In vitro activity of nitrofuran derivatives on growth and morphology of Salmonella enterica serotype Enteritidis.

AIMS: To evaluate the effect of nitrofuran derivatives furazolidone (Fz) and nitrofurantoin (Nf) on Salmonella enterica serovar Enteritidis PT4 in vitro, with regard to cell growth, morphology and ultrastructure. METHODS AND RESULTS: The effects of Fz on the growth rates of Fz resistant (FzR) and sensitive (FzS) strains were assessed by viable counts. Over 24 h incubation, concentrations of <1 microg ml(-1) of Fz were bacteriostatic to the FzS strain. The FzR strain tolerated concentrations up to 16 microg ml(-1) before cell numbers diminished over the same time period. The effect on the growth rate of the FzS strain after 1 h exposure to supra-inhibitory concentrations of Fz, gave a maximum response at 32X minimum inhibitory concentration (MIC) of 4.5 h. Effects on the ultrastructure of bacterial cells by scanning electron and transmission microscopy, and DNA-specific staining with DAPI of the FzS strain exposed to nitrofurans were studied. Abnormalities such as extensive filamentation with sparse, sporadic nucleotide distribution and evidence of extrusions in the cell envelope in the form of blebs were evident. CONCLUSIONS: Nitrofurans exert their bactericidal effect on Salmonella by inducing extensive structural alteration after exposure at sub- or suprainhibitory concentrations, involving inhibition of cell division because of the activated drug causing an intercalating type of binding in DNA. SIGNIFICANCE AND IMPACT OF THE STUDY: These results demonstrate the in vitro activity of the nitrofuran derivatives, furazolidone and nitrofurantoin on Salmonella, defining the pharmacodynamics and physical nature of their action as therapeutic agents.

Microbial cross-contamination by airborne dispersion and contagion during defeathering of poultry.

1. A readily identifiable strain of Escherichia coli K12 was used as a 'marker' organism to determine the sources, routes and patterns of microbial cross-contamination during mechanical defeathering of broiler chicken carcases. 2. Inoculation of scald water with the marker organism led to a relatively even pattern of carcase contamination during subsequent defeathering. Microbial cross-contamination was greater by this route of inoculation than by either surface inoculation of a 'seeder' carcase or oral inoculation of a live bird one day before slaughter. 3. Dispersal of the marker organism was strongly influenced by the mechanical action of the defeathering machines. Forward transmission of the marker occurred by aerosol or large airborne droplets and particulates such as feathers. Moving carcases through the defeathering machines when these were non-operational clearly reduced backward transmission of the marker. 4. Although microbial dispersal was unaffected by increasing the spacing between individual carcases or installing a water curtain at the entry and exit of the defeathering machines, shielding of carcases with aluminium baffles reduced counts of the marker organism from contaminated carcases by > 90%. 5. The results imply that microbial cross-contamination of broiler chicken carcases during defeathering occurs mainly via the airborne route, which could be contained by physical means.

Evaluation of treatment and prophylaxis with nitrofurans and comparison with alternative antimicrobial agents in experimental Salmonella enterica Serovar enteritidis infection in chicks.

The ability of the nitrofuran antimicrobial agents furazolidone and furaltadone to prevent, reduce or eliminate Salmonella enterica serovar enteritidis PT4 infection in artificially challenged day-old chicks was evaluated. Treating the birds with the nitrofurans failed to eliminate established infections with either furazolidone-resistant (FzR) or furazolidone-sensitive (FzS) strains. Simultaneous administration of the nitrofurans to day-old chicks challenged with FzS failed to prevent infection but reduced colonization significantly (p<0.05) compared to unmedicated controls. No reduction of colonization occurred with FzR. Challenging birds with FzS and simultaneous dosing with nitrofurans for 1 week, followed by a second week of continued treatment, resulted in an increase in the level of colonization in the second week rather than a decrease. Dosing with the nitrofurans (200 ppm) for 1 week prior to challenge with FzS and continued medication for a further week prevented colonization of the caecum, liver and spleen. However, cessation of dosing at the time of challenge with salmonella resulted in colonization. Chloramphenicol and tetracycline at concentrations of 200 ppm were both independently capable of preventing colonization by salmonella. Sulphadiazine initially reduced colonization but failed to eliminate the infection. Only when furazolidone was combined with chloramphenicol or when sulphadiazine was combined with trimethoprim, and the combined drugs were administered concurrently with the challenge, was colonization prevented.

Dispersal of micro-organisms in commercial defeathering systems.

1. The extent of cross contamination between carcases and the dispersal of micro-organisms to the environs during defeathering was measured in a commercial processing plant. 2. Defeathering reduced the numbers of a marker organism, a nalidixic acid-resistant strain of Escherichia coli K12, on inoculated carcases but dispersed the organism on to preceding and following carcases. 3. The pattern of microbial dispersal during defeathering was similar for naturally occurring bacteria on the carcase, for example, total aerobic counts and counts of presumptive coliforms, suggesting that the marker organism mimics the natural situation realistically. 4. The majority of feathers, together with micro-organisms, were removed during the first 10 s of the defeathering process, which was completed in 45 s, indicating that control measures to minimise cross contamination would be most effective if applied in the early stages of the process. 5. The method of defeathering used by the machine influenced the pattern of microbial dispersal and the extent of cross contamination to other carcases on the same processing line.

Assessment and development of procedures and apparatus to reduce contamination of lamb carcasses during pelt removal in low-throughput abattoirs.

Two series of experiments were carried out to investigate methods of reducing contamination of lamb carcasses in low-throughput abattoirs, where cradle dressing is normally employed. In the first series, cradle design and pelt removal procedure were investigated, and a method was developed for assessing gross visible contamination. Significant improvements in microbiological and gross visible contamination (P < 0.01) were achieved by procedural changes only; modifications to the cradle design had no effect. In the second series of experiments, two improved methods of pelt removal and the effect of hand washing prior to carcass contact during the pelt removal procedure were investigated. The improved methods comprised a Frame system, in which the pelt was removed in a manner similar to that in a high-throughput inverted line, and a Hybrid system, in which the pelt was removed from the forequarters on a conventional cradle before the carcass was suspended in an "inverted" vertical position for removal of the pelt from the abdomen and hindquarters. The results of microbiological and gross visible contamination from these methods, with and without hand washing, were compared with the conventional Cradle method of pelt removal. Both the Hybrid and Frame systems had significantly less microbiological and gross visible contamination (P < 0.01). However, hand washing had no significant effect on the level of carcass contamination for all three methods of pelt removal. Greatest reductions in microbiological and gross visible contamination were achieved using techniques that minimized hand contact with the carcass during pelt removal by adoption of inverted dressing procedures. Equipment redesign did not reduce carcass contamination.

Infections and intoxications associated with animal feed and forage which may present a hazard to human health.

Animal feed or forage may be the source of a limited number of infections for farm animals that could lead to human illness. Likely organisms include Salmonella enterica, Toxoplasma gondii, Trichinella spiralis and possibly the agent of bovine spongiform encephalopathy. The risk to human health from other infectious agents which may contaminate either feed or forage appear to be either negligible, e.g. Bacillus anthracis and Mycobacterium bovis, or non-existent, e.g. Clostridium botulinum toxin and Listeria monocytogenes. Mycotoxins present in animal feed can result in foods of animal origin also containing them. This risk is well recognized but has yet to be quantified accurately and in some instances the risk may be of theoretical rather than practical importance. Pesticides, agricultural and industrial chemicals, heavy metals and radionuclides may pollute animal feed and forages. The methods available for controlling pollution from these sources are well understood from a technical point of view although the effective implementation of controls can be difficult.

Assessing abattoir hygiene with a marker organism.

A study was made to evaluate the use of a marker organism for assessing whether hygienic slaughter practices were being followed at red meat abattoirs. The organism, a nonpathogenic strain of Escherichia coli K12 that was resistant to nalidixic acid, was detected and counted on a highly specific isolation medium. With beef carcases, the practice of bagging the excised anus reduced, but did not prevent the spread of the organism from an inoculum applied in the anal region before the hide was removed. The carcases of sheep that were processed at a low-throughput abattoir, were contaminated with the marker after the fleece had been inoculated at a single site. The contamination was significantly reduced (P<0.001) when the operative responsible for flaying had cleaned his hands, arms and apron before and during the handling of each carcase, and used a knife which was freshly pasteurised on several occasions. However, the subsequent washing of carcases had little or no effect on the levels of the marker organism. It was concluded that the marker may be of value in assessing hygiene control, improving present practices, and training abattoir staff.

The bacteriological quality of British beef 1. Carcasses sampled prior to chilling.

During a survey of 11 beef abattoirs in England 2200 swab samples were taken from carcasses just before chilling. Geometric mean aerobic plate counts at 30°C on each of four carcass sites ranged from log(10) 2·45 to 4·29cfu cm(2) with the brisket and flank samples tending to be more highly contaminated than those from the fore-rib and groin. Presumptive coliforms were isolated from 24% of the samples and the proportion of positive samples among the abattoirs varied between 1·5% and 43%. Analysis of variance confirmed that the bacteriological status of beef carcasses may be influenced by a number of interacting factors, including abattoir, visit, and sampling site. However, the results showed that working methods alone were not critical factors in the production of beef of superior bacteriological quality.

Effects of fleece soiling and skinning method on the microbiology of sheep carcases.

The fleece of sheep becomes soiled primarily on the abdomen and on the legs. A five-category scoring scale for soiling of the fleece was defined, with score 1 being clean and dry and score 5 being wet and heavily soiled with faecal material. Twenty sheep with each fleece score were slaughtered and dressed on a commercial 'inverted' slaughter line. Swab samples were taken from the shoulder and the abdomen of the carcases immediately after skinning, and total viable counts and the numbers of Enterobacteriaceae were determined. The condition of the fleece significantly affected the microbial load on these parts of the dressed carcase, with the carcases derived from sheep with increasingly dirty fleeces carrying up to 1000 times more microorganisms, and a higher proportion of the carcases being contaminated with Enterobacteriaceae. Modifications to the dressing procedure were made to try to reduce carcase contamination at the shoulder. The application of bulldog clips to prevent direct contact between the fleece and the underlying muscles resulted in higher counts on carcases with a fleece score of 3, probably owing to contamination from the hands of the slaughterman and the problems with ensuring that the clips were in place before contamination had occurred. Delaying the skinning of the shoulder until later in the pelt removal process achieved worthwhile reductions in the contamination of the shoulder of carcases with a fleece score of 4, although the total viable counts were still higher than carcases with a score of 3.

Zoonoses in the meat industry: a review.

Zoonoses are diseases, the infections of which can be transmitted between man and animals. Only a few are of importance with respect to poultry meat and meat from cattle, sheep, horses and goats. Advances in the control of diseases such as tuberculosis, brucellosis and trichinosis in animals have reduced the hazards posed to workers in the meat industry and to consumers of meat. However, inspection of animals ante- and post-mortem cannot detect all infectious agents present. This applies particularly to bacteria such as Campylobacter, Salmonella, verotoxigenic and other pathogenic Escherichia coli and Yersinia. Protection of meat workers from infection depends upon taking normal hygienic precautions, which also protect the meat from contamination from the workers. Consumers are exposed to a smaller range of zoonoses than meat workers because they encounter only meat that has passed inspection. In addition, heavily contaminated parts of the animal, such as the hide, feathers and viscera have been removed. Further advances in making meat safer are likely to result from the introduction of Integrated Quality Assurance systems. These involve identifying, monitoring and keeping records of the disease status and treatment of each animal (or poultry flock) so that its history is known when it reaches the abattoir. They should also include programmes aimed at minimising colonisation by zoonotic bacteria such as camplyobacters, salmonellas and verotoxigenic Escherichia coli.

Evaluation of the influence of supplementing the diet with mannose or palm kernel meal on Salmonella colonisation in poultry.

1. The dietary inclusion of 15 and 25 g/kg mannose was associated with a reduction in the numbers of Salmonella enteritidis (PT4) in the caecal contents of chicks challenged by the food. The same benefit was not recorded for S. infantis, possibly because this strain, unlike S. enteritidis PT4, lacked mannose-sensitive fimbriae. 2. The addition of 25 g/kg palm kernel meal (PKM), but not 20 g/kg desiccated coconut, to the food reduced the degree of salmonella colonisation in the intestinal tract of broiler chicks given diets contaminated with S. kedougou or S. enteritidis from the day of their arrival from the hatchery. 3. The beneficial effect of PKM was also demonstrated at an inclusion rate of 5 g/kg and was similar for preparations with a particle size of either < 150 microns or < 300 microns. 4. Day-old birds challenged orally with S. enteritidis and given food supplemented with 25 g/kg PKM, became clear of infection by 3 weeks of age while birds given unsupplemented food remained infected. 5. These preliminary results suggest that the inclusion of PKM, which contains inter alia, oligosaccharides containing mannose, in the diet of chicks may reduce the extent to which the intestine is contaminated with salmonellas.

Microbial status of chicken portions and portioning equipment.

1. The sources and numbers of microbes contaminating poultry portions, which form an increasing proportion of poultry meat production were investigated. Total aerobic count at 30 degrees C, pseudomonads, coliforms, enterococci and Staphylococcus aureus were enumerated. 2. Samples which had undergone different degrees of portioning, from whole birds to individual retail portions of poultry meat, were collected at points along the portioning line. Equipment surfaces, utensils and hands/gloves of factory personnel were examined. 3. In general, microbial numbers on carcase sampling sites were related to 'exposure factor', that is, the length of time the site was exposed to potential contamination during processing and portioning. 4. When results for individual sampling sites were analysed separately, no significant differences were found between sites on whole birds and the corresponding half bird sample sites. Comparison of numbers on half birds and on portions revealed a more variable situation. 5. Sites touched by rubber gloves when carcases were hung on the automatic portioning lines had greater contamination than other sites. 6. Provided factory and operative hygiene standards are high, portioning does not significantly increase numbers of microorganisms.

Relevance of abattoir hygiene assessment to microbial contamination of British beef carcases.

Eleven beef abattoirs were visited, each on five separate occasions. On each occasion, an audit was carried out according to the official Hygiene Assessment System (HAS) and 10 carcases were sampled at four different sites to assess total viable counts and counts of presumptive coliform bacteria. The HAS scores ranged from 11 to 84 (maximum 100), and the logarithmic mean total viable counts for all sampling sites on each batch of carcases varied between 1.98 and 4.14 colony forming units/cm2. The mean prevalence of coliform contamination ranged from 0 to 85 per cent. There was a significant negative correlation (P < 0.001) between the mean HAS scores and the mean total viable count for each abattoir, but not between the HAS scores and the numbers of coliforms. Within the HAS, the mean scores for all five categories, before weighting, showed a significant correlation with the mean total viable count (P < 0.001); however, the categories concerned with slaughter and dressing, and personnel and practices were of most value in determining trends in carcase contamination. A new advisory classification is proposed for levels of microbial contamination on beef carcases.

Effect of changes in processing to improve hygiene control on contamination of poultry carcasses with campylobacter.

Examination of neck skin and caecal samples taken at a commercial processing plant from 15 randomly chosen poultry flocks showed that all flocks were contaminated initially with thermophilic Campylobacter spp., even in the apparent absence of caecal carriage. During processing, numbers of campylobacter on skin samples were reduced by between 10 and 1000-fold. To improve hygiene control generally, chlorinated-water sprays were used to limit microbial contamination on equipment and working surfaces. In addition, chlorine concentrations in process water were increased and any unnecessary carcass contact surfaces in the processing plant were removed. When comparing flocks before and after the changes, it was found that numbers of campylobacter on packaged carcasses were significantly lower after the changes had been made (P 0.001). In practice, however, the reduction would be likely to have little impact on consumer exposure to campylobacter infection.

Use of a marker organism in poultry processing to identify sites of cross-contamination and evaluate possible control measures.

1. Nine different sites at a poultry processing plant were selected in the course of a hazard analysis to investigate the degree of microbial cross-contamination that could occur during processing and the effectiveness of possible control measures. 2. At each site, carcases, equipment or working surfaces were inoculated with a non-pathogenic strain of nalidixic acid-resistant Escherichia coli K12; transmission of the organism among carcases being processed was followed qualitatively and, where appropriate, quantitatively. 3. The degree of cross-contamination and the extent to which it could be controlled by the proposed measures varied from one site to another.

Microbiological survey of five poultry processing plants in the UK.

1. Neck skin samples were taken from chickens and turkeys at all the main stages of processing to monitor changes in total viable count (TVC) and counts of coliforms and pseudomonads. 2. Processing reduced TVC by up to 100-fold. Geometric mean counts after packaging were log10 4.4 to 5.3 CFU/g whilst corresponding counts of coliforms were 2.7 to 3.8 CFU/g. 3. Increases in mean TVC or coliforms as a result of either defeathering or evisceration did not exceed 0.6 log. 4. Pseudomonads represented only a minor fraction of the initial microflora of the bird and were often reduced by scalding to a figure which could not be detected by direct plating of samples; however, subsequent contamination resulted in means between log10 2.9 and 4.0 CFU/g for packaged carcases. 5. Although Staphylococcus aureus was readily isolated from defeathering equipment, mean counts from defeathered carcases were always below log10 3.0 CFU/g.

Naturally acquired Pasteurella multocida subsp. multocida infection in a closed colony of rabbits: characteristics of isolates.

Twelve litters, comprising 41 rabbits aged 35 to 60 days old, in a closed university colony, were monitored for acquisition of nasal Pasteurella multocida subsp. multocida infection. Isolates from 11 infected rabbits were characterized by colonial morphology, capsular type, biotype and antibiotic resistance. Selected isolates were further characterized by somatic antigen typing. Two major strains of P. multocida subsp. multocida were detected in the colony. One strain had mucoid colonies, fermented few carbohydrates and was serotype A:5, whereas, the other strain had smooth iridescent colonies, non-typeable capsular antigen, type 3 somatic antigen and fermented more than twice as many carbohydrates.

Naturally acquired Pasteurella multocida infection in rabbits: clinicopathological aspects.

A cohort of 41 New Zealand White rabbits, 35 to 60 days old, from twelve litters were followed for twelve weeks for development of pasteurellosis. Eleven of 19 rabbits in five litters acquired Pasteurella multocida infection. The incubation period was difficult to determine as P. multocida infection was detected both before and after the onset of rhinitis. The response of rabbits to infection varied from subclinical infection to death from systemic pasteurellosis. Atrophy of the maxilloturbinates of the nares was detected in rabbits with chronic rhinitis associated with P. multocida infection.

Naturally acquired Pasteurella multocida infection in rabbits: immunological aspects.

Natural infection with P. multocida in New Zealand White rabbits was followed in 2 to 3 weeks by development of immunoglobulin (Ig)M and IgG serum antibodies. The IgM response peaked and returned to lower levels within several weeks after infection, whereas the IgG response progressively increased and remained elevated.