Optimization of an antibiotic sensitivity assay for Mycoplasma hyosynoviae and susceptibility profiles of field isolates from 1997 to 2011.

Mycoplasma hyosynoviae is a common agent responsible for polyarthritis leading to decreased production in swine herds worldwide. Antimicrobial agents are used to combat infections; however breakpoints for M. hyosynoviae have not yet been established. A number of methods have previously been utilized to analyze minimum inhibitory concentrations (MICs) for antibiotics against M. hyosynoviae; however these techniques as currently described are not easily standardized between laboratories. A dry microbroth dilution method was conducted to compare the minimum inhibitory concentrations (MICs) for 18 antibiotics, representative of different classes, against 24 recent isolates (23 field isolates and the type strain) of M. hyosynoviae. The MICs were determined using standard, commercially available 96-well Sensititre(®) plates containing various freeze-dried antibiotics at a range of concentrations appropriate to their potency. Clindamycin (CLI), a lincosamide antibiotic, showed the highest activity and most consistent inhibition for all isolates with an MIC(50) of ≤ 0.12 µg/ml. Tiamulin (TIA), a pleuromutilin derivative, exhibited an MIC(50) of ≤ 0.25 µg/ml. The isolates had similar levels of susceptibility to the quinolones, enrofloxacin (ENRO) and danofloxacin (DANO), exhibiting an MIC(50) of 0.25 µg/ml and 0.5 µg/ml, respectively. For the macrolides, the MIC(50) for tylosin (TYLT) and tilmicosin (TIL) was ≤ 0.25 µg/ml and ≤ 2 µg/ml respectively, but was ≤ 16 µg/ml for tulathromycin (TUL). For the aminoglycosides, the MIC(50) for gentamicin (GEN) was ≤ 0.5 µg/ml, while spectinomycin (SPE) and neomycin (NEO) had an MIC(50) of ≤ 4 µg/ml. The tetracyclines, oxytetracycline (OXY) and chlortetracycline (CTET) both had an MIC(50) of ≤ 2 µg/ml. Florfenicol (FFN) exhibited a MIC(50) of ≤ 1 µg/ml. All isolates were resistant to penicillin (PEN), ampicillin (AMP), ceftiofur (TIO), trimethoprim/sulfamethoxazole (SXT), and sulphadimethoxine (SDM) at all concentrations. Within the isolates tested, there was a range of sensitivity detected, with some isolates being overall more resistant while others appeared more susceptible. Further research is required to demonstrate how this MIC data correlates to clinical efficacy of the various antibiotics in the field.

Evaluation of the ability of tilmicosin to prevent adherence of Mycoplasma hyopneumoniae to cilia using a differentiated swine respiratory epithelial culture system.

Mycoplasmal pneumonia caused by Mycoplasma hyopneumoniae is a serious economic problem for swine producers in the United States. Mycoplasma hyopneumoniae colonizes ciliated respiratory epithelial cells. The organism has been shown to be sensitive to tilmicosin, a synthetic macrolide, in antibiotic sensitivity assays. The efficacy of tilmicosin to inhibit adherence of M. hyopneumoniae to ciliated epithelial cells without direct contact between the antibiotic and the organism was evaluated using in vitro methods. The study demonstrated that tilmicosin inhibited the adherence of M. hyopneumoniae at the highest level tested in the system (2 microg/ml) suggesting that tilmicosin may be efficacious against mycoplasmal pneumonia.

A tissue culture system to study respiratory ciliary epithelial adherence of selected swine mycoplasmas.

An in vitro culture system for swine tracheal epithelial cells was developed to study the adherence of swine mycoplasmas. Swine tracheal epithelial cells were isolated by enzymatic digestion and cultured on microporous membranes. Growth medium was placed under the membrane support to create air-liquid interface feeding resulting in the cells growing cilia and microvilli on the apical surface. Two strains of Mycoplasma hyopneumoniae (pathogenic strain 91-3 and non-pathogenic type strain J) and two strains of Mycoplasma flocculare (type strain Ms42 and field isolate 7160T) were used in this study. The morphology of the cultured tracheal cells was evaluated by transmission electron microscopy. Adherence of M. hyopneumoniae and M. flocculare and damage to the cilia were demonstrated using scanning electron microscopy. The pathogenic M. hyopneumoniae strain 91-3 adhered to cilia inducing obvious damage. The non-pathogenic M. hyopneumoniae strain J did not adhere to mature cilia. Both M. flocculare strains Ms42 and 7160T adhered to mature and budding cilia. No obvious ciliary damage was observed with strain Ms42. Minimal damage consisting of a slight tangling of the cilia occurred after adherence by strain 7160T. This model will enable us to further study the role of adherence of mycoplasmas on the pathogenesis of swine pneumonia.

Phylogenetic relationships of the porcine mycoplasmas Mycoplasma hyosynoviae and Mycoplasma hyopharyngis.

The phylogenetic positions of the porcine mycoplasmas Mycoplasma hyosynoviae and Mycoplasma hyopharyngis were determined by using PCR-amplified 16S rRNA gene sequences. M. hyosynoviae is a member of the Mycoplasma hominis group, while M. hyopharyngis belongs to the Mycoplasma fermentans group of mollicutes. Neither species is closely related to previously characterized porcine mycoplasmas belonging to the Mycoplasma hyorhinis group.

Adaptation of the Sensititre broth microdilution technique to antimicrobial susceptibility testing of Mycoplasma hyopneumoniae.

A broth microdilution technique is described for determining the antimicrobial susceptibility of Mycoplasma hyopneumoniae, using commercially prepared Sensititre plates. Twenty-five field isolates and two reference strains (J & 232), were tested against seven antimicrobials. Field isolates were tested in duplicate and reference strains, four times to estimate reproducibility. Ninety-seven percent of the duplicate MIC results for the field isolates were in agreement, or within one log2 dilution. Similar results were obtained with the reference strains. The isolates were susceptible to lincomycin-spectinomycin, tylosin and oxytetracycline or resistant to amoxycillin, apramycin and erythromycin. Susceptibility to furaltadone varied. This method retains the accuracy and reproducibility of broth MIC determinations, while avoiding the lengthy preparation of antimicrobial dilutions normally associated with more traditional methods.

Hemagglutination and hemagglutination inhibition of turkey red blood cells with Mycoplasma hyopneumoniae.

The ability of Mycoplasma hyopneumoniae to agglutinate RBC was evaluated to develop an in vitro cytadsorption assay. Using swine RBC in a microtitration hemagglutination test, no agglutination or partial agglutination was detected. Comparison of RBC from various other species indicated that improved hemagglutination was obtained with RBC from turkeys. This hemagglutination was detected only when mycoplasma cells used in the assay had been frozen and thawed, heated at 50 C for 30 minutes, or treated with trypsin. Treatment of RBC with trypsin or neuraminidase enhanced hemagglutination. Possible surface lectin activity in M hyopneumoniae was evaluated by use of carbohydrates in a blocking assay; hemagglutination was not inhibited by any of 13 carbohydrates evaluated. Mycoplasma hyopneumoniae convalescent porcine serum and monoclonal antibodies against 2 M hyopneumoniae immunogens of molecular weights of 64,000 and 41,000 inhibited hemagglutination.

Isolation of Mycoplasma salivarium from swine.

Mycoplasma salivarium, a common human oropharyngeal mycoplasma, was isolated from nasal and pharyngeal secretions of 14 of 284 swine in a barrier-maintained, disease-free herd. M. salivarium was recovered from one boar 6 times over a 26-month period and one time only from 13 other swine. Human isolates of M. salivarium were compared with the swine isolates by DNA-DNA hybridization and SDS-PAGE of the cell proteins and the strains were shown to be closely related. One of eight of the swine from which M. salivarium was isolated had complement-fixing antibodies and another culture-positive animal had metabolic-inhibiting antibodies to M. salivarium. Overt disease was not associated with the organism. These results support previous findings that mycoplasmas closely related to M. salivarium may be isolated from the nasopharynges of swine and they further indicate that the organism can establish persistently in swine without evidence of overt disease.

Serotyping of Haemophilus pleuropneumoniae by rapid slide agglutination and indirect fluorescent antibody tests in swine.

One hundred and forty-one isolates of Haemophilus pleuropneumoniae from Iowa and Illinois swine were characterized morphologically and biochemically and serotyped by rapid slide agglutination (RSA) and indirect fluorescent antibody (IFA) tests. Hyperimmune antisera were produced in rabbits using inactivated whole-cell suspensions of the reference strains for H pleuropneumoniae serotypes 1 to 7 and strain 202, representing the taxon "minor group." Cross testing of the reference strains and reference antisera indicated the antisera to be essentially serotype-specific, although reactivity of some antisera with heterologous strains was observed. Cultures of the 141 isolates formed adherent or smooth colonies or mixtures of these colony forms. Adherent and smooth colony types were found in all serotypes identified. Microscopic and biochemical characteristics of all isolates were typical of those previously described for H pleuropneumoniae. The overall incidence of H pleuropneumoniae serotypes was serotype 5, 55.3%; serotype 1, 34.0%; serotype 7, 7.8%; and nontypeable, 2.8%. Comparing the 2 test procedures, 87.2% of the isolates could be typed by RSA, and 66.0% could be typed by IFA. Cross-reactions between serotype 4 antisera and serotype 5 and 7 isolates were common with the IFA test. The reactions with serotype 7, but not serotype 5, were eliminated by cross adsorption of serotype 4 antisera. There was good correlation between the 2 test procedures, but RSA was judged to be more specific and sensitive than IFA.