形体的分离与鉴定

从北京和呼和浩特市4个火鸡场的95只火鸡、50只火鸡胚和42份精液样品中分离到15个分离物,用国际霉形体分类分会推荐的生化、遗传学和血清学方法证明其中有两个分离物是两种霉形体混合物,共17株霉形体。所有分离物在无抑菌剂的培养基上连续传代5次未出现细菌形态、对毛地黄皂苷敏感、不水解尿素、DNA的G C含量介于26-34mol％之间。生长抑制试验和间接表面免疫荧光技术的试验结果将其中的15个株分属于霉形体属的5个种,即鸡毒霉形体(Mycoplasma gallisepticum)4株、滑液霉形体(M.synoviae)2株、衣阿华霉形体(M.iowae)1株、鸡霉形体(M.gallinarum)和雏鸡霉形体(M.pullorum)各4株。其余2株也属霉形体属,但尚未鉴定到种。     

Prevalence of mycoplasmas in the respiratory tracts of pneumonic calves.

The prevalence of mycoplasmas in the respiratory tracts of 148 pneumonic calves originating from 25 herds in the Netherlands is reported. Four types of culture media were used to isolate mycoplasmas: solid modified Edward medium, 2 types of Friis media, and A7B differential agar medium. Mycoplasmas were isolated both from nasal swab specimens and lung lavage fluids collected from live calves and from nasal mucosa and lung tissue specimens collected post mortem. All of the mycoplasma strains isolated could be identified as either Ureaplasma diversum (isolated from 80% of 25 herds), Mycoplasma dispar (92%), M. bovirhinis (88%), M. bovis (20%), M. bovigenitalium (4%), M. arginini (16%), or M. canis (12%). Isolation rates of M. dispar and U. diversum were considerably higher from lung lavage fluids than from nasal swab specimens. M. bovis was detected only in fattening herds and not in dairy herds. The respiratory tracts of 75% of the calves examined contained at least 2 mycoplasma species. In total, 25 different combinations of mycoplasma species were detected in specimens collected from noses and lungs. The pathogenic species U. diversum and M. dispar had not been isolated before in the Netherlands.     

Guidelines and recommendations for antimicrobial minimum inhibitory concentration (MIC) testing against veterinary mycoplasma species. International Research Programme on Comparative Mycoplasmology

The absence of standardised procedures for minimum inhibitory concentration (MIC) testing of antimicrobial agents against veterinary mycoplasma and ureaplasma species (Mollicutes) has made it difficult to compare results originating from different laboratories. This report, prepared on behalf of the International Research Programme on Comparative Mycoplasmology (IRPCM), offers guidelines and recommendations for veterinary MIC testing of these organisms in an effort to rectify this problem. The subjects discussed include suitable media for broth and agar MIC assays, storage and preparation of antimicrobial agents, standardisation of mycoplasma inocula for MIC tests, validation of equipment, incubation conditions, and determination of MIC end points. A standard medium for all veterinary mycoplasma MIC tests cannot currently be recommended, owing to the diversity of nutritional requirements of different mycoplasma species. Instead mycoplasma broths or agars giving optimal growth of specific mycoplasmas or ureaplasmas are recommended, as suboptimal growth may lead to falsely low MIC results. The importance of using standardised mycoplasma inocula, for assays using either solid or liquid media is stressed. The growth phase may be less important as lag phase and logarithmic phase cultures of Mycoplasma gallisepticum, M. synoviae, M. bovis and M. hyopneumoniae have given very similar results in liquid MIC assays. The liquid method of Tanner and Wu and the agar method described by Hannan et al. are compared and described in detail. Methods for calculating MIC50s and MIC90s are described and the interpretation of results discussed. Methods for assessing mycoplasmacidal (MMC) activity of antimicrobial agents are also described. Adoption of these guidelines should lead to more consistent MIC results being obtained between laboratories.     

Enzootic Pneumonia in Pigs: Propagation of a Causative Mycoplasma in Cell Cultures and in Artificial Medium

Three strains of a new species of mycoplasma were recovered from pneumonic pig lungs, known free of Mycoplasma hyorhinis, by prolonged incubation in pig testicle cell cultures. The three strains produced a characteristic cytopathic effect in the cell cultures. A highly enriched meat-infusion-broth medium was evolved and permitted regular propagation of these organisms. Pneumonia could consistently be produced by intratracheal inoculation of pigs with the mycoplasma propagated in the enriched broth medium or in cell cultures. The mycoplasma were recovered from the lungs of experimentally infected pigs by inoculation into the broth medium. Comparative studies of the pneumonia producing mycoplasma and of M. hyorhinis were carried out in cell cultures, broth media, and in pigs infected experimentally by different routes. The morphological characteristics of the mycoplasma, grown in the different media, are described and illustrated.     

Experience in identification and control of mycoplasma in dairy cattle stock under routine laboratory conditions

Exacting demands have to be met by milk testing routine laboratories where mycoplasma species are to be cultured from milk samples. Cleanliness of sampling, immediate cold storage, and no-delay transport of the milk samples to the testing centre are of greatest importance to the informative value of all mycoplasma tests. In the summer season, it is recommended to freeze the milk samples immediately after sampling. Mycoplasma broth should not be inoculated to animals of the herd from which the samples had been taken. A modified mycoplasma culturing medium is described in this paper. It will enable wider introduction of mycoplasma diagnosis by more Regional Institutes of Veterinary Medicine. Also reported in this paper are diagnosis and successful control of enzootic mastitis caused by Acholeplasma laidlawii and Acheloplasma axanthum. Repeated bacteriological testing of milk and secretion of all cows in the herd helped in picking out all bacteriologically positive animals and isolating them from the negative individuals. All animals that had produced positive responses to bacteriological testing were killed, notwithstanding clinical udder and general milk findings. Definite success of any control action undertaken against mycoplasmic mastitis will depend strongly on no-delay bacteriological testing of milk and secretion samples from all cows of the given herd, as early as in the acute phase of mastitis caused by mycoplasma.     

Comparative Study of Agar Mediums for Propagation of Ruminant Mycoplasma

A brain heart infusion agar supplemented with 16.7% rabbit serum (BHIR) was found the most suitable for the culturing of ruminant mycoplasma. Gourlay medium and Perreau medium (4, 5) were not suitable for growth of Mycoplasma mycoides var. mycoides or M. agalactiae, but were satisfactory for M. mycoides var. capri.

Four strains of M. mycoides var. mycoides, three strains of M. agalactiae and three strains of M. mycoides var. capri were grown in our laboratory.

     

Studies of bovine Mycoplasma mastitis. 2. Testing of various culture media and culture methods for the isolation of Mycoplasma from milk samples]

The recipes Medium-I broth, Medium-B broth, and Weissenseemycoplasma broth as well as Medium-I agar, Medium-B agar, and MRL agar were tested for their applicability to culturing mycoplasma from milk samples, using the direct and indirect techniques. No dependable information on the occurrence of mycoplasma was obtainable from tested material unless several nutritive media and techniques were combined. Medium I, for which almost no imported substances were needed, was in no way inferior to common international nutritive substrates. Its use in conjunction with the indirect culturing technique is recommended for routine diagnosis.     

Mycoplasms of the swine--A review]

The mycoplasmas constitute a group of microorganisms placed between bacteria and virus. The name, Mycoplasma, is derived from the mycelial morphology of the organisms. The minimal reproductive unit, the elementary body, measures 0.2-0.5 mum. Unlike bacteria, mycoplasmas are not confined by a rigid cell wall, but just by a thin membrane. For their cultivation, though common bacteriological technique is adequate, especially enriched media are required. Antibiotics, as a rule penicillins, are added to the medium for inhibition of bacteria. Up to the present, 5 porcine species of mycoplasma are known: Mycoplasma suipneumoniae, Mycoplasma hyorhinis, Mycoplasma hyosynoviae, Mycoplasma flocculare, and Acholeplasma granularum. The 4 species first mentioned are very common among swine in Denmark. A. granularum has not been demonstrated so far. Occasionally, other species of mycoplasma are found in swine. M. suipneumoniae is by far the most important porcine mycoplasma, being to-day regarded as the primary etiologic agent in porcine enzootic pneumonia. A pure mycoplasma infection usually results in only weak clinical signs of pneumonia, but the disease may be aggravated by secondary factors as bacteria, parasites, and bad housing conditions. Enzootic pneumonia is usually prevalent only in fattening units, where it tends to persist indefinitely. The mycoplasma infection is practically incurable. Control of the disease is attempted by the SPF-program launched by the Danish Meat Research Institute, Roskilde. In this connexion the high sensitivity of mycoplasmas to physico-chemical influence is of advantage, because it results in a low rate of survival of the organisms outside the host. A further advantage is afforded by the fast that M. suipneumoniae is a definitely swine-specific organism. The rest of the porcine mycoplasmas are of far lesser importance. Yet, M. hyorhinis may produce a sero-fibrinous inflammation of serous cavities and joints in pigs less than 10 weeks old, and M. hyosynoviae may produce arthritis in fattening pigs.     

Culturing anomalies associated with Mycoplasma recovered from the tissues of chicks and turkey poults experimentally infected with Mycoplasma gallisepticum or Mycoplasma gallinarum.

Tissues of mycoplasma infected chicks and turkey poults were cultured and subcultured on mycoplasma agar. Usually, colonies which grew on the agar initially inoculated could be subcultured, but sometimes they could not. At other times, colonies were not seen on the agar initially inoculated but appeared on the subcultured plate.     

Mycoplasma canis and urogenital disease in dogs in Norway

Mycoplasmas identified as Mycoplasma canis were isolated from nine dogs with clinical signs of urogenital disease in Norway over a period of 20 months. Some of the dogs had been treated unsuccessfully with antibiotics, and three were euthanased as a result of severe persistent disease. Seven of the dogs had a urinary tract infection, one had chronic purulent epididymitis and one had chronic prostatitis. Overt haematuria was frequently observed among the dogs with cystitis. M canis was isolated in pure culture from seven of the dogs and in mixed culture from the other two. In three cases the mycoplasma was cultivated only from urinary sediment, and it was typically obtained in smaller numbers than would be considered indicative of a urinary tract infection. In contrast with most mycoplasmas, the M canis isolated from all the dogs grew on ordinary blood agar plates used for routine bacteriological cultivation. Specific mycoplasma media were not used and the presence of other Mycoplasma or Ureaplasma species cannot be excluded.     

3种支原体培养基对猪肺炎支原体CJ株培养效果的比较分析

通过猪肺炎支原体CJ株接种3种猪肺炎支原体培养基后的生长活性研究发现,与其他2种培养基相比,接种改良Friis培养基培养2~3 d含菌量可达到1.0×109~10CCU/m L,具有生长迅速、含菌量高的特点,可用于猪肺炎支原体CJ株的培养。     

Efficacy trials in turkey poults with tylosin tartrate against Mycoplasma gallisepticum and Mycoplasma meleagridis.

Tylosin tartrate, administered in the drinking water at a concentration of 0.55 g/litre for the first three days after hatching, was highly effective in controlling the adverse consequences of a Mycoplasma gallisepticum infection, established by air sac injection at one day of age, in turkey poults. Tylosin was ineffective in controlling M meleagridis infections established in embryo or at one day of age when administered in the drinking water of poults. Both mycoplasma isolates used were inhibited in vitro by a tylosin concentration of 0.1 mug/ml.     

Detection of mycoplasmas in cell cultures, using L6M, containing 6-methylpurine deoxyriboside

Methods for the detection of mycoplasmas in cell cultures with 6MPDR are rapid and inexpensive and do not require special skill in the mycoplasma culturing. Properties of L6M, a dry, lyophilised, sterilised, and thermostabile reagent containing 6MPDR, are described. Its diagnostic value is comparable to that of the liquid, 6MPDR containing reagent MycoTect requiring storage at -70 degrees C.     

Mycoplasma hyorhinis in Taiwan: diagnosis and isolation of swine pneumonia pathogen

This study attempted to determine whether one multiplex polymerase chain reaction (PCR) is an effective adjunct method for diagnosing Mycoplasma hyopneumoniae and Mycoplasma hyorhinis infection, and whether M. hyorhinis should be considered as an enzootic pneumonia or porcine respiratory disease complex pathogen in Taiwan. To our knowledge, this study is the first to isolate and identify M. hyorhinis as a porcine pathogen in Taiwan. A novel isolation method and a multiplex PCR test were applied to detect and isolate M. hyorhinis. The correlation of M. hyorhinis with swine pneumonia was also examined using a challenge test. Based on weight, 18 pigs were assigned to three groups and housed throughout the study in a specific-pathogen-free (SPF) facility and provided with aseptic feed and water. Groups 1 (n=6) and 2 (n=6) were challenged with 5mL M. hyorhinis culture via tracheal intubation on day 1. The M. hyorhinis strains ATIT-1, -3, and-7 were used to infect group 1 and the strain ATCC 27717 was used for group 2. Culture medium was replaced by phosphate-buffered saline in group 3 (n=6). All pigs were slaughtered on day 28, and their lungs were removed for examination of lesions. Of the six pigs in group 1 challenged with wild-type strains, two had typical mycoplasma pneumonia lesions. No gross lung lesions were observed in groups 2 and 3. Although further examination is necessary to confirm that wild-type strains can cause pneumonia, it appears that M. hyopneumoniae is no longer the only mycoplasma pathogen implicated in the diagnosis of swine enzootic pneumonia (SEP).     

Comparison of media used for the primary isolation of Mycobacteriurm bovis by veterinary and medical diagnostic laboratories

Veterinary and medical laboratories engaged in the cultural diagnosis of bovine or human tuberculosis were requested to supply samples of the media that they routinely use for the primary isolation of M. bovis. Fourteen laboratories supplied 7 basic media types; these were Lowenstein-Jensen, Stonebrink's, modified Middlebrook 7H11 agar, tuberculosis bovine blood agar, egg yolk agar, Gerloff's egg and Herrold's egg yolk. Two strains of M. bovis were used to test the media, strain AN5, a glycerol-tolerant laboratory strain and M86/90 a glycerol-sensitive wildtype strain. AN5 grew well on all media with the exception of Herrold's and strain M86/90 did not grow on media containing glycerol and grew poorly on Herrold's medium. It is recommended that Lowenstein-Jensen with pyruvate (but without glycerol), Stonebrink's, modified Middlebrook 7H11 and tuberculosis bovine blood agar should be considered the media of choice for the primary isolation of M. bovis. Egg yolk agar also proved adequate for this purpose in the trial. This medium may be suitable for routine use but to date experience with its use is limited.     

Separating Mycoplasma gallisepticum field strains from nonpathogenic avian mycoplasmas

Mycoplasma gallisepticum (MG) has repeatedly emerged as a serious problem in U.S. broiler, layer, and turkey industries. Tracing the source of an outbreak is essential if MG control is to be accomplished. Amplified fragment length polymorphism (AFLP), random amplification of polymorphic DNA (RAPD), and restriction fragment length polymorphism (RFLP) are valuable tools used to study MG epidemiology, allowing diagnosticians to determine the source of MG infections. In some past outbreaks, AFLP, RAPD, and RFLP fingerprinting, which require pure MG cultures, were not successful because of contaminating nonpathogenic mycoplasmas from field samples. The objective of this research was to develop a method to separate rapidly growing nonpathogenic avian mycoplasma species from slower-growing MG field strains. Mixtures of MG and three separate nonpathogenic avian mycoplasmas were inoculated onto chick embryo fibroblasts cells (CEF) allowing MG to penetrate the CEF cells. Later, gentamicin sulphate was added to the culture, eliminating the nonpathogenic mycoplasmas and allowing MG to be isolated in pure culture. Mixtures of Mycoplasma synoviae (MS) and MG could not be separated in this assay. However, removal of nicotinamide adenine dinucleotide and cysteine hydrochloride during serial passage in Frey broth medium successfully eliminated growth of MS.     

A synthetic, selective culture medium for Pseudomonas aeruginosa

Selective and differentiating media consisting of simple chemical components have been developed, both in solid and in liquid form, for culturing Pseudomonas aeruginosa from foodstuffs. These media work on the basic principle that Gram-negative bacteria can utilize ammonia as inorganic nitrogen source. This principle has already been utilized for developing a culture medium for coliform bacteria (Szita and Biró, 1986; Szita et al., 1988). P. aeruginosa can produce the ammonia needed for its growth by decomposing acetamide. The liquid synthetic medium was compared with the nitrofurantoin broth and the solid one with cetrimide agar by parallel inoculation of 60 raw milk samples and 20 P. aeruginosa pure cultures. The main advantages of the synthetic media are their high selectivity, high sensitivity and rapidity. Owing to their advantageous properties, the new media can be recommended for replacing cetrimide agar and nitrofurantoin broth.     

Sensitivity of mycoplasmas of the respiratory tract of pigs and horses to erythromycin and its use in selective media

The ability of erythromycin in liquid medium to suppress the growth of eight species of acholeplasma and of 13 species of mycoplasma was tested. The Acholeplasma spp and two glycolytic Mycoplasma spp from horses--a slow glucose-metabolising (SGM) mycoplasma and a strain N3, related to M mycoides--were sensitive to erythromycin. Thus the growth of acholeplasmas can be suppressed when attempts are made to isolate pathogens from the porcine respiratory tract, but, in the case of horses, erythromycin would suppress not only Acholeplasma spp but also two Mycoplasma spp of unknown pathogenicity in the equine respiratory tract.     

Characterization and identification of caprine, genital mycoplasmas

A total of 55 mycoplasma strains, isolated from the vagina of goats, were examined. Three strains, being arginine positive and glucose negative, could not be finally classified. Five isolates were identified as Acholeplasma laidlawii. Fourty-seven strains were phosphatase positive, glucose and arginine negative. Nine of these formed “film and spots” on standard growth medium, and reduced tetrazolium aerobically. Serological examination identified 7 as M. agalactiae, while 2 were M. bovis. The remaining 38 isolates did not reduce tetrazolium aerobically, and did not produce “film and spots.” on standard growth medium. All these except one were identified as M. bovigenitalium by immunofluorescence. Their relationship to group 11 otf Al-Aubaidi is discussed.