Rapid isolation of Brachyspira hyodysenteriae and Brachyspira pilosicoli from pigs

The aim of this study was to compare and evaluate the time required to isolate Brachyspira hyodysenteriae and Brachyspira pilosicoli from porcine faeces. This was done using previously described selective media (spectinomycin) S400, (colistin, vancomycin and spectinomycin) CVS and (spectinomycin, vancomycin, colistin, spiramycin and rifampin with swine faecal extract) BJ, compared with the method based on blood agar modified medium, with spectinomycin and rifampin (BAM-SR), including a pre-treatment step. Fourteen spirochaetal strains were obtained in pure cultures after 5 days (48 h in BAM-SR primary plate and three passages every 24 h in brain heart infusion (BHI) without antibiotics) pre-treating simulated samples in brain heart infusion broth with spectinomycin (400 microg/ml) and rifampin (15 microg/ml), before streaking on the selective BAM-SR medium. Spirochaetes from samples in S400, CVS and BJ, with and without pre-treatment, were obtained in pure cultures only after repeatedly transferring on plates of the same selective medium requiring 15-18 days according to the strain. BAM-SR used after the pre-treatment step showed a detection limit ranging from 3.5 x 10(2) to 6.7 x 10(7) cells/g faeces and was the only method able to support the growth of spirochaetes after 48 h.     

Evaluation of a selective medium for isolation of Haemophilus pleuropneumoniae.

Crystal violet, lincomycin, spectinomycin and bacitracin were evaluated as selective agents in media for isolation of Haemophilus pleuropneumoniae. No single antimicrobial agent or combination of two or more inhibited all non-Haemophilus strains (Escherichia coli, Pasteurella haemolytica, Pasteurella multocida, Streptococcus faecalis, Streptococcus equisimilis and Staphylococcus aureus) without marked suppression of 16 H. pleuropneumoniae strains. A medium containing 1 micrograms/mL of crystal violet, 1 microgram/mL of lincomycin, 8 micrograms/mL of spectinomycin and 128 micrograms/mL of bacitracin inhibited one E. coli strain and the Gram-positive strains while H. pleuropneumoniae strains were suppressed to a minor degree only. Haemophilus pleuropneumoniae was isolated on the selective medium on three occasions from the nose or pharynx of two out of eight experimentally inoculated pigs. Haemophilus pleuropneumoniae was recovered from the nose of only two pigs at necropsy and from tonsil of one, whereas the lower airways in most pigs and the lung lesions in all pigs were positive. There was no advantage to using the selective medium for the recovery of H. pleuropneumoniae at necropsy from these eight experimentally infected pigs, probably because other bacteria were absent or present in very low numbers in the tissues with H. pleuropneumoniae. The isolation rate on selective medium was higher than the rate on non-selective medium (p less than or equal to 0.1; chi 2 test) when the airways of slaughtered pigs were cultured. This was likely due to a high degree of contamination. Dry swabs placed in tryptone yeast extract with nicotinamide-adenine-dinucleotide gave a significantly higher recovery rate than commercial Culturette swabs in modified Stuart's transport medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective medium for isolation of Haemophilus somnus from cattle and sheep

Incorporation of vancomycin (5 micrograms/ml), neomycin (5 micrograms/ml), sodium azide (50 micrograms/ml), nystatin (100 iu/ml) and cyclohexamide (100 micrograms/ml) into 5 per cent horse blood agar results in a selective medium for the primary isolation of Haemophilus somnus from cattle and sheep. Addition of thiamine monophosphate (1 microgram/ml) to the medium enhanced growth of this bacterium. Gram-positive bacteria did not grow on the medium and colonies of many Gram-negative bacteria were eliminated or reduced in numbers and size. Colonies of H somnus were larger on the selective medium than on sheep blood agar but retained typical morphology. Recovery of 18 laboratory strains was 73 to 166 per cent (mean 112) on selective medium compared to sheep blood agar. H somnus was isolated from the vagina of a total of 136 (28.6 per cent) of 476 cows surveyed, 79 (16.6 per cent) on sheep blood agar and 129 (27.1 per cent) on selective medium. The selective agents and thiamine were stable indefinitely as a freeze dried mixture while prepared plates were stable for two weeks.     

Development of improved methods for the transport and isolation of Haemophilus somnus

The survival of several strains of Haemophilus somnus under various simulated transport conditions was investigated. Recovery of H somnus from alginate swabs kept at room temperature was possible for up to 27 hours after sampling, but this period could be extended to 72 hours if the swabs were refrigerated. Storage of swabs in transport media did not prolong survival time significantly but did increase the number of bacterial contaminants, thus making recovery of H somnus less likely. The sensitivity of several strains of H somnus to a number of dyes, antibiotics and other antimicrobial agents was determined and a selective isolation medium then formulated. This medium which consisted of brain heart infusion agar supplemented with 5 per cent ovine blood, 5 per cent equine serum, 0.5 per cent yeast extract, cycloheximide (100 micrograms ml-1) and lincomycin (3 micrograms ml-1), facilitated the isolation of H somnus from contaminated material. However, while this medium was effective against many contaminants, it did not prevent swarming by Proteus species.     

Detection of Streptococcus suis type 2 in tonsils of slaughtered pigs using improved selective and differential media

New selective and differential media were devised for the isolation and identification of Streptococcus suis type 2. The selective medium (NNCC) agar) was Todd-Hewitt broth containing 1.5% Bactoagar and 5% defibrinated sheep blood with addition of sodium azide (50 micrograms/ml), nalidixic acid (25 micrograms/ml), colistin (12.5 micrograms/ml) and crystal violet (2 micrograms/ml). The differential medium consisted of heart infusion agar and antiserum specific only for S. suis type 2. In a total of 291 pigs tested by a combination of these media, S. suis type 2 was isolated and confirmed from 40 of them (13.7%).     

In vitro comparison of the activity of various antibiotics and drugs against new Taiwan isolates and standard strains of avian mycoplasma

Twenty-nine antibiotics or drugs were incorporated individually into mycoplasma agar to evaluate their inhibitory activity against avian mycoplasmas: 100 recent Taiwan isolates of 7 serotypes and 10 standard strains of 7 serotypes were tested. All of the standard strains were very sensitive to erythromycin, chlorotetracycline, doxycycline, minocycline, and tetracycline, but the local isolates were highly resistant to these antibiotics. The drugs or antibiotics that possessed an MIC90 of 50 micrograms/ml or less against the local isolates were tiamulin (less than 0.4 micrograms/ml), lincospectin (2.7), josamycin (2.7), lincomycin (3.0), spectinomycin (4.8), tylosin (6.0), kanamycin (6.0), chloramphenicol (6.0), gentamicin (7.5), apramycin (24.5), doxycycline (27.4), minocycline (29.0), spiramycin (30.0), colistin (44.3), leucomycin (45.0), and streptomycin (50.0). The MIC90 of the other antibiotics or drugs was greater than 50 micrograms/ml. None of the isolates or strains were sensitive to nalidixic acid, ronidazole, penicillin, ampicillin, cephalexin, carbadox, or four sulfa drugs at a concentration about 5 times the therapeutic level.     

Characterisation of some Ornithobacterium rhinotracheale strains and examination of their transmission via eggs

The biochemical characteristics and antibiotic susceptibility of 12 Ornithobacterium rhinotracheale strains isolated from chickens and turkeys suffering from respiratory clinical signs and the survival of some isolates on egg-shell and within chicken eggs during hatching were examined. All O. rhinotracheale strains showed typical biochemical characteristics. Among the 16 drugs examined, penicillin G, ampicillin (MICs ranging from < or = 0.06 microgram/ml to 1 microgram/ml), ceftazidim (with MICs from < or = 0.06 microgram/ml to 0.12 microgram/ml), erythromycin, tylosin, tilmicosin (with some exceptions MICs ranged from < or = 0.06 microgram/ml to 1 microgram/ml) and tiamulin (MICs varied from < or = 0.06 microgram/ml to 2 micrograms/ml) were the most effective. Lincomycin, oxytetracycline and enrofloxacin also gave good inhibitions, but with most strains in a higher concentration (MICs ranged in most cases from 2 micrograms/ml to 8 micrograms/ml). The other antibiotics inhibited the growth of O. rhinotracheale only in very high concentrations (colistin) or not at all (apramycin, spectinomycin, polymyxin B). At 37 degrees C, O. rhinotracheale did not survive on egg-shell for more than 24 hours, while upon inoculation into embryonated chicken eggs it killed embryos by the ninth day, and from the 14th day post-inoculation no O. rhinotracheale could be cultured from the eggs at all. These results suggest that O. rhinotracheale is not transmitted via eggs during hatching.     

Comparison of selective culture and serologic agglutination of Treponema hyodysenteriae for diagnosis of swine dysentery

Samples of faeces and of serum were collected from pigs of various ages on 21 farms. Faecal samples were cultured on trypticase soy agar containing 5 per cent citrated bovine blood and 400 micrograms per ml spectinomycin, incubated at 42 degrees C in Gaspak jars under an atmosphere of 80 per cent hydrogen: 20 per cent carbon dioxide. Antibody titres to Treponema hyodysenteriae were determined by a microtitration agglutination method using merthiolate-inactivated whole cell antigen prepared from a beta-haemolytic isolate. Results indicated that mean titres in pigs from which beta-haemolytic T hyodysenteriae was isolated were significantly higher than in pigs which yielded isolates of weak beta-haemolytic T innocens or in culturally negative pigs (P less than 0.0225). Mean titres of herds where beta-haemolytic T hyodysenteriae was isolated were significantly higher (P less than 0.005) than the mean titres of either of the other two groups. However, mean titres of herds where no isolates were obtained were not significantly different from mean titres of herds where weak beta-haemolytic T innocens was isolated.     

A novel method for isolation of Brachyspira (Serpulina) hyodysenteriae from pigs with swine dysentery in Italy

Brachyspira (Serpulina) hyodysenteriae was isolated from 10 of 11 pigs with clinically suspected swine dysentery in six herds in northern Italy. All strains were successfully isolated in the selective blood agar modified medium with spectinomycin and rifampin (BAM-SR) currently used in our laboratory to isolate B. (S.) pilosicoli of human origin, after pre-treatment of intestinal material with spectinomycin and rifampin in foetal calf serum. Isolates had phenotypic characteristics typical of B. (S.) hyodysenteriae.     

A selective procedure for the field isolation of pathogenic Streptococcus spp. of rainbow trout (Salmo gairdneri)

A procedure established for the selective isolation of the species of Streptococcus responsible for rainbow trout streptococcosis in South Africa, consisted of the inoculation of samples into nutrient broth which had been supplemented with 100 micrograms/ml of nalidixic acid, 160 micrograms/ml of oxolinic acid or 200 micrograms/ml of sodium azide. After incubation, the sample was plated onto tetrazolium agar on which the rainbow trout pathogenic Streptococcus species grew as a red colony. The colonies were isolated from the tetrazolium agar and identified as rainbow trout pathogenic isolates by biochemical and serological tests. In the laboratory the selective procedure is capable of detecting about 2 bacteria per ml. This procedure was used in the field and biochemically identical Streptococcus species were found in the mud and a freshwater crab from the water source of a site with a history of streptococcosis.     

In vitro sensitivity of Hungarian Actinobaculum suis strains to selected antimicrobials

In vitro antimicrobial sensitivity of 12 Hungarian isolates and the type strain ATCC 33144 of Actinobaculum suis to different antimicrobial compounds was determined both by the agar dilution and by the disc diffusion method. By agar dilution, MIC50 values in the range of 0.05-3.125 micrograms/ml were determined for penicillin, ampicillin, ceftiofur, doxycycline, tylosin, pleuromutilins, chloramphenicol, florfenicol, enrofloxacin and lincomycin. The MIC50 value of oxytetracycline and spectinomycin was 6.25 and 12.5 micrograms/ml, respectively. For ofloxacin, flumequine, neomycin, streptomycin, gentamicin, nalidixic acid, nitrofurantoin and sulphamethoxazole + trimethoprim MIC50 values were in the range of 25-100 micrograms/ml. With the disc diffusion method, all strains were sensitive to penicillin, cephalosporins examined, chloramphenicol and florfenicol, tetracyclines examined, pleuromutilins, lincomycin and tylosin. Variable sensitivity was observed for fluoroquinolones (flumequine, enrofloxacin, ofloxacin), most of the strains were susceptible to marbofloxacin. Almost all strains were resistant to aminoglycosides but most of them were sensitive to spectinomycin. A strong correlation was determined for disc diffusion and MIC results (Spearman's rho 0.789, p < 0001). MIC values of the type strain and MIC50 values of other tested strains did not differ significantly. Few strains showed a partially distinct resistance pattern for erythromycin, lincomycin and ampicillin in both methods.     

The growth of Treponema hyodysenteriae and other porcine intestinal spirochaetes in a liquid medium.

A new simple method for the preparation of a liquid medium containing rabbit serum for the propagation of Treponema hyodysenteriae and other porcine intestinal spirochaetes is described. The medium, when dispensed in shallow layers and sealed under 10 per cent CO2 in nitrogen, had a redox potential not greater than -125mV and an initial pH of about 6.9 when buffered with bicarbonate. Growth of T hyodysenteriae developed more rapidly and viable counts reached higher levels at 42 degrees C than at 37 degrees C. Viable counts increased at least 10,000-fold after two to five days' incubation, depending on the temperature. Growth could be initiated from small inocula that failed to produce colonies on blood agar. Using a 1 per cent inoculum, the medium supported the growth of two strains of T hyodysenteriae through 10 serial passages.     

Selective media for isolation of Brucella abortus strain RB51

Brucella abortus strain RB51 (SRB51) is the standard vaccine used to protect cattle against brucellosis and is currently being used to vaccinate bison in the United States (US). Currently available media for culture of Brucella have not been evaluated for their ability to support growth of SRB51. In this study, five selective media for isolating brucellae, four commercially available media for gram-negative bacteria, and tryptose agar with 5% bovine serum (TSA) were compared to two SRB51 selective media developed in this study (rifampin brucellae medium (RBM), and malachite green brucellae medium (MGB)), for their ability to support growth and enhance recovery of SRB51. Four of the five media currently used for isolation of brucellae and two of the four media used for other Gram-negative bacteria did not support growth of SRB51. Modified Kuzdas and Morse (MKM), Brilliant Green, Skirrow's, RBM, and MGB supported growth of SRB51 in a manner similar to TSA. Recovery of SRB51 from tissues of SRB51-vaccinated bison was attempted on TSA, MKM, RBM, and MGB. From a total of 436 samples, SRB51 was isolated from 9.6, 4.3, 5.5, and 9.0% on TSA, MKM, RBM, or MGB media, respectively. Strain RB51 was recovered on only one medium (nine on TSA; three on RBM; and 9 on MGB) from 21 samples. Overgrowth of contaminating bacteria prevented potential detection of SRB51 from 9. 4, 5.5, 0.07, and 5.9% of samples on TSA, MKM, RBM, or MGB, respectively. These data suggest that the use of RBM and MGB, in combination with TSA, enhances the ability to recover SRB51 from tissue samples.     

Diversity in nicotinamide-adenine dinucleotide requirement for the growth of different strains of Mycoplasma synoviae

The type strain WVU 1853 and field strains SG, N26 and A642 of Mycoplasma synoviae were examined for their requirement for nicotinamide-adenine dinucleotide (NAD) for in vitro growth. All the strains grew and could be repeatedly passaged in Frey broth medium supplemented with filter-sterilised NAD. In modified Frey broth medium from which NAD was omitted and broth medium for which the supplements including yeast extract and NAD were autoclaved, only strains N26 and A642 could be grown and passaged. The growth curves of strain N26 determined in broth media with and without NAD were similar. These results indicated that there are differences in NAD-requirement for in vitro growth among strains of M synoviae.     

Treponema hyodysenteriae growth under various culture conditions

The influence of various culture conditions on the growth of Treponema hyodysenteriae was determined. Six different anaerobically prepared culture broths were tested for the ability to support growth of strains B78, B204 and B169. Each medium contained glucose (0.2%) and 10% (v/v, final concn.) heat-inactivated fetal calf serum. Brain-heart infusion (BHIS), heart infusion (HS) and veal infusion (VS) broths gave the highest cell yields of the spirochete with the shortest incubation times. Vigorous mixing of the cultures and the introduction of O2 (1%, final concn.) into the culture atmosphere were necessary for optimum growth. Although BHIS broth was found to be the best for routine cultivation of the 3 strains, HS broth was more suitable for investigating the physiology of growing cells, inasmuch as cell growth in this medium was limited unless a growth substrate was added. Glucose, fructose, sucrose, galactose, trehalose, N-acetyl-glucosamine, glucosamine, mannose, maltose and pyruvate were growth substrates for all 3 strains. During the growth of B204 cells in HS broth under N2:O2 (99:1), glucose and O2 were consumed and CO2, H2, acetate and butyrate were produced. In HS agar-containing medium, cells of strains B78 and B204 formed spreading colonies typical in appearance to those of other spirochetes.     

Comparative studies on enrichment and selective media for isolation of Salmonellae from faecal specimens.

Enrichment media (tetrathionate, selenite and Rapp ap ort broths) and selective media (desoxycholate citrate agar and brilliant green agar) were tested in different combinations to ascertain their capacity for isolation of salmonella bacteria. The material consisted of 299 samples of cattle faeces from two herds infected with salmonella (Table 1), and of 111 artificially contaminated samples of pig faeces (Table 3). The tetrathionate and selenite broths were equally useful for the material as a whole, whereas the results varied between different species of salmonella which is of great practical interest. The number of salmonella isolations was much lower when enrichment with Rappaport broth was used. The rate of salmonella isolations can often be increased by parallel enrichments with two different media. Of the selective agar media tested, brilliant green agar was superior to desoxycholate citrate agar.     

The occurrence of Treponema in fecal samples from dogs and cats with and without intestinal diseases

6 (6.9%) of 87 examined dogs without diarrhoea proved to be carriers of Treponema (1x T. hyodysenteriae, 5x T. innocens), whereas in fecal samples from 62 dogs with enteric symptoms no isolation of Treponema succeeded. 5 fecal samples (3.7%) of cats without signs of diarrhoea were found to contain Treponema (1x T. hyodysenteriae, 4x T. innocens), whereas the fecal samples of 31 cats with diarrhoea didn't show any growth of Treponema by cultural investigations. Due to the results of these investigations the conclusion can be drawn that Treponema belong to the usual bacteria of dogs' and cats' intestines and cannot be suspected to cause diarrhoea in these animals primarily.     

A selective medium for the rapid isolation of pseudomonads associated with poultry meat spoilage.

1. A new selective medium (CFC) has been developed for the rapid isolation of pigmented and non-pigmented pseudomonads associated with the spoilage of poultry meat held under chill conditions. It comprises Difco Heart Infusion Agar supplemented with 50 microgram cephaloridine, 10 microgram fucidin and 10 microgram cetrimide/ml. 2. CFC medium was found to be more selective than three other media which have been used for isolating pseudomonads from foods, when tested with pure cultures of 28 reference organisms. 3. CFC supported the growth of a higher proportion of pseudomonads from freshly-eviscerated carcasses and processing equipment when the organisms were present only in low numbers relative to other genera.     

Antibiotic resistance of Escherichia coli strains isolated from chickens with colisepticaemia in Morocco

Sixty-two strains of Escherichia coli were isolated in 58 farms from broiler chickens showing respiratory signs and lesions characteristic of avian colibacillosis. Serological examination of these strains showed that the types 078, 01 and 02 (for the somatic antigen) and K1 (for the capsular antigen) were the most frequently found. Newcastle disease virus was also isolated in two cases. All the strains of E. coli isolated were sensitive to colistin, flumequine and gentamicin. A few strains were resistant to neomycin, nalidixic acid and trimethoprim. The frequency of strains resistant to nitrofurans, sulfonamides, chloramphenicol, spectinomycin and ampicillin was intermediate. Most strains were resistant to tetracycline. Multiple resistance was common.     

Isolation and characterization of avian rotaviruses

Rotaviruses were isolated from intestinal contents obtained from flocks of turkey poults and pheasant poults with diarrhea and from different age groups of chickens showing various signs of intestinal disorders. The incorporation of 5 micrograms trypsin/ml in the inoculum and medium was essential for virus isolation in chicken kidney cells. All isolates were identified as rotaviruses by fluorescent-antibody technique using a National Institutes of Health reference rotavirus antiserum against human rotavirus strain "D," Type 2. Negative-staining and transmission electron microscopy showed the presence of rotavirus particles. Furthermore, polyacrylamide gel electrophoresis pattern of viral RNA segments of our isolates confirmed that they are rotaviruses. Seven-day-old turkey poults could be infected with turkey and chicken rotavirus isolates; in contrast, chicks of the same age were refractory.