Effect of ampicillin, cephalexin, ceftiofur and tetracycline treatment on selection of resistant coliforms in a swine faecal microcosmos.

AIMS: To analyse and compare the effect of selection power for antimicrobial resistance (AMR) in coliforms of two kinds of ß-lactams-aminopenicillins; ampicillin (Amp) and cephalosporins; cephalexin (Cpn) and ceftiofur (Cef)-and tetracycline (Tet) using an approach based on a swine faecal microcosmos. METHODS AND RESULTS: Sixteen faecal samples from 32 pigs (mixed two by two) were treated with Amp, Cpn, Cef and Tet for 6 h (T6h) at concentrations expected to reach the animals gut when using in vivo standard doses. Controls (no drug added) were also tested. Next, samples were 1 : 100 diluted and left under the same conditions (no antimicrobial added) for further 20 h (T20h). The proportion of resistant coliform bacteria (R coliforms) to each antimicrobial was analysed just before starting the treatment (T0), at T6h and at T20h. Coselection was also studied by replica plating. Treatment for 6 h yielded significant increase in proportion of R coliforms, regardless of the drug and lack of selection pressure showed different effects at T20h depending on the antimicrobial used. Selective pressure was associated with the type of the ß-lactam with Amp selecting for significantly higher numbers of R coliforms than cephalosporins. CONCLUSIONS: AMR development was observed following short treatment, and for Amp and Tet treatment, resistance persisted 20 h beyond the interruption of treatment. An association between kind of ß-lactam and power of selection was found. SIGNIFICANCE AND IMPACT OF THE STUDY: AMR represents a threat to human health globally and antimicrobial treatment of livestock has a direct impact on this problem. Through our approach based on a swine faecal microcosmos, we demonstrated the effect on AMR development of several drugs commonly used in livestock. Cephalosporins, representing last-line antimicrobials in human medicine, exerted lower selective pressure than Amp under the conditions used and yielded higher proportion of multidrug-R strains.

Isolation of vancomycin-resistant Enterococcus from apparently healthy human animal attendants, cattle and cattle wastes in Tanzania.

AIM: The study aimed to isolate and characterize Enterococcus species from apparently healthy waste attendants, cattle and cattle waste in Tanzania. Emphasis was given to antimicrobial resistance and in particular occurrence of vancomycin (VA)-resistant enterococci. METHODS AND RESULTS: Faecal samples were collected from healthy cattle, cattle waste attendants and cattle house wastes, and isolation of Enterococcus species was performed using Slanetz Bartley agar. Isolates were characterized with regard to species, antimicrobial susceptibility and presence of VA resistance genes. Enterococcus faecalis was the most prevalent species from all sources of isolation (43·5%), followed by Enterococcus faecium (38·4%). Isolates of E. faecium showed a higher number of phenotypic antimicrobial resistance than isolates of E. faecalis. Fifty-eight isolates, which showed resistance or intermediate resistance to VA by disc diffusion test, were analysed for VA-resistant Enterococcus (VRE) by PCR. The vanA gene was detected in 14 isolates of E. faecium and 12 isolates of E. faecalis, while vanB was detected in three isolates. No isolates were found to carry vanC1-gene. CONCLUSION: VRE was detected in both human and cattle samples, despite no known use of antimicrobial agents that can select for VRE in livestock in Tanzania. Enterococcus faecalis was the most commonly isolated species from cattle and humans. SIGNIFICANCE AND IMPACT OF THE STUDY: The study provides information on the prevalence of VRE in human and nonhuman samples in Tanzania calling for further studies on the origin of VRE in such isolates, since no selection mechanism in Tanzania are known.

Prevalence and antimicrobial resistance of Salmonella serovars isolated from poultry in Ghana.

Poultry are possible sources of non-typhoidal Salmonella serovars which may cause foodborne human disease. We conducted a cross-sectional study to determine the prevalence of Salmonella serovars in egg-laying hens and broilers at the farm level and their susceptibility to antimicrobials commonly used in the poultry industry in Ghana. Sampling of faeces by a sock method (n = 75), dust (n = 75), feed (n = 10) and drinking water (n = 10) was performed at 75 commercial egg-laying and broiler farms in two regions of Ghana and skin neck (n = 30) at a local slaughterhouse from broilers representing different flocks. Salmonella was detected in 94/200 (47%) samples with an overall flock prevalence of 44·0%. Sixteen different serovars were identified with S. Kentucky (18·1%), S. Nima (12·8%), S. Muenster (10·6%), S. Enteritidis (10·6%) and S. Virchow (9·6 %) the most prevalent types. The predominant phage type of S. Enteritidis was PT1. All strains were susceptible to cefotaxime, ceftazidime and cefoxitin. Fifty-seven (60·6%) strains were resistant to one or more of the remaining nine antimicrobials tested by disk diffusion, of which 23 (40·4%) showed multi-resistance (resistance to ⩾3 classes of antimicrobials). Of the resistant strains (n = 57), the most significant were to nalidixic acid (89·5%), tetracycline (80·7%), ciprofloxacin (64·9%), sulfamethazole (42·1%), trimethoprim (29·8%) and ampicillin (26·3%). All S. Kentucky strains were resistant to more than two antimicrobials and shared common resistance to nalidixic acid or ciprofloxacin and tetracycline, often in combinations with other antimicrobials. PFGE analysis using XbaI of S. Kentucky demonstrated one dominant clone in the country. In conclusion, poultry produced in Ghana has a high prevalence of multi-resistant Salmonella and the common finding of clonal S. Kentucky in the Kumasi area warrants further investigations into the epidemiology of this serovar. There is an urgent need for surveillance and control programmes on Salmonella and use of antimicrobials in the Ghanaian poultry industry to protect the health of consumers.

UV-B and temperature enhancement affect spring and autumn phenology in Populus tremula.

Perennial plants growing at high latitudes synchronize growth and dormancy to appropriate seasons by sensing environmental cues. Autumnal growth cessation, bud set and dormancy induction are commonly driven by the length of photoperiod and light quality, and the responses are modified by temperature. However, although ultraviolet (UV)-B radiation is well known to affect plant growth and development, information on the effects on bud phenology is scarce. We examined the separate and combined effects of enhanced temperature and UV-B on autumnal bud set and spring bud break in female and male clones of Populus tremula in an outdoor experiment in Joensuu, Eastern Finland. Enhancements of UV-B and temperature were modulated to +30% and +2 °C, respectively, from June to October 2012. Enhanced UV-B accelerated bud set, while increased temperature delayed it. For both UV-B and temperature, we found sex-related differences in responsiveness. Temperature increase had a stronger delaying effect on bud maturation in male compared with female clones. Also, male clones were more responsive to UV-B increase than female clones. Increasing autumnal temperature enhanced bud break in spring for both sexes, while UV-B enhanced bud break in male clones. In conclusion, we found that UV-B affected phenological shifts in P. tremula, and that temperature and UV-B affected genders differently.

Genetic relatedness of commensal Escherichia coli from nursery pigs in intensive pig production in Denmark and molecular characterization of genetically different strains.

AIMS: To determine the genetic relatedness and the presence of virulence and antibiotic resistance genes in commensal Escherichia coli from nursery pigs in Danish intensive production. METHODS AND RESULTS: The genetic diversity of 1000 E. coli strains randomly picked (N = 50 isolates) from cultured faecal samples (N = 4 pigs) from five intensive Danish pigs farms was analysed by repetitive extragenic palindromic-PCR (REP-PCR) and 42 unique REP-profiles were detected (similarity <92%). One profile was dominant (67.2% of strains) but farms differed significantly in the diversity of commensal E. coli: between eight and 21 different profiles per farm were detected. One to three strains representing each REP-profile were characterized by multilocus typing scheme-typing, as well as for presence of antimicrobial and virulence genes and serogrouping through microarray analysis. The 42 REP-profiles were classified into 22 different sequence types (ST) with ST10 being the most common, encompassing 10 REP-profiles. Resistance and virulence genes were detected in most of the isolates. Genes encoding AmpC-ß-lactamases and quinolone resistance were found in one and three isolates, respectively. Toxin-producing genes were observed in 20 isolates. CONCLUSIONS: A low genetic diversity was found in commensal gut E. coli from nursery pigs in Denmark. No correlation was observed between REP-profiles, ST-types and resistance/virulence patterns. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study analysing in depth the genetic variability of commensal E. coli from pigs in Danish intensive pig production. A tendency for higher diversity was observed with in nursery pigs that were treated with zinc oxide only, in absence of other antimicrobials. Strains with potential to disseminate virulence and antibiotic resistance genes to pathogenic subgroups of E. coli were found to be wide-spread.

Antimicrobial resistance in faecal samples from buffalo, wildebeest and zebra grazing together with and without cattle in Tanzania.

AIM: The aim of this study was to determine whether the practice of co-grazing with cattle and wild life constitutes a risk of transmission of antibiotic resistant bacteria to wild ungulates. METHODS AND RESULTS: Faecal samples were collected from buffalo (n = 35), wildebeest (n = 40), zebra (n = 40) and cattle (N = 20) from Mikumi National Park, Tanzania (MNP), where cattle is prohibited and from Ngorongoro Conservation Area (NCA) where co-grazing is practiced. The number of coliforms and enterococci resistant to selected antibiotics was determined. Wild life generally harboured higher number of resistant Escherichia coli and Enterococci than cattle, but with no general influence in wild life of co-grazing with cattle. Vancomycin-resistant Enterococci were detected in wild life samples, and E. coli resistant to cefotaxime and enrofloxacin were observed among isolates from all wild life, but not from cattle. Culture independent estimates of the number of sulII gene copies obtained by qPCR did not differ between wild life from the two sample sites, while tetW was significantly higher in samples from MPN than from NCA. CONCLUSIONS: Antibiotic resistant bacteria were not more frequently found in ungulates grazing together with cattle than ungulates without this interaction. SIGNIFICANCE AND IMPACT OF THE STUDY: This study did not indicate that transmission of antibiotic resistant bacteria is a frequent event following co-grazing of wild life and cattle.

Demonstration of persistent contamination of a cooked egg product production facility with Salmonella enterica serovar Tennessee and characterization of the persistent strain.

AIMS: The aim of this study was to investigate whether continuous contamination of light pasteurized egg products with Salmonella enterica serovar Tennessee (S. Tennessee) at a large European producer of industrial egg products was caused by persistent contamination of the production facility and to characterize the persistent strains. METHODS AND RESULTS: Seventy-three S. Tennessee isolates collected from products over a 3-year period with intermittent contamination, and 15 control strains were compared by pulsed field gel electrophoresis (PFGE) using two enzymes. Forty-five case isolates distributed throughout the full period were shown to belong to one profile type. Isolates representing different PFGE profiles were all assigned to ST 319 by multilocus sequence typing (MLST). The case isolates did not show a higher ability to form biofilm on a plastic surface than noncase isolates. Characteristically, members of the persistent clone were weak producers of H2 S in laboratory medium. S. Tennessee isolated from the case was able to grow better in pasteurized egg product compared with other serovars investigated. CONCLUSIONS: It was concluded that the contamination was caused by a persistent strain in the production facility and that this strain apparently had adapted to grow in the relevant egg product. SIGNIFICANCE AND IMPACT OF THE STUDY: S. Tennessee has previously been associated with persistence in hatching facilities. This is the first report of persistent contamination of an egg production facility with this serovar.

Automated identification of abnormal infant movements from smart phone videos.

Cerebral palsy (CP) is the most common cause of physical disability during childhood, occurring at a rate of 2.1 per 1000 live births. Early diagnosis is key to improving functional outcomes for children with CP. The General Movements (GMs) Assessment has high predictive validity for the detection of CP and is routinely used in high-risk infants but only 50% of infants with CP have overt risk factors when they are born. The implementation of CP screening programs represents an important endeavour, but feasibility is limited by access to trained GMs assessors. To facilitate progress towards this goal, we report a deep-learning framework for automating the GMs Assessment. We acquired 503 videos captured by parents and caregivers at home of infants aged between 12- and 18-weeks term-corrected age using a dedicated smartphone app. Using a deep learning algorithm, we automatically labelled and tracked 18 key body points in each video. We designed a custom pipeline to adjust for camera movement and infant size and trained a second machine learning algorithm to predict GMs classification from body point movement. Our automated body point labelling approach achieved human-level accuracy (mean ± SD error of 3.7 ± 5.2% of infant length) compared to gold-standard human annotation. Using body point tracking data, our prediction model achieved a cross-validated area under the curve (mean ± S.D.) of 0.80 ± 0.08 in unseen test data for predicting expert GMs classification with a sensitivity of 76% ± 15% for abnormal GMs and a negative predictive value of 94% ± 3%. This work highlights the potential for automated GMs screening programs to detect abnormal movements in infants as early as three months term-corrected age using digital technologies.

The importance of motility and chemotaxis for extra-animal survival of Salmonella enterica serovar Typhimurium and Dublin.

AIMS: This study investigated the importance of flagella and motility of Salmonella enterica serovar Typhimurium and Dublin in models of extra-animal survival. METHODS AND RESULTS: The study was performed using transposon mutants in flagella genes fliC and fljB and in chemotaxis genes cheA, cheB and cheR. Flagella and chemotaxis were found to be of minor importance for attachment to plant leaves, survival in liquid manure and interaction with the nematode C. elegans, while differences were observed between the fliC mutant and the wild-type strain of S. Dublin in interactions with amoebae. CONCLUSIONS: The study shows that flagella and chemotaxis play a minor role in extra-animal survival of these two serovars of Salmonella under the conditions tested. SIGNIFICANCE AND IMPACT OF THE STUDY: Extra-animal survival is important in the full infection cycle for zoonotic salmonellae. Such serovars are motile. Even though the current study was only based on the characterization of two serovars, it strongly suggests that motility and chemotaxis are of minor importance during the spread of Salmonella from one animal to the next through the external environment.

A new real-time PCR method for the identification of Salmonella Dublin.

AIMS: Development of a real-time PCR method for the specific detection of Salmonella Dublin. METHODS AND RESULTS: The method was directed towards a Salm. Dublin-specific sequence of the vagC gene on the Salmonella virulence plasmid (pSDV) and towards Salmonella genus-specific sequence of the invA gene, serving as an internal amplification control. The method showed 100% inclusivity and exclusivity when tested on a strain collection containing 50 serotyped S . Dublin strains, 20 strains of other Salmonella serotypes and 10 non- Salmonella strains. The method also showed 100% inclusivity and 99% exclusivity in a collaborative study comprising eight laboratories, where each laboratory received ten different S . Dublin strains and 10 other Salmonella serotypes. CONCLUSIONS: The method showed excellent performance both when validated in the laboratory and in the collaborative study. SIGNIFICANCE AND IMPACT OF THE STUDY: Application of the present method in food control, for example at slaughterhouses, can improve the contamination control of this veterinary and clinically important Salmonella serotype.

Time-course investigation of infection with a low virulent Pasteurella multocida strain in normal and immune-suppressed 12-week-old free-range chickens.

Twelve-week-old indigenous chickens, either immune-suppressed using dexamethasone (IS) or non-immune-suppressed (NIS), were challenged with a low virulent strain, Pasteurella multocida strain NCTC 10322(T), and developed clinical signs and pathological lesions typical of chronic fowl cholera. NIS birds demonstrated much more severe signs of fowl cholera than IS birds. With few exceptions, signs recorded in IS and NIS birds were of the same types, but significantly milder in the IS birds, indicating that immune suppression does not change the course of infection but rather the severity of signs in fowl cholera. P. multocida signals by fluorescent in situ hybridization (FISH) were observed between 1 h and 14 days in the lungs, trachea, air sacs, liver, spleen, bursa of Fabricius and caecal tonsils, while signals from other organs mostly were observed after 24 h. More organs had FISH signals in NIS birds than in IS birds and at higher frequency per organ. Many organs were positive by FISH even 14 days post infection, and it is suggested that these organs may be likely places for long-term carriage of P. multocida following infection. The present study has demonstrated the spread of P. multocida in different tissues in chickens and distribution of lesions associated with chronic fowl cholera, and pointed to a decrease of pathology in IS birds. Since dexamethasone mostly affects heterophils, the study suggests that these cells play a role in the development of lesions associated with chronic fowl cholera in chickens.

Survival of Salmonella on cuts of beef carcasses subjected to dry aging.

AIMS: The aim of this study was to determine the survival of 15 different strains of Salmonella of selected serotypes during prolonged cold storage of beef. METHODS AND RESULTS: Fifteen strains of eight different serotypes of Salmonella were spiked onto fresh cuts beef portions, and the survival was followed during storage in a laboratory cooling system. Over a 14-day period, all strains were reduced significantly in numbers; however, strains of Salmonella Typhimurium DT104 and Salmonella Enteritidis PT4 and PT8 survived significantly longer than strains of the serovars Dublin, Derby, Infantis and Newport. For five selected strains, the observations were verified in a pilot plant cooling facility mimicking industrial cooling. No significant differences in reduction were found between the two cooling methods. CONCLUSIONS: A significant reduction in Salmonella can be obtained by dry aging of beef during cold storage but the survival is strain dependent. SIGNIFICANCE AND IMPACT OF THE STUDY: From a hygienic point of view, cold storage of unpacked beef, which is still performed in small slaughterhouses, is a good alternative to vacuum packaging.

Rapid quantification of viable Campylobacter bacteria on chicken carcasses, using real-time PCR and propidium monoazide treatment, as a tool for quantitative risk assessment.

A number of intervention strategies against Campylobacter-contaminated poultry focus on postslaughter reduction of the number of cells, emphasizing the need for rapid and reliable quantitative detection of only viable Campylobacter bacteria. We present a new and rapid quantitative approach to the enumeration of food-borne Campylobacter bacteria that combines real-time quantitative PCR (Q-PCR) with simple propidium monoazide (PMA) sample treatment. In less than 3 h, this method generates a signal from only viable and viable but nonculturable (VBNC) Campylobacter bacteria with an intact membrane. The method's performance was evaluated by assessing the contributions to variability by individual chicken carcass rinse matrices, species of Campylobacter, and differences in efficiency of DNA extraction with differing cell inputs. The method was compared with culture-based enumeration on 50 naturally infected chickens. The cell contents correlated with cycle threshold (C(T)) values (R(2) = 0.993), with a quantification range of 1 x 10(2) to 1 x 10(7) CFU/ml. The correlation between the Campylobacter counts obtained by PMA-PCR and culture on naturally contaminated chickens was high (R(2) = 0.844). The amplification efficiency of the Q-PCR method was not affected by the chicken rinse matrix or by the species of Campylobacter. No Q-PCR signals were obtained from artificially inoculated chicken rinse when PMA sample treatment was applied. In conclusion, this study presents a rapid tool for producing reliable quantitative data on viable Campylobacter bacteria in chicken carcass rinse. The proposed method does not detect DNA from dead Campylobacter bacteria but recognizes the infectious potential of the VBNC state and is thereby able to assess the effect of control strategies and provide trustworthy data for risk assessment.

Demonstration of persistent strains of Campylobacter jejuni within broiler farms over a 1-year period in Lithuania.

AIMS: The aim of the study was to investigate the flock prevalence of Campylobacter jejuni and Campylobacter coli in broiler farms in Lithuania and to identify possible persistent strains of Camp. jejuni using amplified fragment length polymorphism (AFLP) typing method. METHODS AND RESULTS: During 1 year, 42 broiler flocks from 9 broiler farms were examined to determine the prevalence of Campylobacter-positive broiler flocks in Lithuania. Among 42 broiler flocks examined, 31 flocks (73.8%) were positive for Camp. jejuni and 17 flocks (40.48%) for Camp. coli. Campylobacter jejuni isolates were genotyped by AFLP method using BspDI and BglII restriction enzymes. Typing of 190 isolates generated 50 AFLP genotypes with the highest diversity of strains found in the summer season. Each farm showed one or more predominant AFLP types, and one AFLP type (A32) was found in five broiler farms over a 1-year period. CONCLUSIONS: Campylobacter jejuni and Camp. coli are highly prevalent in broiler farms in Lithuania. Farm-specific genotypes were identified in all farms examined. Type A32 was present and persisted in different broiler farms, and a common source of transmission of Camp. jejuni was suspected. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time, Camp. jejuni in broiler flocks has been genetically characterized in Lithuania. Persistent strains of Camp. jejuni were detected over one period at the beginning of broiler meat production chain and, therefore, the identification of contamination source of such strains and the mechanism of their particular ability to persist are crucial to establish effective control measures against Camp. jejuni infection in broiler farms.

Evaluation of novel multiplex qPCR assays for diagnosis of pathogens associated with the bovine respiratory disease complex.

Bovine respiratory disease complex is the most common disease requiring the use of antimicrobials in industrial calf production worldwide. Pathogenic bacteria (Mannheimia haemolytica (Mh), Pasteurella multocida (Pm), Histophilus somni (Hs), and Mycoplasma bovis) and a range of viruses (bovine respiratory syncytial virus, bovine coronavirus, bovine parainfluenza virus type 3, bovine viral diarrhea virus and bovine herpesvirus type 1) are associated with this complex. As most of these pathogens can be present in healthy and diseased calves, simple detection of their presence in diseased calves carries low predictive value. In other multi-agent diseases of livestock, quantification of pathogens has added substantially to the predictive value of microbiological diagnosis. The aim of this study was to evaluate the ability of two recently developed quantitative PCR (qPCR) kits (Pneumo4B and Pneumo4V) to detect and quantify these bacterial and viral pathogens, respectively. Test efficiencies of the qPCR assays, based on nucleic acid dilution series of target bacteria and viruses, were 93-106% and 91-104%, respectively, with assay detection limits of 10-50 copies of nucleic acids. All 44 strains of target bacteria were correctly identified, with no false positive reactions in 135strains of non-target bacterial species. Based on standard curves of log10 CFU versus cycle threshold (Ct) values, quantification was possible over a 5-log range of bacteria. In 92 tracheal aspirate samples, the kappa values for agreement between Pneumo4B and bacterial culture were 0.64-0.84 for Mh, Pm and Hs. In an additional 84 tracheal aspirates, agreement between Pneumo4B or Pneumo 4V and certified diagnostic qPCR assays was moderate (0.57) for M. bovis and high (0.71-0.90) for viral pathogens. Thus Pneumo4 kits specifically detected and quantified the relevant pathogens.

Investigations of the occurrence of Avibacterium paragallinarum infections in Uganda.

Investigations were conducted to determine the occurrence of Avibacterium paragallinarum in poultry in Uganda. A total of 710 each of bacteriologic and serum samples were taken from chickens and turkeys for demonstration of A. paragallinarum and antibodies. Samples for isolation of A. paragallinarum were also subjected to direct polymerase chain reaction (PCR) for demonstration of the organism's presence. Antibodies to A. paragallinarum were demonstrated in the sera using the hemagglutination inhibition test. A total of five isolates were recovered from two out of five commercial layer chicken farms investigated where suspected cases of infectious coryza were reported, and all of them belonged to Page's serovar C. PCR detected more positive samples (11/68) than did culture (5/68). Isolates were not recovered from free-range poultry nor were there any positive samples by PCR. The overall seroprevalence was 40.5% and the seroprevalence to serovars A, B, and C were 18%, 0.5%, and 22%, respectively. Antibodies to all Page's serovars A, B, and C were demonstrated in free-range chickens but only serovar C antibodies were demonstrated in commercial chickens. No antibodies were demonstrated in turkeys. This is the first time infectious coryza has been confirmed in Uganda and the causative agent, A. paragallinarum, isolated. A high seroprevalence observed in free-range chickens seems to indicate a subclinical infection under extensive village management conditions.

Isolation of Campylobacter spp. from a pig slaughterhouse and analysis of cross-contamination.

The purpose of this study was to establish the prevalence and possible contamination routes of Campylobacter spp. in a pig slaughterhouse. Swab samples were taken from the last part of rectum, from the carcasses surface before meat inspection and from slaughter line surface from 4 different pig herds during slaughtering. Identification of Campylobacter isolates was determined by the use of phase-contrast microscopy, hippurate hydrolysis, indoxyl acetate hydrolysis tests and PCR based restriction fragment length polymorphism method (PCR-RFLP). Pulsed-field gel electrophoresis (PFGE) typing using two macro-restriction enzymes SmaI and SalI was applied to in-slaughterhouse contamination analysis of pig carcasses. The study showed that 28 (63.6%) of the 44 samples collected at slaughterhouse were contaminated by Campylobacter spp. Up to 5 different colonies were obtained from each swab sample and a total of 120 different isolates were collected. 23.4% (28 of 120) isolates were identified as C. jejuni (19 from carcasses and 9 from slaughter line surfaces) and 76.6% (92 of 120) isolates as C. coli (28 from faeces, 47 from carcasses and 17 from slaughter line surfaces). The typing results showed identity between isolates from successive flocks, different carcasses, and places in the slaughterhouse in contact with carcasses. The results suggest that cross-contamination originated in the gastro-intestinal tract of the slaughtered pigs and that cross-contamination happened during the slaughter process.

Survival of Escherichia coli and Salmonella Typhimurium in slurry applied to clay soil on a Danish swine farm.

A pilot study was carried out on a Danish swine farm infected with multi-resistant Salmonella Typhimurium DT104 (MRDT104). We aimed to (1) investigate to which degree the decline of Escherichia coli and Salmonella in swine slurry applied to farmland depended on the application method; (2) estimate the survival times of E. coli and Salmonella in the soil surface following deposition of naturally contaminated pig slurry; and (3) simulate survival of Salmonella in different infection levels using E. coli data as input estimates. Slurry was deposited by four different methods: (1) hose applicator on black soil followed by ploughing and harrowing; (2) hose applicator on black soil followed only by harrowing; (3) hose applicator on a field with winter-wheat seedlings without further soil treatment; (4) slurry injector on a field with winter-wheat seedlings without further soil treatment. E. coli and Salmonella could not be detected at all in soil following treatment 1. Following the other treatments, E. coli was not detected in soil samples after day 21 and Salmonella was no longer detected after day 7. Simulation results showed that clinical (4 log CFU g(-1)) and sub-clinical Salmonella levels (2500 CFU g(-1)) would fall below the detection limit within 10 or 5 days, respectively. Analysis of samples from 62 Danish MRDT104-infected swineherds showed that nearly 75% of these herds had low levels of MRDT104 (< 10 CFU g(-1)) in their slurry. Our results show that ploughing and harrowing of soil amended with contaminated pig slurry was an effective means to reduce environmental exposure to E. coli and Salmonella on this clay-soil farm.

Human urokinase receptor expression is inhibited by amiloride and induced by tumor necrosis factor and phorbol ester in colon cancer cells.

The modulation of urokinase plasminogen activator receptor (uPAR) gene expression by tumor necrosis factor alpha (TNF alpha), phorbol ester (PMA) and amiloride was studied in three colon cancer cell lines. uPAR mRNA and protein were induced by TNF alpha and by PMA but were inhibited by amiloride at concentrations of 0.1 to 1 mM in the presence or absence of TNF alpha and PMA. Nuclear run-on transcription assay indicated that the effects of amiloride and TNF alpha were mediated at least in part at the transcriptional level, whereas PMA may act in part via a posttranscriptional mechanism. These results suggested that uPAR gene expression is modulated by multiple signal transduction pathways.

Virulence characterization of a strain of Salmonella enterica subspecies houten (subspecies IV) with chromosomal integrated Salmonella plasmid virulence (spv) genes.

The Salmonella plasmid virulence genes (spv) are commonly found on plasmids contained in a small number of serotypes of Salmonella belonging to subspecies I, where they are important for survival within macrophages and the establishment of successful systemic infection. However, in this study, spv genes were detected by the polymerase chain reaction in the chromosome of a plasmid-free strain of S. IV 16:z4, z32:- (Salmonella subspecies IV). The full range of spv genes (spvR, spvA, spvB, spvC and spvD) was demonstrated, but a 216-bp deletion, accompanied by an insertion of 59-bp cryptic DNA, was present in spvA. S. IV 16:z4, z32:- was avirulent in mice and did not become virulent with the introduction of a fully functionally serotype-associated virulence plasmid (SAP) from S. typhimurium. By use of an spvRAB'-chloramphenicol acetyl transferase fusion gene, it was demonstrated that S. IV 16:z4, z32:- did not express the spv genes. Salmonella subspecies IV is monophasic, and in phylogenetic analyses it clusters distantly to Salmonella subspecies I, where all the serotypes that normally carry SAPs are found. The mechanisms by which spv genes have been transferred to this serotype remain unknown.