Comparison of selenite F, Muller-Kauffmann tetrathionate and Rappaport's medium for the isolation of salmonellas from sewage-polluted natural water using a pre-enrichment technique.

Three enrichment broths, selenite F, Muller-Kauffmann tetrathionate and Rappaport, were examined for their efficiency in salmonella isolation. The three media, prepared from single ingredients in the laboratory, were compared with their commercial equivalents. Laboratory-prepared media were more efficient for isolating salmonellas from sewage-polluted natural water samples. A pre-enrichment stage using buffered peptone water was employed throughout the investigation. The size of inoculum from the pre-enrichment medium was relevant to successful salmonella isolation. Inocula studied were 1 ml and one loopful (3 mm diameter loop). The smaller inoculum gave better results with Rappaport, the larger with selenite and tetrathionate. Using the optimal inocula, Rappaport was the most efficient enrichment broth of the three fluid media in this study.     

Comparison of selenite F, Muller-Kauffmann tetrathionate and Rappaport's medium for salmonella isolation from chicken giblets after pre-enrichment in buffered peptone water.

Six hundred and eighty three samples of chicken giblets were examined for salmonellas. Three hundred and forty nine of these were neck and crop specimens and 224 were combined liver and heart samples. Two hundred and ten, in all, contained salmonellas. The technique of examination included pre-enrichment in buffered peptone water at 37 degrees C for 18 h and subculture to three enrichment media: Muller-Kauffmann tetrathionate, selenite F and Rappaport''s magnesium chloride malachite green broth. Inocula from buffered peptone water to 10 ml of tetrathionate and selenite were 1 ml in each case. The inoculum from the pre-enrichment medium to 10 ml of Rappaport was 0.005 ml. Tetrathionate and selenite were incubated at 43 degrees C for 48 h. Rappaport''s medium was incubated at 37 degrees C for 48 h. Subcultures from all three enrichment broths were made at 24 h and 48 h to brilliant green MacConkey agar. Selective agars were incubated at 37 degrees C for 24 h. The most successful technique for salmonella isolation used Rappaport''s medium, which was significantly more efficient than either tetrathionate or selenite. This finding reinforces results obtained using sewage polluted natural water as test material and it is suggested that routine examination of environment samples for salmonellas could be based on Rappaport''s medium alone. If S. typhi, S. dublin or subgenus III salmonellas were likely to be present in the sample, the technique described here would require modification.     

The prevalence of Salmonella enteritidis and other Salmonella sp. among Canadian registered commercial chicken broiler flocks

A nation-wide survey was conducted to estimate the prevalence of Salmonella enteritidis and other salmonellas among Canadian commercial broiler flocks. Environmental (litter and/or water) samples from 226 of 294 (76.9%) randomly selected flocks were contaminated with salmonellas. Litter samples were more often contaminated with salmonellas than water samples (47.4 v. 12.3%). Fifty different salmonella serovars were isolated. The most prevalent serovars were S. hadar, S. infantis, and S. schwarzengrund; they were isolated from samples of 98/294 (33.3%), 26/294 (8.8%), and 21/294 (7.1%) flocks, respectively. Feed samples of 39/290 (13.4%) flocks were contaminated with salmonellas. Salmonella enteritidis was isolated from the environmental samples of 9/294 (3.1%) flocks. Salmonella enteritidis phage type (PT) 8 was isolated from seven flocks, and PT 13a from two flocks.     

The prevalence of Salmonella enteritidis and other Salmonella spp. among Canadian registered commercial layer flocks

A survey was conducted to estimate the prevalence of Salmonella enteritidis and other salmonellas among Canadian commercial egg producing flocks. Environmental (faecal and eggbelt) samples from 152 of 295 (52.9%) randomly selected flocks were contaminated with salmonellas. Thirty-five different salmonella serovars were isolated. Eggbelt samples were more often contaminated with salmonellas than faecal samples (25.7 v. 10.1%). The most prevalent serovars were S. heidelberg, S. infantis, S. hadar, and S. schwarzengrund; they were isolated from samples of 59/295 (20%), 18/295 (6.1%), 17/295 (5.8%), and 15/295 (5.1%) flocks, respectively. Feed samples of 21/295 (7.2%) flocks were contaminated with salmonellas. Salmonella enteritidis was isolated from the environmental samples of 8/295 (2.7%) flocks. Salmonella enteritidis phage type (PT) 8 was isolated from 5 flocks, PT 13a from 2 flocks, and PT 13 from 1 flock.     

Salmonella infection in chicks following the consumption of artificially contaminated feed

Poultry feed was contaminated artificially with either Salmonella kedougou or S. livingstone using a two-stage mixing process. Intestinal infection became established in a small proportion of birds when feed containing between 0.1 and 0.3 salmonellas/g was given continuously for 2 or 3 weeks from the day of their purchase as 'day-olds' while nearly all birds became infected when the feed contained 100-300 salmonellas/g. Between these limits a dose response was demonstrated in that the proportion of birds becoming colonized with salmonellas increased as the numbers of salmonellas added to feed was increased. Nalidixic acid-resistant strains of both serotypes were used to facilitate the recovery of organisms. The isolation rate was higher from dilutions of caecal contents inoculated directly onto brilliant green agar supplemented with nalidixic acid than it was from swabs of cloacal faeces, even when an enrichment technique was used. This observation confirms that the incidence of salmonella carriage in flocks will be under-estimated if only cloacal faeces are cultured. Of the two serotypes used S. kedougou proved the more efficient colonizer although for both serotypes variation in infection rates was demonstrated in different groups of birds given feed containing comparable numbers of salmonellas.     

The isolation of salmonellas from British pork sausages and sausage meat.

Between 1969 and 1974, 1467 packets (3309 samples) of pork sausages and sausage meat produced by two large and two medium sized manufacturers and several local butchers were examined for the presence of salmonellas. Of these, 435 packets (786 samples) were found to contain salmonellas, but there was a wide variation in the isolation rates according to the producer. The salmonella incidence in samples from several small and two medium sized producers was low (0-11%) while the results from the two large producers investigated showed a striking difference, the rate of salmonella contamination in the product of one was low (about 2%) and in that of the other consistently high (40-60%). A comparison of liquid enrichment media, incubation temperatures and selective agar media was also carried out to determine the most efficient combination for the isolation of salmonellas from minced meat products. The results showed that (a) incubation of enrichment cultures at 43 degrees C. yielded a consistently greater number of salmonella isolations that at 37 degrees C., regardless of plating medium, (b) tetrathionate broth A (Rolfe) was superior to selenite broth as en enrichment medium at both 37 and 43 degrees C. and (c) brilliant green agar gave better results than deoxycholate citrate sucrose agar and bismuth sulphite agar as a selective medium.     

Salmonella isolation with Rappaport's medium after pre-enrichment in buffered peptone water using a series of inoculum ratios

The ability of malachite green/magnesium chloride broth (Rappaport's medium) to isolate salmonellas from 25 ml quantities of sewage-polluted natural water was investigated. Samples were first pre-enriched in buffered peptone water and varying volumes of inoculum from the pre-enrichment culture were inoculated into Rappaport's broth. Inoculum ratios in the range 1:2000 to 1:10 were examined. The inoculum ratio denotes the ratio of the volume of inoculum to the volume of fluid medium into which it is introduced. Optimum results were obtained with the 1:2000 ratio, although the salmonella isolation rate was only slightly less with the 1:500 and 1:100 ratios. The 1:2000 inoculum ratio was obtained with a graduated loop holding approximately 0.005 ml of fluid. Use of a loop for inoculation has advantages in speed of performance and safety of manipulation.     

Salmonella isolation with Rappaport's medium after pre-enrichment in buffered peptone water using a series of inoculum ratios.

The ability of malachite green/magnesium chloride broth (Rappaport''s medium) to isolate salmonellas from 25 ml quantities of sewage-polluted natural water was investigated. Samples were first pre-enriched in buffered peptone water and varying volumes of inoculum from the pre-enrichment culture were inoculated into Rappaport''s broth. Inoculum ratios in the range 1:2000 to 1:10 were examined. The inoculum ratio denotes the ratio of the volume of inoculum to the volume of fluid medium into which it is introduced. Optimum results were obtained with the 1:2000 ratio, although the salmonella isolation rate was only slightly less with the 1:500 and 1:100 ratios. The 1:2000 inoculum ratio was obtained with a graduated loop holding approximately 0.005 ml of fluid. Use of a loop for inoculation has advantages in speed of performance and safety of manipulation.     

Evolution of antimicrobial susceptibility in salmonellas from bovine origin in France.

Animals are a wide source of salmonellas for humans and their environment. Domestic ruminants play an important role because of their high susceptibility to salmonellas infection giving an acute disease with massive salmonella excretion and mortality if lack of treatment. As in human medicine use of antibiotics at therapeutic doses in animals can lead to the selection of resistant strains may be pathogenic for humans by direct contact or through environment and food. Taking into account the importance of salmonellosis on calves in rearing units, CNEVA Lyon has set up since 1982 a national network for antimicrobial resistance monitoring of main bacterial pathogens in bovine. This network allowed to detect a recent evolution of antimicrobial susceptibility in salmonella from dairy cows related with new problem of salmonellosis in cattle.     

Further research into the possibility of salmonella-free fattening and slaughter of pigs.

At a pig-fattening farm in the south-western Veluwe which was infected with salmonellas it was sought to achieve salmonella-free fattening in a specially adapted piggery. The test piggery was thoroughly cleaned and disinfected and measures were taken to exclude birds, insects and rodents. An attempt was also made to obtain salmonella-free piglets. Clean clothing, special footwear and disinfectants were used when entering the piggery. During the experiment an infection was detected in the test piggery caused by the same salmonella serotypes as had only been found immediately before the test at the breeding farm. Other salmonella serotypes occurring at the fattening farm did not find their way into the test piggery, and therefore it can be concluded that after the pigs had been brought in all the hygienic barriers functioned adequately. The test showed that the hygienic measures taken had a beneficial effect on growth performance, even though salmonellas were not entirely excluded. After fattening the pigs were slaughtered in two groups. The first group was slaughtered in the usual way, but with the second group extra care was taken with the individual singeing of the carcasses and the careful removal of the intestines. Tests on the carcasses showed that 46% of the pigs in the first group were contaminated with salmonellas as against only 7% in the second. From this it can be concluded that slaughter need not lead to further contamination by salmonellas present in the intestines; indeed, carefully carrying out the slaughter process can even reduce the contamination of the surface of pig carcasses by salmonellas.     

Salmonella dublin infection of calves: use of small doses to simulate natural infection on the farm.

Small numbers of Salmonella dublin were used to infect calves in an attempt to simulate natural infection on the farm. Twenty calves were exposed to S. dublin by one or more of the following methods: Sucking cows which were excreting S. dublin in their faeces (less than 10(2)-10(5) organisms/g). Housing on S. dublin contaminated bedding. Drinking S. dublin contaminated water (10(2)-10(4) organisms/ml). During this experiment some calves were given therapeutic does of oxytetracycline. After exposure the calves were examined for faecal excretion of S. dublin (in some instances mouth swabs and blood samples were also examined) and for clinical signs of illness. Most of the calves became infected with S. dublin but excretion was usually sporadic and the numbers of salmonellas excreted were small. No clinical signs of salmonellosis were observed by S. dublin was isolated from one calf at post-mortem. Another six calves, dosed orally with either 10(6) or 10(8) S. dublin, showed signs of mild illness and although three calves had diarrhoea excretion of salmonellas was intermittent. S. dublin was isolated from one of these calves at post-mortem.     

Competitive exclusion of salmonellas from the chick caecum using a defined mixture of bacterial isolates from the caecal microflora of an adult bird.

Colonization of the caeca of newly hatched chicks by Salmonella typhimurium was prevented by oral administration of a mixture of cultures comprising 48 different bacterial strains originating from an adult bird known to be free from salmonellas. The treatment conferred protection to the same degree as that obtained previously with a suspension of adult caecal contents or an undefined anaerobic culture from the same source and was demonstrated in four separate laboratory trials. Examination of the caecal microflora of chicks one day after being given the protective treatment showed that the presence of high levels of lactobacilli and Bacteroides spp. which are not found usually at two days of age in chicks produced under commercial conditions was indicative of the successful establishment of an adult-type microflora. Although the usual method of administering the protective organisms was to dose the chicks directly into the crop, it was also found possible to incorporate the organisms in the drinking water given to the birds at dilutions up to one in five, the maximum tested. When chicks were given the bacterial mixture via the crop and fed on a diet containing 10 mg kg-1 nitrovin and 100 mg kg-1 monensin, the bacteroides failed to establish in the caeca and the birds were not protected against salmonella colonization. However, when the bacterial cultures were incorporated in the drinking water and the chicks given the same feed, normal protection was obtained; possible reasons for these observations are discussed.     

The effect of feeding diets containing avoparcin on the excretion of salmonellas by chickens experimentally infected with natural sources of salmonella organisms

Chickens were readily infected with salmonella organisms when fed diets containing unsterilized bone-meal or provided with drinking water containing a suspension of natural salmonella infected chicken faeces. When fed diets containing avoparcin at concentrations of 10 or 100 mg/kg chickens infected in these ways excreted larger numbers of salmonellas for longer periods than did chickens fed a nonmedicated diet.     

The effect of feeding diets containing avoparcin on the excretion of salmonellas by chickens experimentally infected with natural sources of salmonella organisms.

Chickens were readily infected with salmonella organisms when fed diets containing unsterilized bone-meal or provided with drinking water containing a suspension of natural salmonella infected chicken faeces. When fed diets containing avoparcin at concentrations of 10 or 100 mg/kg chickens infected in these ways excreted larger numbers of salmonellas for longer periods than did chickens fed a nonmedicated diet.     

Influence of multiple plating from fluid media on salmonella isolation from animal feeding stuffs.

The influence of multiple plating of fluid cultures on salmonella isolation from animal feeding stuffs was examined. Four plating were made from broth culture after 24 h at 37 degrees C and four platings from selenite enrichment from 24 h at 43 degrees C. Selenite enrichment followed broth culture which was used as a pre-enrichment stage. Brilliant green MacConkey agar plates were employed for broth subculture and brilliant green MacConkey and desoxycholate citrate agars for selenite subculture. The eight brilliant green plates subcultured from broth and selenite were examined for salmonellas after incubation for 24 h at 37 degrees C. The four desoxycholate citrate agars after 24 h at 37 degrees C were used for motility enrichment. The food sample size was a single 100 g instead of 4 x 25 g cultured in an earlier study. This pooling of samples aimed at technical economy. Quadruple plating played an important part in salmonella isolation from 100 g specimens. The combination of multiple plating with motility enrichment was the most successful technique used.     

The epidemiology of Salmonella infection of calves: the role of dealers

Salmonellas were detected in the environment of 10 of the 12 calf dealers' premises studied. The cleaning and disinfection routines were often ineffective and salmonellas were isolated from 7.6% and 5.3% of the wall and floor samples before disinfection and 6.8% and 7.6% afterwards. Eight different salmonella serotypes were detected, of which the commonest were Salmonella typhimurium, predominantly phage type DT204C, and S. dublin. Plasmid profiles were used to fingerprint S. typhimurium DT204C and the results indicated that with the exception of one of the premises, prolonged salmonella-persistence in the environment was not occurring. Three separate epidemics of salmonellosis in calves were studied by use of plasmid profile analysis. The results illustrated the role of delers, and their subcontractors, in the dissemination of salmonellas. The study concludes with suggestions for methods to reduce the spread of salmonellas in the calf marketing chain.     

Salmonellas in Danish pigs: a comparison of three isolation methods.

Caecal samples from 350 Danish bacon pigs were investigated for salmonella using three methods of isolation. (1) Direct inoculation of 1 g of faeces into 10 ml of Muller-Kaufmann medium (MK medium) with addition of 0.3% Teepol 610 and subculture on Brilliant Green lactose sucrose phenol-red agar (BLSF agar) with 0.3% Teepol 610. (2) Pre-enrichment of 5 g of faeces into buffered peptone water with addition of 1% Teepol 610 followed by enrichment of 1 ml in 10 ml MK medium with 1% Teepol 610 and subculture on BLSF agar with 0.3% Teepol. (3) Incubation of 0.1 ml of the pre-enrichment (2) into 10 ml Rappaport-Vassiliadis medium (RV 10 medium) incubated at 43 degrees C, subculture on BLSF agar. The MK media with and without pre-enrichment yielded higher findings than the RV 10 media. In total, 28 (8%) of the pigs were found positive, representing 11 (7.4%) of a total of 142 herds investigated. Lymph glands were collected at a later date from six of the positive herds. Five of the herds were found positive. The number of salmonellas in the glands was low, probably less than ten per gram.     

Salmonellas in Danish pigs: a comparison of three isolation methods

Caecal samples from 350 Danish bacon pigs were investigated for salmonella using three methods of isolation. (1) Direct inoculation of 1 g of faeces into 10 ml of Muller-Kaufmann medium (MK medium) with addition of 0.3% Teepol 610 and subculture on Brilliant Green lactose sucrose phenol-red agar (BLSF agar) with 0.3% Teepol 610. (2) Pre-enrichment of 5 g of faeces into buffered peptone water with addition of 1% Teepol 610 followed by enrichment of 1 ml in 10 ml MK medium with 1% Teepol 610 and subculture on BLSF agar with 0.3% Teepol. (3) Incubation of 0.1 ml of the pre-enrichment (2) into 10 ml Rappaport-Vassiliadis medium (RV 10 medium) incubated at 43 degrees C, subculture on BLSF agar. The MK media with and without pre-enrichment yielded higher findings than the RV 10 media. In total, 28 (8%) of the pigs were found positive, representing 11 (7.4%) of a total of 142 herds investigated. Lymph glands were collected at a later date from six of the positive herds. Five of the herds were found positive. The number of salmonellas in the glands was low, probably less than ten per gram.     

Quality control tests of two salmonella enrichment media using different inocula.

Inocula of polluted water naturally infected with salmonellas effectively distinguished six brands of selenite and six brands of tetrathionate enrichment media into satisfactory and unsatisfactory categories. Minimal inocula of pure cultures differentiated the tetrathionates, but not the selenites. Inocula of naturally infected chicken giblets suggested that there was a difference between two comparable brands of tetrathionate, but this was not statistically significant. The difference was, however, clearly demonstrated by minimal inocula of pure cultures. Intensive investigation of two inferior tetrathionates revealed inhomogeneity in the distribution of brilliant green in one bottle of one brand. The importance of the salmonella serotype and even the colonial variant used for the pure culture inoculum was also demonstrated.     

Salmonella colonization in commercial pet turtles (Pseudemys scripta elegans)

An epidemiological survey was conducted on two commercial turtle farms in southern Louisiana to determine the reason for an apparent increase in the prevalence of Salmonella spp. in turtle hatchlings at the time of pre-export certification examination. Pond water was consistently found to be contaminated (6/36 samples) with either Salmonella newport, S. arizonae, or S. poona. Environmental specimens obtained from eggs and turtle hatcheries (204 specimens) failed to yield Salmonella spp. A sample comprising 197 hatchlings, derived from a batch previously demonstrated to be contaminated, showed a salmonella prevalence of 12%, with S. arizonae and S. poona the only serotypes isolated. Four serotypes of Salmonella sp. isolated by a certifying laboratory in 1988, and 20 salmonella isolates obtained from hatchling turtles, were all resistant to gentamicin. The emergence of gentamicin resistance in Salmonella spp. isolated from turtles will reduce the effectiveness of preventive measures in use in Louisiana since 1984.