NEWSLETTER
COMMUNITY REFERENCE LABORATORY FOR
SALMONELLA
VOL. 11 NO. 2
Juni 2005
ISSN 1572-3836
Produced by
Community Reference Laboratory for Salmonella
National Institute of Public Health and the Environment
P.O. Box 1, 3720 BA Bilthoven, The Netherlands
phone:
(31) 30-274 3537 (Kirsten Mooijman)
(31) 30-274 4263 (Hans Korver: contact person newsletter)
fax:
(31) 30-274 4434
e-mail address:
[email protected]
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Editorial Note
1
For Information
2
Literature Information
4
Address-list NRLs 6DOPRQHOOD
34
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Bilthoven, 1 July 2005
Dear colleagues,
Two months have already passed since we last met at the yearly CRL-6DOPRQHOOD workshop.
We can look back to an interesting and stimulating meeting and we hope you enjoyed it too.
Last month we have sent you an evaluation form by which we hope to hear your views
(positive and negative) on the workshop. Your suggestions/remarks can be very useful to
improve the future workshops. We are presently working on the report of the meeting. A draft
version of the report will soon be sent to the EC DG-Sanco. The final report will be made
available in hard copy for all NRLs-6DOPRQHOOD in the near future. As you have been informed
earlier, all presentations of the workshop are available at the CRL-6DOPRQHOOD website:
http://www.rivm.nl/crlsalmonella/workshop/index.html
From 13 to 17 June, meetings took place of CEN/TC275/WG6 and ISO/TC34/SC9 in Warsaw
(Poland). Shortly before the meeting the information was received that members of
ISO/TC34/SC9 had voted positive on the new work item proposal for the detection of
6DOPRQHOOD in animal faeces and samples of the primary production stage. The main
comments on draft Annex D of ISO 6579 were discussed at the meeting, on which agreement
was found. It was agreed that I would amend draft Annex D by including the accepted (small)
comments. In the mean time I have already sent a new version of Annex D to the secretariat of
ISO/TC34/SC9. Soon the document will be sent around for voting as Draft International
Standard (DIS). If you do not obtain the document in the coming 1-2 months via your national
standardisation body, please let me know. I will then forward to you the DIS version of Annex
D. Voting and comments should be sent via the official route (ISO secretariat).
In this newsletter the time frame for the next interlaboratory comparison study on the
detection of 6DOPRQHOOD is included. As you may read, we plan to do an earlier (short)
reporting of the total results and a follow-up in case of problems. To be able to do so, we need
your co-operation by respecting the deadlines.
I would like to wish you all nice and sunny summer holidays!
Best regards,
Kirsten Mooijman
1
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Week
41
43
Date
10 – 14
October
24 – 28
October
44
31 October –
4 November
45
7 - 11
November
21 – 25
November
47
48
49
51
28 November –
2 December
5–9
December
19 -23
December
Topic
Mailing of the protocol, standard operating procedure and
test report to the NRLs
Mailing of the parcels to the NRLs as diagnostic specimens
by door-to-door courier service.
Immediately after arrival of the parcels at the laboratory:
Check for any serious damages (GR QRW DFFHSW GDPDJHG
SDFNDJHV;
Check for completeness;
Remove the electronic temperature recorder from the parcel
(leave it in the plastic bag with labcode) and return it to
CRL-6DOPRQHOOD using the return envelope;
6WRUHWKHGXVWDQGIDHFHVVDPSOHVDWDWƒ&“R&
6WRUHWKHFDSVXOHVDWƒ&“R&.
If you did not receive the parcel at 28 October, do contact the
CRL immediately.
Preparation of:
1. Non selective pre-enrichment medium (see SOP 6.1)
2. Selective enrichment media (see SOP 6.2)
3. Solid selective plating media (see SOP 6.3)
4. Confirmation media (see SOP 6.4)
Performance of the study, following the instructions as given
in the protocol and the SOP of study IX (2005).
Completion of the test report and faxing or e-mailing it to
the CRL. The original test report will be sent to CRL.
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1RYHPEHU
Data input at CRL-6DOPRQHOOD and sending these data by
CRL to NRLs for checking
Checking the results by the National Reference Laboratories
Sending of the final results to the NRLs together with a short
report. As a follow-up, actions will be undertaken for those
NRLs which scored below the average results of all NRLs.
In the time table of the interlaboratory comparison study IX on the bacteriological detection of
6DOPRQHOOD, to be organised in fall 2005, a VWULFWGHDGOLQH for sending the results to the CRL6DOPRQHOOD is indicated.
The reason for setting this strict deadline (on 25 November 2005) is that we want to prepare a
short report to inform all NRLs within 1 to 2 months after the study on the overall results. In
earlier studies the NRLs received only their own results immediately after the study. The
2
information on how they performed in comparison with the other NRLs was given ca half a
year after the study (at the workshop). This is considered very late and with this short report in
December we try to improve the information on the study. We will start the first overall
analyses immediately after the deadline. Results which will be received after the deadline can
not be used in the analyses for the short report
It may still be possible to use late results in the analyses for the final report but results
received after publishing the short report will not be incorporated in the final report.
&KDQJHV15/8QLWHG.LQJGRP
Contact person Dr. Stanley McDowell moved to other work areas and
the new contact person of NRL United Kingdom will be:
Dr. Sam Strain, email: [email protected]
3
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Literature related to 6DOPRQHOOD and directive 92/117/EEC
WinSPIRS week 14 to 26, 2005
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:25/'6328/75<6&,(1&(-2851$/0$5 Intact eggs can become contaminated with Salmonella as a result of infections of the
reproductive tissues of the laying hens but also by penetration through the shell. In
this paper, the penetration of Salmonella through the shell of hen eggs is reviewed. A
description is given of the advantages and disadvantages of the various methods used
to study bacterial penetration through the shell and membranes. The possibility of
Salmonella contamination of the shell after lay is included because shell
contamination is the first requisite for penetration. Various factors affect the
probability of bacterial penetration. Both the intrinsic and extrinsic factors are
highlighted. For the extrinsic factors, the influence of bacterial strain and number of
organisms, temperature, moisture and immersion and storage conditions on the
probability of Salmonella penetration through the shell is described. With regard to
intrinsic factors, the presence of cuticle, shell characteristics (shell quality, porosity,
shell defects) and membrane properties are summarized.
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An epidemiological investigation of a calf rearing premises and a closely associated
dairy herd was carried out after the isolation of Salmonella enterica serovar Paratyphi
B variant Java phage type 3b variant 2 from clinically diseased calves on the
premises. The isolate was resistant to ampicillin, chloramphenicol, streptomycin,
sulphonamides, tetracyclines, trimethoprim and cefoperazone. The organism was
widespread on the calf unit and was also recovered from the dairy premises, mainly
from groups of weaned calves. The investigation was extended to 10
epidemiologically linked farms but no S Java was isolated from any of the 40 to 60
samples collected from each premises. Molecular studies showed that the S Java
isolates were genetically most similar to isolates from cases of human disease
associated with ornamental fish tanks or feed. Long PCR and resistance gene
profiling identified a resistance island which was indistinguishable from the human
’fish tank’ strain of S Java and animal and human epidemic strains of S Typhimurium
DT104. The isolates were clearly distinguished from multi-resistant S Java strains
commonly associated with continental poultry. This is the first report of S Java with
this resistance pattern in Great Britain.
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Eggs that harbor Salmonella in their edible contents pose a significant risk of
transmitting disease to consumers. Although Salmonella deposition inside yolks does
not usually occur at a high frequency in naturally contaminated eggs, bacterial
penetration through the vitelline membrane could lead to rapid and extensive
multiplication in the nutrient-rich yolk contents. The present study used an in vitro
egg contamination model to assess the ability of Salmonella strains to penetrate the
vitelline membrane and multiply inside yolks. An S. enteritidis strain and 2
Salmonella heidelberg strains, initially inoculated onto the outside of the vitelline
membrane, were able to enter the yolk contents (at frequencies ranging from 10 to
25% of experimentally contaminated eggs) during 24 h of incubation at 30 degrees C.
Variants of these parent strains, obtained by in vivo passage into eggs laid by infected
hens, penetrated the yolk membrane at significantly higher frequencies. These results
demonstrate that pathogens such as S. enteritidis and S. heidelberg can penetrate into
and begin to multiply inside the yolks of contaminated eggs during the first day of
storage at warm temperatures.
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Worldwide, the use of antimicrobials in food production has been associated with
drug resistance in foodborne pathogens such as Salmonella. However, little is known
about the efficaciousness of fluorequinolone treatment on Salmonella Typhimurium
T104 infections in pig breeding herds. A combined eradication procedure with
enrofloxacin application on sows and piglets, feeding of encapsulated organic acids
to sows, disinfection with peracetic acid, separation of the growers from the sows and
serological discrimination using a new whole-cell-based enzyme-linked
immnosorbent assay (ELISA) was evaluated for the suitability to eradicate and to
control endemic S. Typhimurium DT104 infections in a closed herd. Thirty-seven
sows and their piglets were treated everyday from day 14 ante partum until the day of
weaning. Eighteen sows and their piglets served as controls. From the first day of life
until day 168 after birth, faecal samples (n=1671) of all piglets were analysed for
Salmonella shedding. In parallel, systemic antibody responses were monitored by
whole-cell-based isotype-specific ELISA systems. From birth to weaning the
prevalence in both groups was between 2% and 9%. After weaning, intermittent
shedding could be observed in both groups, and salmonellae could be found in up to
7.7% of the faecal samples. As a result, a dramatic increase in Salmonella-infected
growers was observed, as of day 115 after birth, 47.4% of the animals of the treated
group were tested positive for S. Typhimurium. Our results indicate that despite longterm antibiosis treatment and optimized hygiene measures, shedding of S.
Typhimurium by the sows and the Subsequent infection of their offspring could not
be effectively prevented. Although it could be not shown that elimination of S.
Typhimurium DT104 infection was achieved, the disinfection procedures described
and the diagnostic test used are effective instruments to decrease the Salmonella load
and to identify individual infected animals. Both of these are important factors for an
improved consumer protection.
5
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The diagnostic accuracy of a PCR used to identify horses shedding Salmonella spp.
in their feces during hospitalization was estimated, relative to bacterial culture of
serially collected fecal samples, using longitudinal data. Five or more fecal samples
were collected from each of 116 horses admitted as inpatients, for reasons other than
gastrointestinal disease, between July 26, 2001 and October 25, 2002. All 873 fecal
samples collected were tested with a PCR based on oligonucleotide primers defining
a highly conserved segment of the histidine transport operon gene of Salmonella
typhimurium, and each sample was cultured for Salmonella spp. One or more
samples from 87 (75%) horses were PCR positive, and Salmonella was cultured from
1 or more samples from 11 (9.5%) horses. All culture-positive horses had at least 1
PCR-positive result, whereas only 29 (28%) culture-negative horses were PCR
negative on all fecal samples tested. The PCR was most specific, relative to bacterial
culture of serially collected fecal samples, when used to test samples from
Quarterhorse or breeds other than Thoroughbred or Standardbred, or from clinical
(vs. healthy, accompanying horses) cases. Overall, the PCR had the greatest
agreement (70%), compared with bacterial culture of serially collected fecal samples,
using a cutoff of 2 or more positive PCR test results to define a Salmonella-positive
horse. The reasons why some fecal samples, from which Salmonella organisms
cannot be isolated, are PCR positive need to be determined before the PCR can be
incorporated into Salmonella surveillance programs for hospitalized equine
populations.
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In 2003, 14 cases of multidrug-resistant Salmonella Newport infections were
reported. This is the first documented foodborne outbreak of multi-resistant S.
Newport in France. The bla(CMY) gene was present in all isolates. All cases reported
having eaten horse meat from a common wholesaler. The country of origin of the
imported meat could not be identified.
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The Finnish salmonella control program (FSCP) for beef production is based on both
randomized and selective testing of herds and animals. Sampling of individual
animals in abattoirs is randomized. Herds are selectively tested for salmonella on the
basis of clinical symptoms and/or other factors. Risk assessment of FSCP is
inherently difficult due to the complexity of the complete data set, especially if the
detailed labeling of the testing types is lost. However, for a risk assessment of the
whole production chain, methods for exploiting all available data should be
considered. For this purpose, a hierarchical Bayesian model of true salmonella
6
prevalence was constructed to combine information at different levels of aggregation:
herds in geographical regions and individual animals arriving for slaughter. The
conditional (municipality specific) probability of selection of a herd for testing was
modeled given the underlying true infection status of the herd and information about
the general sampling activity in the specific region. The model also accounted for the
overall sensitivity of the sampling methods, both at the herd and at the animal level.
In 1999, the 95% posterior probability intervals of true salmonella prevalence in the
herd population, in individual cattle, and in slaughter animal populations were
[0.54%, 1.4%] (mode 0.8%), [0.15%, 0.39%] (mode 0.2%), and [0.12%, 0.36%]
(mode 0.2%), respectively. The results will be further exploited in other risk
assessments focusing on the subsequent parts of the beef production chain, and in
evaluation of the FSCP.
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Foodborne salmonellosis continues to be a public health issue of considerable
concern. Animal feed has been a major link in pre-harvest food animal production.
Although monitoring systems and control measures are available to limit Salmonella
spp. contamination on animal feeds detection methodology is relatively time
consuming in the context of time inputs for feed processing and mixing. Current
cultural methods of Salmonella spp. detection in feeds require several days for
confirmation. This amount of time represents significant problems if control
measures are to be effectively implemented in a fashion that keeps feed processing
costs low. Molecular methods offer improved sensitivity and potential reduction in
assay time. In particular, several commercial polymerase chain reaction (PCR)
assays, and combined PCR-hybridization assays have been suggested as possible
means to implement more rapid detection of Salmonella spp. extracted from animal
feeds. It has now become possible to rapidly detect and confirm the presence of
foodborne Salmonella spp. in feed matrices by commercial amplification detection
systems. The primary challenges remaining are to develop more reliable recovery and
extraction procedures for routine processing of samples from a wide variety of feed
matrices and apply molecular techniques for assessing physiological status of
Salmonella spp. contaminants in animal feeds.
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The purpose of this study was to examine potential prophylactic effects of a coarse
feed structure and/or potassium diformate (KDF as feed additive) addition to diets on
the microflora of the digestive tract in reared piglets experimentally infected with
Salmonella Derby. The results show that coarse grinding as well as KDF addition are
able to influence positively the intestinal flora and are capable to reduce Salmonella
excretion of infected piglets. Coarse grinding of main ingredients (e.g. cereals) led to
an increased number of lactobacilli as well as to higher counts of Gram-positive
7
coccoid bacteria in the colon chyme, while KDF resulted in a tendency towards lower
counts of Escherichia coli within the digestive tract. Moreover, a combination of both
treatments influenced the composition of the gastrointestinal flora quite positively.
Furthermore, the combination of these dietetic measures resulted in a reduced
Salmonella excretion rate, shorter Salmonella shedding period and a reduced
translocation of Salmonella within the infected piglets. The positive effects of
combining both treatments led to a significantly reduced spreading of Salmonella
within the group of pigs, which might be used to diminish Salmonella prevalence in
pig production.
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Objective: A survey of Salmonella contamination of poultry droppings used as
manure, retail fresh beef, fresh beef retailers’ aprons and fresh beef retail tables, was
carried, out.
Design: A total of 120 samples of poultry droppings collected from five poultry
farms, 96 fresh beef samples, 96 beef retailers’ aprons and 96 fresh beef retail tables
were examined for the presence of Salmonella species.
Results: Different Salmonella serotypes were isolated from all the sources.
Salmonella paratypi A had an isolation rate of 12.5% from poultry droppings, 4.2%
from fresh beef, and 2.1% and 4.2% from meat retailers’ aprons and tables
respectively. Other serotypes isolated from the sources included S. typhimurium, S.
enteriticlis, S. gallinarum, S. pullorum, S. typhi and S. agama. Salmonella typhi was
not isolated from poultry droppings throughout the survey.
Conclusion: There is a need to create more environmental and personal hygiene
awareness among the Nigerian populace, especially among food vendors. (c) 2004
International Society for Infectious Diseases. Published by Elsevier Ltd. All rightsreserved.
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Salmonella-induced enterocolitis is the leading food-borne illness with a lethal
outcome and causes millions of cases of gastroenteritis each year. We examined
genomic variation among 12 environmental, veterinary, and clinical Salmonella
enterica serovar Dublin, Agona, and Typhimurium strains isolated in Ireland between
2000 and 2003, as well as two clinical isolates from Canada and four archival
isolates, which belonged to serovars Dublin and Agona. Using DNA-DNA
hybridization to a microarray consisting of most of the predicted protein-encoding
sequences of the S. enterica serovar Typhimurium LT2 genome, we identified a
number of genomic regions that were absent in one or more serovars. The 34
genomic regions encoded proteins involved in sugar metabolism, transport, fimbrial
and phage biogenesis, and transcriptional regulation, as well as inner and outer
membrane-associated proteins. Two of the four prophages identified in strain LT2,
8
prophages Gifsy-1 and Gifsy-2, were present in all six serovar Typhimurium strains
examined. Prophage Fels-1 was absent from all 18 isolates examined, and Fels-2 was
completely absent from the serovar Typhimurium isolates and the Salmonella
Reference Collection B serovar Dublin strain Du2. All five Salmonella pathogenicity
islands were present in all isolates. Plasmid pSLT was absent from all serovar Agona
isolates, and only homologues of the spv genes were present in eight of the nine
serovar Dublin strains. Only limited intraserovar diversity was found among the nine
serovar Dublin, three serovar Agona, and six serovar Typhimurium isolates examined
even though these isolates had extensive geographic, temporal, and source
differences.
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Typhoid fever is a significant cause of morbidity and mortality worldwide, causing
an estimated 16 million cases and 600,000 deaths annually. Although overall rates of
the disease have dramatically decreased in the United States, the number of travelrelated infections has increased in recent decades. Drug resistance among Salmonella
enterica serotype Typhi strains has emerged worldwide, making antimicrobial
susceptibility testing an important function in public health laboratories. Pulsed-field
gel electrophoresis (PFGE) subtyping of food-borne and waterborne pathogens has
proven to be a valuable tool for the detection of outbreaks and laboratory-based
surveillance. This retrospective study examined the distribution of PFGE patterns of
S. enterica serotype Typhi isolates from patients with a history of international travel.
Isolates were collected as part of a passive laboratory-based antimicrobial
susceptibility surveillance study. Isolates were PFGE subtyped by using the
restriction enzyme XbaI to restrict the total genomic DNA. Isolates indistinguishable
with XbaI were further characterized using the restriction enzyme BlnI. A total of
139 isolates were typed, representing travel to 31 countries. Restriction fragment
patterns consisted of 14 to 18 fragments ranging in size from 580 to 40 kbp. Seventynine unique PFGE patterns were generated using XbaI. Isolates from the same
geographic region did not necessarily have similar PFGE patterns. Of the 139
isolates, 46 (33 %) were resistant to more than one antimicrobial agent (multidrug
resistant [MDR]). Twenty-seven (59 %) of 46 MDR isolates had indistinguishable
PFGE patterns with both XbaI and BlnI. It appears that MDR S. enterica serotype
Typhi has emerged as a predominant clone in Southeast Asia and the Indian
subcontinent.
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The molecular epidemiology of 98 isolates of Salmonella serovar Agona (n = 27), S.
Montevideo (n = 42) and S. Senftenberg (n = 29) from wild-living gulls, fish-meal
factories, feed factories, humans and domestic animals was investigated using
pulsed-field gel electrophoresis (PFGE) and computerized numerical analysis. Two
9
of the S. Agona profiles were identified both in gulls and in two of the factories. In
addition, one of these profiles was detected in two infected poultry farms. Two of the
S. Montevideo profiles were also identified both in gulls and in two of the factories,
and one of these profiles was observed in a human isolate. Four factories shared an
identical S. Senftenberg profile. The S. Senftenberg profile found in gulls was not
identified in any other source investigated. The presence of isolates with identical
PFGE profiles indicates potential epidemiological links between different factories,
as well as between gulls and factories.
1LHOVHQ/5 (UVEROO$. )DFWRUV DVVRFLDWHG ZLWK YDULDWLRQ LQ EXONWDQNPLON 6DOPRQHOOD
'XEOLQ (/,6$ 2'& LQ GDLU\ KHUGV 35(9(17,9(9(7(5,1$5<0(',&,1( 0$< Our objective was to determine factors that contribute to variation in bulk-tank-milk
Salmonella Dublin enzyme-linked immunosorbent assays (ELISA) corrected opticdensity measurements (ODC%) in dairy herds. We constructed hierarchical mixed
models with repeated bulk-tank-milk ELISA ODC% in 31 Danish dairy herds. Four
models included different combinations of explanatory factors, and we compared
how well these models described the variation in the data. Herd was included as a
random effect nested within Salmonella status and barn type.
Detection of Salmonella Dublin or Salmonella Typhimurium by bacteriological
culture of individual faecal samples or of slurry samples was associated with higher
bulk-tank-milk ELISA ODC%, as was apparent Salmonella prevalence, the mean
ELISA ODC% or mean-yield-corrected ELISA ODC% in milk samples collected
from all individual cows. However, combinations of risk factors that included
number or prevalence of cows with a very high ELISA ODC% provided better
models, indicating that the effect of the cow-level explanatory variables on the bulktank-milk ELISA ODC% was related to the activity of the infection in the herd. Barn
type (loose housing or tie stalls) was not associated with the variation in bulk-tankmilk ELISA ODC% in these models, which might be useful in planning of
surveillance programs and intervention strategies. (c) 2005 Elsevier B.V. All rights
reserved.
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Salmonella enterica serovar Pullorum causes persistent infections in laying hens.
Splenic macrophages are the main site of persistence. At sexual maturity, numbers of
bacteria increase and spread to the reproductive tract, which may result in vertical
transmission to eggs or chicks. In this study we demonstrate that both male and
female chickens may develop a carrier state following infection but that the increases
in bacterial numbers and spread to the reproductive tract are phenomena restricted to
hens, indicating that such changes are likely to be related to the onset of egg laying.
The immunological responses during the carrier state and through the onset of laying
in hens were determined. These indicate that chickens produce both Immoral and Tcell responses to infection, but at the onset of laying both the T-cell response to
Salmonella and nonspecific responses to mitogenic stimulation fall sharply in both
10
infected and noninfected birds. The fall in T-cell responsiveness coincided with the
increase in numbers of Salmonella serovar Pullorum and its spread to the
reproductive tract. Three weeks after the onset of egg laying, T-cell responsiveness
began to increase and bacterial numbers declined. Specific antibody levels changed
little at the onset of laying but increased following the rise in bacterial numbers in a
manner reminiscent of a secondary antibody response to rechallenge. These findings
indicate that a nonspecific suppression of cellular responses occurs at the onset of
laying and plays a major role the ability of Salmonella serovar Pullorum to infect the
reproductive tract, leading to transmission to eggs. The loss of T-cell activity at the
point of laying also has implications for Salmonella enterica serovar Enteritidis
infection and transmission to eggs, along with its control by vaccination offering a
"window of opportunity" in which infection may occur.
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The genetic diversity of the Derby serotype of Salmonella enterica in Spain was
examined by pulsed-field gel electrophoresis (PFGE). Out of 24 identified PFGE
profiles, a major clone was detected in 19% of strains from humans, 52% from food,
and 62% from swine. This clone (clone 1) was isolated from pork products,
suggesting swine as its source.
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Infection with Salmonella Typhimurium is seldom associated with clinical disease in
pigs. However, control is important as the public is concerned about the human
health impact. The producers, the abattoirs and the authorities are interested in
implementing procedures to mitigate this risk. To evaluate the effect of different
procedures, a stochastic risk model was developed to simulate the prevalence of
Salmonella infection during the production process from the live pig on the farm, to
the final carcass. This paper describes the model and findings of simulating different
control scenarios. The variables with maximum effect on the Salmonella prevalence
on the final carcass were (1) number of herds with a high prevalence of Salmonella,
(2) singeing efficiency, (3) contamination and cross-contamination at degutting and
(4) cross-contamination during handling. However, improvement of any single factor
in isolation had a limited impact upon the level of contamination. The largest
reduction was observed when several factors were improved concurrently. (c) 2005
Elsevier B.V. All rights reserved.
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Today the latent salmonellosis presents the most frequent infection with salmonellae
in pigs. Approximately 20% of food related human infections with salmonellae can
be attributed to the pig. The control of salmonella infection is an important task of
11
the veterinarian practitioner and an effective contribution to the protection of
consumers. The salmonellae monitoring established in Germany on a voluntary basis
within the quality assurance system "QS" for pigs provides the veterinarian with
important data concerning the salmonellae status on farms. Without doubt these data
must be used in order to establish a strategic zoonosis control programme. A
practical example may demonstrate the possibilities for controlling latent salmonella
infection in a pig herd.
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The pork tonsils are an important carrier of Salmonella, which could be involved in
the contamination of pork products during the slaughter process.
This paper reports a 23S rRNA fluorescent in situ hybridization (FISH) method used
as a rapid screening tool for Salmonella detection in tonsils of slaughtered pigs and
its comparison with the conventional culture method. As a rapid screening method,
the FISH technique would reduce the high volume of negative samples that are
routinely analyzed, indicating presumptive positive samples in a real and practical
time.
The use of a Sal3 probe allowed the rapid (7 h) Salmonella detection in 16 (34%) of
47 naturally contaminated tonsils, without pre-enrichment. Salmonella was isolated
by the culture method in six samples that were also FISH-positive samples, and FISH
failed to identify only one of the culture-positive samples.
The results indicate the potential of this technique as a rapid screening method for
detecting Salmonella in tonsils from pork slaughtered for consumption
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In the USA, control of food-borne salmonellosis associated with meat consumption
has been predominantly focused at slaughter and processing. It is expected that
standards at slaughter and processing will become more stringent, creating pressure
to reduce prevalence of Salmonella-positive food animals through on-farm
interventions. The aim of this study was to compare traditional fecal culture and the
Danish Mix-ELISA (DME) for determination of Salmonella prevalence pre-harvest
in swine. In Trial 1, five cohorts of individually identified pigs were longitudinally
sampled during the growing period to compare the kinetics of prevalence as
estimated by fecal culture and the DME. In Trial 2, the correlation between fecal
prevalence and seroprevalence was estimated pre-marketing in 49 groups of pigs. In
Trial 1, fecal prevalence and seroprevalence showed similar kinetics, with a tendency
of a higher 013% cut-off to more closely approximate fecal prevalence. In Trial 2,
correlations between fecal culture and the DME were 0.40, 0.36, 0.43, and 0.43 (p <
0.001) for OD% cut-offs >= 10, 20, 30, and 40, respectively. Based on these results, a
higher OD% cut-off would be recommended if more approximate estimation of fecal
prevalence is desired and longitudinal sampling would be suggested for evaluating
the impact of on-farm interventions for Salmonella reduction whether utilizing fecal
12
culture or the DME. Further evaluation of the impact of Salmonella serovar present
on farms on seroprevalence and the relationship of on-farm seroprevalence with food
safety risk are needed prior to utilizing the DME for pre-harvest Salmonella
diagnostics in the US swine herd. (c) 2005 Elsevier B.V. All rights reserved.
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A putative link between Salmonella persistence in the agricultural sector and
resistance to disinfectants has been sparsely investigated. Therefore, minimum
inhibitory concentration (MIC) tests against five disinfectants commonly used in
poultry premises (formaldehyde, glutaraldehyde/benzalkonium chloride compound,
oxidising compound, tar oil phenol, iodophor) were performed on 286 Salmonella
isolates, including 256 from Danish broiler houses, altogether representing nine
serotypes. Six of these isolates were used for adaptation and de-adaptation studies
involving the five disinfectants. Amongst 60 of these isolates selected for growth
studies in cyclohexane (possibly associated with up-regulated efflux), only one
isolate grew. From this isolate and the six isolates used in the adaptation and deadaptation studies, mutants highly resistant to triclosan (a disinfectant linked with
mar-type resistance) were selected. In addition, adaptation and de-adaptation studies
with triclosan were performed.
For the 286 isolates, the small variations in MICs could not be associated with
Salmonella persistence in Danish broiler houses or previous use of relevant
disinfectants. Adaptation and de-adaptation did not alter MICs to the five farm
disinfectants. Compared to the parent isolates, MICs for the triclosan adapted and deadapted isolates and the triclosan mutants were significantly increased to triclosan,
but not to the five disinfectants. Moreover, most of the triclosan adapted and deadapted isolates grew in cyclohexane. Thus, there was no correlation between
triclosan and cyclohexane resistance on one hand and resistance to the five
disinfectants on the other, suggesting that triclosan resistance is not linked with
resistance to these disinfectants. (c) 2005 Elsevier B.V. All rights reserved.
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A cross-sectional study was undertaken to determine the prevalence and distribution
of Salmonella serotypes from apparently healthy slaughtered sheep and goats from
November 2002 to May 2003 at Debre Zeit abattoir, Ethiopia. From a total of 107
slaughtered animals (60 goats and 47 sheep), 642 samples (feces, mesenteric lymph
node, spleen, liver, abdominal and diaphragmatic muscle) were collected aseptically.
Of 107 animals examined one or more of the samples were Salmonella positive in 13
(12.1%) of the animals of which 10 (9.3%) were goats and 3 (2.8%) were sheep.
Thirty-three (5.1 %) Salmonella isolates were collected from the 642 samples
analyzed. Of the 282 and 360 different samples analyzed from slaughtered sheep and
goats, 10 (3.5%) and 23 (6.4%) were Salmonella positive, respectively. Salmonella
was detected in 2 (1.9%), 3 (2.8%), 5 (4.7%), and 7 (6.5%) of each 107 samples of
spleen, feces, liver, and mesenteric lymph nodes, respectively. Of the diaphragmatic
13
and abdominal muscles examined, 8.4% (9/107) and 6.5% (7/107) were Salmonella
positive. Out of the 33 Salmonella isolates, nine different serotypes were identified of
which S. infantis was predominant (45.5%) followed by S. butantan (24.2%), S.
braenderup and S. kingabwa (each 6.1%). Other serotypes identified were S.
zanzibar, S. anatum, S. typhimurium, S. kottbus, and S. hadar (each 3%). Results of
the present study indicated that Salmonella is common in apparently healthy
slaughtered sheep and goats. It also showed the presence of a wide range of
Salmonella serotypes in sheep and goats, which are of veterinary and public health
significance in Ethiopia. (c) 2004 Elsevier B.V. All rights reserved.
0D]XUHN- +ROEHUW/ 3DUULVK0. 6DOHKL( 5DZ HJJV /HVVRQV OHDUQHG IURP DQ
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-2851$/2)38%/,&+($/7+0$1$*(0(17$1'35$&7,&( 0$<-81 Objectives: We investigated a salmonellosis outbreak related to a cafeteria to
determine its extent and to identify illness risk factors. Methods: A case was defined
as isolation of Salmonella Group D or serotype Enteritidis from the stool of a person
who ate at the cafeteria during June 22-July 10, 2003, and developed diarrhea in 3
days or less. Food histories of case patients (n = 11) were compared with those of
their well meal companions (n = 16). Results: Consumption of coconut meringue pie
was associated with illness (odds ratio = 150.0; 95% confidence interval = 6.46901.4). Meringue was made with raw shell eggs and was baked to an internal
temperature of 83 degrees F. Conclusions: Restaurant operators and public health
officials should be alert for recipes containing raw shell eggs. Food service operators
should use pasteurized egg products in meringue recipes if meringue will not be
cooked to the required temperature of 155 degrees F For clarification purposes,
policy makers should consider adding "meringue" to the Food Safety Rule that lists
foods in which pasteurized eggs should be substituted for raw shell eggs.
5RVV,/+HX]HQURHGHU0:'LVFULPLQDWLRQZLWKLQSKHQRW\SLFDOO\FORVHO\UHODWHGGHILQLWLYH
W\SHV RI 6DOPRQHOOD HQWHULFD VHURYDU W\SKLPXULXP E\ WKH PXOWLSOH DPSOLILFDWLRQ RI SKDJH
ORFXV W\SLQJ WHFKQLTXH -2851$/2)&/,1,&$/0,&52%,2/2*< $35 Multilocus sequence typing (MLST) is a relatively new high-resolution typing system
employed for epidemiological studies of bacteria, including Salmonella.
Discrimination based on MLST of housekeeping genes may be problematical, due to
the high identity of gene sequences of closely related Salmonella species. The
presence of genomic sequences derived from stable temperate phages in Salmonella
offers an alternative for MLST of Salmonella. We have used MLST of prophage loci
in Salmonella enterica serovar Typhimurium to discriminate closely related isolates
of serovar Typhimurium. We have compared these results to MLST of five
housekeeping genes, as well as pulsed-field gel electrophoresis (PFGE). The
presence or absence of prophage loci in the 73 serovar Typhimurium isolates tested,
as well as allelic variation as detected by sequencing, provided greater discrimination
between isolates than either MLST of housekeeping genes or PFGE. Amplification of
prophage loci alone separated serovar Typhimurium isolates into 27 groups
comprising multiple isolates or individual strains. Sequencing of isolates found
14
within the clusters separated isolates even further. By contrast, PFGE could only
divide the 73 isolates into five distinct groups. MLST using housekeeping genes did
not provide any significant separation of isolates in comparison to amplification or
MLST of prophage loci. The results demonstrate that the amplification and
sequencing of prophage loci provides a high-resolution, objective method for the
discrimination of closely related isolates of serovar Typhimurium. It is proposed that
multiple amplification of phage locus typing may provide sufficient discrimination
for epidemiological purposes without recourse to MLST.
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A formalized Salmonella Toxoid vaccine against avian salmonellosis was developed
from toxins of a strain of Salonella Weltevreden (BM/1643) and was evaluated for
the efficacy in respect of protection against homologous and heterologous challenges
in poultry. The birds were vaccinated subcutaneously at 3rd and 5th week of age. The
immunized birds was challenged intraperitoneally and orally with homologous (S.
Weltevreden) and heterologous (S. Typhimurium and S. Gallinarum) serovars at 4th
week post-primary vaccination. The vaccine afforded 100% protection with S.
Weltevreden and S. Typhimurium respectively. However, the cross protection was
83.33% with S, Gallinarum. The protective index of the vaccine was 94.00%. The
challenged immunized birds shed less salmonellae than the non-immunized birds.
The peak serum antibody titre was observed at 4th week post-primary vaccination,
which declined gradually thereafter till the 9th week of observation.
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Salmonella Scriftenberg was detected in the coastal areas of Galicia (NW Spain) in
1998, where it remained the predominant serovar for the next four years. Although
the overall incidence of this serovar in the zone was lower than 1%, contamination by
Salmonella serovar Senftenberg was located in very specific areas of the Ria de
Arousa, where it persisted for more than five years. A total of 60 Salmonella serovar
Senftenberg isolates, originating from surveillance activities in marine environments,
was subjected to molecular characterization by pulsed-field gel electrophoresis
(PFGE). PFGE analysis of the marine isolates allowed the differentiation of three
main PFGE types, which contained the majority of the isolates, each type showing a
specific spatial distribution in the coastal waters. The most prevalent pulse types
persisted for more than four years, emphasizing their capacity to adapt and survive in
marine environments. Using PFGE analysis, marine isolates were compared with
Salmonella serovar Senftenberg isolates from neighbouring mussel-processing
facilities and to other epidemiologically unrelated isolates from human, animal and
feed sources. Comparison of the restriction patterns showed that indistinguishable
PFGE types were present in the isolates from mussel-processing facilities and their
surrounding marine areas, suggesting that the mussel processing is the main source
for contamination with Salmonella Senftenberg in these marine environments. A
15
molecular fingerprinting relationship was established between three shellfish isolates
and a human isolate, which could be considered as preliminary evidence of infection
caused by Salmonella Senftenberg associated with molluscan consumption. (c) 2004
Federation of European Microbiological Societies. Published by Elsevier B.V. All
rights reserved.
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Since Turkey currently lacks a national reference center for Salmonella infections,
the present study was conducted to document the distribution of serotypes and
antimicrobial resistance patterns among Salmonella enterica isolates recovered from
clinical samples in ten Turkish provinces over a 2-year period. Among the 620
Salmonella enterica isolates recovered between 1 July 2000 and 30 June 2002, strains
belonging to the serotypes Enteritidis (47.7%), Typhimurium (34.7%), Paratyphi B
(6.0%), Typhi (2.9%), Paratyphi A (0.2%) and serogroup C (8.5%) were found.
Resistance to multiple antimicrobial agents was particularly high among Salmonella
Typhimurium isolates (76.7%), and resistance or decreased susceptibility to
ciprofloxacin (MIC >= 0.125 mg/l) was demonstrated in Salmonella Paratyphi B,
Salmonella Typhimurium and Salmonella Enteritidis strains. All of the Salmonella
Typhi isolates were susceptible to ciprofloxacin. The results indicate that decreased
susceptibility to ciprofloxacin is an emerging problem in Salmonella enterica in
Turkey, particularly in multiresistant strains.
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Quinolone-resistance determining region (QRDR) of Gyrase A gene was sequenced
in 54 Salmonella strains of pig origin that have different quinolone-resistance
patterns. Those strains accounted for 12 different serotypes. Mutations at Ser83 or
Asp87 were predominant in the studied isolates. However, for serotypes Anatum and
Virchow, resistance to quinolones seemed to be linked to specific mutations, namely,
Ser83 -> Tyr and Ser83 -> Phe, respectively. Other mutations found in different
positions did not seem to have clinical significance except for changes at Asp82. (c)
2005 Elsevier B.V. All rights reserved.
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To study the effect of processing and storage parameters on the survival of
Salmonella on fresh Italian parsley, parsley bunches were dipped for 3 or 15 min in
suspensions that were preequilibrated to 5, 25, or 35 degrees C and inoculated with
Salmonella transformed to express enhanced green fluorescent protein. Loosely
attached and/or associated, strongly attached and/or associated, and internalized
16
and/or entrapped Salmonella cells were enumerated over 0, 1, and 7 days of storage
at 25 degrees C and over 0, 1, 7, 14, and 30 days of storage at 4 degrees C using
surface-plating procedures. Leaf sections obtained from samples after 0, 1, and 7
days of storage were examined using confocal scanning laser microscopy.
Temperature of the dip suspension had little effect on the attachment and survival of
Salmonella cells on parsley. Regardless of the temperature or duration of dip,
Salmonella was internalized. Immersion for longer times resulted in higher numbers
of attached and internalized cells. Microscopic observations supported these results
and revealed Salmonella cells near the stomata and within cracks in the cuticle.
Storage temperature had the greatest impact on the survival of Salmonella cells on
parsley. When stored at 25 degrees C, parsley had a shelf life of 7 days, and
Salmonella populations significantly increased over the 7 days of storage. For parsley
stored at 4 degrees C, numbers of Salmonella cells decreased over days 0, 1, and 7.
After 7 days of storage, there were no viable internalized Salmonella cells detected.
Storage temperature represents an important control point for the safety of fresh
parsley.
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Salmonella serotypes are important foodborne pathogens of humans that can be
acquired through consumption of contaminated meat and dairy products. Salmonella
infection also can be a significant animal health issue. As part of a national study of
U.S. dairy operations conducted between March and September 2002, fecal samples
were collected from representative cows in 97 dairy herds in 21 states and were
cultured to determine the prevalence of Salmonella shedding. Salmonella was
recovered from the feces of at least one cow in 30.9% of the herds. Overall, 7.3% of
fecal samples were culture positive for Salmonella. The three most frequently
recovered serotypes were Salmonella Meleagridis (24.1%), Salmonella Montevideo
(11.9%), and Salmonella Typhimurium (9.9%). The susceptibilities of Salmonella
isolates recovered were determined using a panel of 16 antimicrobial drugs.
Salmonella isolates recovered from dairy cows had relatively little resistance to these
antimicrobial agents; 83.0% of the isolates were susceptible to all antimicrobials
tested. This study provides updated information on the prevalence and susceptibility
patterns of Salmonella in dairy herds and on cow and herd characteristics. These data
contribute to our understanding of the ecology of Salmonella in the dairy farm
environment.
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Studies were conducted to compare the effect of sodium hypochlorite (SH) versus
monochloramine (MON) on bacterial populations associated with broiler chicken
17
carcasses. In study 1, nominal populations (6.5 to 7.5 log CFU) of Escherichia coli,
Listeria monocytogenes, Pseudomonas fluorescens, Salmonella serovars, Shewanella
putrefaciens, and Staphylococcus aureus were exposed to sterilized chiller water
(controls) or sterilized chiller water containing 50 ppm SH or MON. SH at 50 ppm.
eliminated all (6.5 to 7.5 log CFU) viable E. coli, L. monocytogenes, and Salmonella
serovars; 1.2 log CFU of P. fluorescens; and 5.5 log CFU of S. putrefaciens. MON
eliminated all (6.5 to 7.5 log CFU) viable E. coli, L. monocytogenes, S. putrefaciens,
and Salmonella serovars and 4.2 log CFU of P. fluorescens. In study 2, chicken
carcasses were inoculated with P. fluorescens or nalidixic acid-resistant Salmonella
serovars or were temperature abused at 25 degrees C for 2 It to increase the
populations of naturally occurring E. coli. The groups of Salmonella serovarinoculated or temperature-abused E. coli carcasses were immersed separately in pilotscale poultry chillers and exposed to tap water (controls) or tap water containing 20
ppm SH or 20 ppm MON for 1 h. The P. fluorescens-inoculated group was immersed
in pilot-scale poultry chillers and exposed to tap water (controls) or tap water
containing 50 ppm SH or 50 ppm MON for I h. Carcasses exposed to the SH
treatment had nominal increases (0.22 log CFU) in E coli counts compared with
controls, whereas exposure to MON resulted in a 0.89 log reduction. Similarly,
average nalidixic acid-resistant Salmonella serovar counts increased nominally by
34% (41 to 55 CFU/ml) compared with controls on carcasses exposed to SH,
whereas exposure to MON resulted in an average nominal decrease of 80% (41 to 8
CFU/ml). P. fluorescens decreased by 0.64 log CFU on carcasses exposed to SH and
decreased by 0.87 log CFU on carcasses exposed to MON. In study 3, SH or MON
was applied to the chiller in a commercial poultry processing facility. E. coli counts
(for carcass halves emerging from both saddle and front-half chillers) and Salmonella
prevalence were evaluated. Data from carcasses exposed to SH during an 84-day
historical (Hist) and a 9-day prepilot (Pre) period were evaluated. Other carcasses
were exposed to MON and tested during a 27-day period (Test). E. coli counts for
samples collected from the saddle chiller were 25.7, 25.2, and 8.6 CFU/ml for Hist,
Pre, and Test, respectively. E. coli counts for samples collected from the front-half
chiller were 6.7, 6.9, and 2.5 CFU/ml for Hist, Pre, and Test, respectively.
Salmonella prevalence was reduced from 8.7% (Hist + Pre) to 4% (Test). These
studies indicate that MON is superior to SH in reducing microbial populations in
poultry chiller water.
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The combined effect of ultrasonic waves and heat treatment applied simultaneously
was evaluated on the survival of strains of Salmonella Senftenberg on shells of intact
eggs. This combined process has a higher killing effect than heat treatment alone.
The decimal reduction times (D-values) were decreased by 65.2 to 11.1% in the
temperature range studied (57.8 to 67 degrees C). In contrast to the effect on
Salmonella enterica serovar Enteritidis in a previous study, thermoultrasonication
had no important advantage for elimination of Salmonella Senftenberg. However,
because 52 degrees C is a nonlethal temperature for Salmonella Senftenberg, the
conditions used for the elimination of Salmonella Enteritidis (52 degrees C for 12
min) in the previous study would be equivalent to ultrasonic treatment alone in the
18
present study. This thermoultrasonication treatment may result in a 100-fold greater
reduction of Salmonella Senftenberg than that achieved by common in-shell egg
pasteurization (60 degrees C for 3.5 min).
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This study was carried out to determine the prevalence of some virulence
characteristics associated with Salmonella isolates recovered from processed turkey
carcasses in the Midwestern region of the United States. A total of 94 Salmonella
isolates recovered from turkey carcasses from two processing plants (A and 13) were
examined to determine the prevalence of invA, pagC, and spvC genes. Bioassays also
were used to evaluate aerobactin and colicin production. All isolates (100%) were
positive for the presence of invA and pagC but were negative for spvC. Overall, 19. 1
% of all isolates tested were positive for aerobactin production, and 25.5% of all
isolates were positive for colicin. Aerobactin and colicin production differed among
isolates recovered from the two plants; more isolates from plant B produced these
compounds. The Salmonella isolates examined in this study possess significant
potential for causing human illness.
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The presence of antiadhesive component(s) in the hen egg yolk against foodborne
pathogens was anticipated from results of a previous animal study conducted by the
authors. The previous work showed egg yolk powder without specific antibodies is
effective in controlling Salmonella enteritidis, Salmonella typhimurium, and
Escherichia coli 0157:1-17 colonization in laying hens. Therefore, this study was
necessary to locate the activity and identify the effective component(s). In vitro
experiments were conducted using confluent Caco-2 cell monolayers. S. enteritidis,
S. typhimurium, and E coli 0157:H7 were investigated against the various extracted
granule and plasma fractions in three different assays: adhesion elimination, adhesion
prevention, and antimicrobial. This study revealed original findings and identified the
protective yolk fraction against the foodborne pathogens as the granule component,
high-density lipoproteins (HDL). The protective activity conveyed by HDL was
confirmed to remain intact despite peptic and tryptic enzymatic digestion and to have
antiadhesive but not antimicrobial effect.
*RZ6:DOGQHU&5RVV&7KHHIIHFWRIWUHDWPHQWGXUDWLRQRQZHDQLQJZHLJKWVLQDFRZ
FDOIKHUGZLWKDSURWUDFWHGVHYHUHRXWEUHDNRIGLDUUKHDLQFDOYHV&$1$',$19(7(5,1$5<
-2851$/5(98(9(7(5,1$,5(&$1$',(11( 0$< The objective of this study was to describe a multiyear outbreak of calf diarrhea in an
Alberta cow-calf herd and the impact of severe diarrhea on calf productivity. A
19
retrospective analysis was performed through the use of detailed individual animal
records and laboratory reports. The most significant laboratory finding was
Salmonella Typhimurium isolated from the fecal samples of 2 ill members of the
treatment crew and from 3 calves on postmortem examination. In 2002, at the peak
of the outbreak, 90.3% (325/360) of the calves required treatment and 8.9% (32/360)
of the calves died. In 2003, the severity of the problem had declined with only 20.9%
(47/225) of the calves requiring treatment and 3.1% (7/225) of the calves dying. In
both years, the weaning weights of treated calves were significantly reduced
compared with those of nontreated calves. For calves weaned in 2002, after adjusting
for the effect of calf sex, calves treated more than 6 times had 200-day adjusted
weaning weights that were 15.2 kg (95% CI; 5.8 to 24.4 kg) lighter than of those
calves treated only once or not at all (P = 0.0015, n = 321). For calves weaned in
2003, calves were 5.5 kg (95% CI; 0.23 to 10.8, P = 0.04, n = 207) lighter per
treatment day with electrolytes, when calf sex and dam age were controlled for.
Assuming that increased days of treatment or number of treatments is representative
of disease severity, long-term calf performance is negatively affected by severe
calfhood disease. The estimated lost revenue and treatment expenses, excluding the
cost of labor, cow feed, and maintenance, was $22 800 in 2002 and $1589 in 2003.
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An experiment was conducted with three hundred and twenty broiler chickens to
evaluate the influence of supplementation of probiotic on growth, microbiological
status and carcass quality of chickens. The probiotic contained similar proportions of
six strains of variable organisms namely Lactobacillus acidophilus, Lactobacillus
casei, Bifidobacterium bifidum, Aspergillus oryzae; Streptococcus faecium and
Torulops-is sps and was fed at 100mg/kg diet. The body weight and feed conversion
of probiotic fed groups were superior (p < 0.05) compared to the control group in the
4(th), 5(th) and 6(th) weeks. The chickens fed the diet with probiotic had lower (p <
0.05) numbers of coliforms and Campylobacter than chickens fed the control diet. All
chickens’ carcasses on the control diet were positive for Salmonella while only 16 of
the 40 carcasses were positive from chickens fed diets containing probiotic. The leg
and breast meat of probiotic fed chickens were higher (p < 0.05) in moisture, protein
and ash, and lower in fat as compared to the leg and breast meat of control chickens.
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The authors investigated the prevalence of Salmonella spp. in 205 wild birds and
mammals belonging to 45 species during the years 2001 and 2002 in the Basque
Country (Spain). Salmonella was isolated from 16 (7.8%) animals. The prevalence
was 8.5% (7/82) in birds, and 7.2% (9/123) in mammals. Nine serotypes, all of them
belonging to the species Salmonella enterica, were identified: two isolates of
Typhimurium (from 1/3 griffon vultures [Gyps fulvus], and 1/5 sparrowhawks
[Accipiter nisus]); one of 6,14:z4, z23: (subsp. houtenae, 1/1 common kestrel [Falco
tinnunculus]); one of Muenchen (1/1 captive Harris’s hawk [Parabuteo unicinctus]);
20
two of Enteritidis (1/5 tawny owls [Strix aluco], and 1/14 foxes [Vulpes vulpes]);
one of Give, Newport and Umbilo and one untyped islolate (4/22 badgers [Meles
meles]); two of Worthington and one of 38:IV:z35 (subsp. arizonae, 3/40 wild boars
[Sus scrofa]); and three other untyped isolates (1/1 northern fulmar [Fulmarus
glacialis], 1/11 buzzards [Buteo buteo], 1/4 genets [Genetta genetta]). Salmonella
isolation was never associated with macroscopic or microscopic lesions. The results
of this study confirm the importance of wildlife as a Salmonella reservoir and as a
potential risk for humans and livestock.
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DQG VHGLPHQW E\ D QHVWHGPXOWLSOH[ SRO\PHUDVH FKDLQ UHDFWLRQ DVVD\ 5(6($5&+,1
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From 1995 to 2002, 53 serovars of Salmonella were isolated in the Seine estuary
(France). The 3 serovars most frequently found were S. enterica serovar
Typhimurium, S. enterica serovar Infantis and S. enterica serovar Virchow. A nested
multiplex PCR (nm-PCR) assay was developed to detect the presence of Salmonella
in estuarine water and sediment samples. The target gene used was the phase 1
flagellin fliC chromosomal gene, present in all Salmonella serovars. A set of 4
primers was first used to amplify an 890-bp sequence of the fliC gene, and then a
second set of 3 primers was used for the nested PCR. The nmPCR method has been
successfully tested for 2:3 serovars, 13 of which are of epidemiological significance.
The detection limit of the assay, without any pre-enrichment step, was estimated at 1
CFU in deionized water, and at 4-5 CFU in the reaction mixture when tested on
estuarine water seeded with a Salmonella strain. When the nmPCR was used together
with the classical culture method in environmental samples, it gave additional
positive results for 11.3% of the sediment samples and 20% of the water samples
despite a high background of other bacteria. Overall, the results demonstrated that
this molecular approach informed us about the contamination by Salmonella of
estuarine water and sediment samples. Positive amplifications suggested the presence
of Salmonella DNA and could thus provide information about a recent (culturable) or
past (non-culturable, released DNA) contamination of environmental samples by this
pathogenic bacteria. (c) 2005 Elsevier SAS. All rights reserved.
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FKDUDFWHUL]DWLRQ RI D QHZ VHURYDU RI 6DOPRQHOOD ERQJRUL = LVRODWHG IURP D
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Three Salmonella strains isolated from a lizard (Gallotia shnoni) in the ’’ Isla del
Hierro ’’ (Canary Islands, Spain) were serotyped as Salmonella bongori serotype
13,22:z(39):-, which has not been described in the Kauffmann-White scheme of
Salmonella serovars. In order to shed light on the assignment of those strains to the S.
bongori species, several genes were amplified and/or sequenced. The iroB gene has
been reported to be present only in S. enterica, while the invA gene has been
described as being a helpful tool in distinguishing Salmonella from other bacterial
species. Both genes were amplified and, as expected, only invA could be amplified.
The fliC gene, encoding the phase 1 flagellin fljB gene, encoding phase 2 flagellin,
and the gapA gene, which is believed to present polymorphic alleles among different
subspecies, were amplified and sequenced. The sequence obtained from fliC(z39)
21
matched with the sequences fliC(z39) obtained from other serovars. The sequence
obtained from gapA clustered into the S. bongori group when it was compared to
others previously described. We conclude that these three isolates are members of the
S. bongori species representing a new serovar that will be described in the next
supplement to the Kauffmann-White scheme. (c) 2005 Elsevier SAS. All rights
reserved.
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The genetic bases of antimicrobial drug resistance (R) of 79 Salmonella enterica
serotype Hadar clinical isolates (recovered during 1995-2001 in a Spanish region)
was investigated. The isolates showed a limited genomic variation, as demonstrated
by PFGE analysis using XbaI (three profiles, S >= 0.77) and BlnI (seven profiles, S
>= 0.49; with 95% of the isolates falling into two clusters, S >= 0.75). Thirteen Rprofiles, ranging from susceptible to multidrug resistant, were recognized. All
susceptible isolates (14%) were recovered before or during 1998, when multidrug
resistance (MDR) was still uncommon (20% from 1995-1998). In later years, the
percentage of MDR increased considerably (92% in 2001). Resistance to nalidixic
acid, tetracycline, streptomycin and ampicillin-cefalotin, encoded by gyrAAsp87/Asn, tet(A), strA/B, and bla(TEM) genes, respectively, were the most
common, appearing together in 38% of the isolates. In all tetracycline- and
streptomycin-resistant isolates, strA/B and tet(A) were chromosomally located,
whereas blaTEM was plasmid-born. Five different blaTEM plasmids (pUO-ShR1 to
pUO-ShR5, of about 9.4, 23, 30, 45, and 95 kb, respectively) were identified. pUOShR3 and pUO-ShR5 harbored additional R-genes: [dfrA1] and [acc(3)IV-strA/B],
respectively. pUO-Sh2, pUO-Sh3, pUO-ShR4, and pUO-Sh5 were self-transferable,
and the latter could also mobilize pUO-ShR1. The reported data constitute a useful
background for further epidemiological studies of MDR in S. Hadar.
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A study was undertaken to determine the persistence of Escherichia coli O157:H7
and salmonellae in the gut of a free-living nematode. Caenorhabditis elegans, as
affected by temperature and relative humidity and to determine if infected worms
transmit Salmonella enterica serotype Newport to progeny and uninfected worms.
Worms were fed cells of a non-pathogenic strain of E. coli (OP50), E. coli 0157:H7,
S. enterica serotype Newport, and S. enterica scrotype Poona, followed by incubating
at 4, 20, or 37 degrees C for up to 5 days. initial populations of ingested pathogens
significantly increased by up to 2.93 log(10) cfu/worm within 1 day at 20 degrees C
on K agar and remained constant for an additional 4 days. When worms were placed
on Bacto agar, Populations of ingested pathogens remained constant at 4 T, decreased
significantly at 20 T, and increased significantly at 37 degrees C within 3 days.
22
Wortris fed E. coli OP50 or S. Newport were incubated at 4 or 20 degrees C at
relative humidities of 33%. 75%, or 98% to determine survival characteristics of
ingested bacteria. Fewer cells of the pathogens survived incubation at 33% relative
humidity compared to higher relative humidities. Populations of ingested E. coli
OP50 and S. Newport decreased by LIP to 1.65 and 3.44 log(10) cfu/worm,
respectively, in worms incubated at 20 degrees C and 33% relative humidity.
Placement together on K agar of adult worms, labeled with green fluorescent protein
(gfp) in the pharynx area, that had ingested gfP-labeled S. Newport and uninfected
wild type worms resulted in transfer of the pathogen to gut of wild type worms. S.
Newport was isolated from C elegans two generations removed from exposure to the
pathogen. Results of these studies show that C. elegans may serve as a temporary
reservoir of foodborne pathogens, and could perhaps, be a vector for contaminating
preharvest fruits and vegetables, thus potentially increasing the risk of enteric
infections associated with consumption of raw produce. (c) 2005 Elsevier B.V. All
rights reserved.
+HOPV0 (WKHOEHUJ6 0ROEDN. ,QWHUQDWLRQDO 6DOPRQHOOD 7\SKLPXULXP '7
LQIHFWLRQV (0(5*,1*,1)(&7,286',6($6(6 -81 The incidence of multidrug-resistant (MDR) Salmonella Typhimurium infections in
humans, and in particular MDR definitive phage type 104 (DT104), has increased
substantially in many countries in the last 2 decades, often associated with increased
illness. To examine the magnitude of this problem, a survey was conducted among
countries with available antimicrobial resistance or phage typing surveillance data. A
total of 29, primarily industrialized, countries participated in the survey, which
covered the years 1992-2001. Overall, the incidence of MDR S. Typhimurium and
DT104 increased continuously during this period, although the problem affected
primarily Europe and North America. The increase appeared to have peaked in the
United Kingdom but not in other countries. Also, the incidence of quinolone-resistant
S. Typhimurium was increasing. This survey implies that MDR S. Typhimurium
constitutes an increasing public health problem in large parts of the world and
emphasizes the importance of surveillance and control programs.
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DQWLPLFURELDO UHVLVWDQFH LQ 6DOPRQHOOD RXWEUHDNV (0(5*,1*,1)(&7,286
',6($6(6-81
Few studies have evaluated the health consequences of anti microbial-resistant
Salmonella strains associated with outbreaks. Among 32 outbreaks occurring in the
United States from 1984 to 2002, 22% of 13,286 persons in 10 Salmonella-resistant
outbreaks were hospitalized, compared with 8% of 2,194 persons in 22 outbreaks
caused by pansusceptible Salmonella strains (p<0.01).
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We report the prevalence and characteristics of Salmonella strains resistant to
ciprofloxacin and extended-spectrum cephalosporins in Taiwan from January to May
2004. All isolates resistant to extended-spectrum cephalosporins carried bla(CMY-2),
23
and all ciprofloxacin-resistant Salmonella enterica serotype Choleraesuis isolates
were genetically related.
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,PUH$ 2ODV]) 1DJ\% 'HYHORSPHQW RI D 3&5 V\VWHP IRU WKH FKDUDFWHULVDWLRQ RI
6DOPRQHOOD IODJHOOLQ JHQHV $&7$9(7(5,1$5,$+81*$5,&$ Analysis of flagellin genes was carried out on strains of Salmonella Typhimurium,
Salmonella Hadar, Salmonella Abortusequi, Salmonella Enteritidis and Salmonella
Gallinarum serovars, using a PCR system designed in this study. The purpose of
these studies was to explore the flagellin genes of biphasic and monopliasic
Salmonellae for future targeted genetic interventions. The PCR primers were
designed for two different structural genes of flagellin (fliC, fljB), for the repressor of
fliC (fljA), for the operator region of fliC, and for the invertase system responsible
for phase variation in Salmonella (hin, hixL, hixR). PCR analysis revealed that all of
the examined genes (fliC, fliC-operator, fljB, fliA, hin, hixL, hixR) were present in
all S. Typhimurium (n = 10) and S. Hadar (n = 10) strains tested. The results obtained
on S. Typhimurium and S. Hadar strains confirmed their biphasic character at DNA
level. However, the S. Enteritidis (n = 46) and S. Gallinarum (n = 5) strains lacked
the invertase system (hin, hixL, hixR) as well as the fljA and fljB genes, while fliC
and its operator were detectable. Consequently, the S. Enteritidis strains could only
express fliC gene resulting in phase H 1 flagellin. The examined S. Gallinarum
strains were also demonstrated to have a cryptic flagellin gene (MC). On the other
hand, PCR results on S. Abortusequi (n = 2) indicated that both flagellin genes (fliC,
fljB) and the whole phase variation system were present in both strains tested but
only the H2 phase gene (fljB) was expressed. The phenotype of these strains could be
clarified by motility test and/or by classical flagellar serology. The findings are also
substantiated by the results of serovar-specific PCR for S. Typhimurium and S.
Enteritidis. In conclusion, the PCR system developed in this study proved to be
suitable for characterisation of Salmonella flagellin genes and confirmed serological
results regarding all S. Typhimurium, S. Hadar and S. Enteritidis strains. This system
could also identify cryptic flagellar genes of S. Abortusequi and S. Gallinarum.
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FRQWURO RI DQ RXWEUHDN RI VDOPRQHOORVLV FDXVHG E\ PXOWLGUXJUHVLVWDQW 6DOPRQHOOD
W\SKLPXULXP LQ DSRSXODWLRQRIKRVSLWDOL]HGKRUVHV9(7(5,1$5<0,&52%,2/2*<0$<
An outbreak of salmonellosis in a population of hospitalized horses resulted in the
closure of a teaching hospital for a period of 10 weeks. Fecal samples were collected
from suspected cases and cultured for Salmonella. Salmonella isolates were
characterized using antimicrobial susceptibility testing, pulsed-field gel
electrophoresis (PFGE) and phage typing. Thirty-three cases of infection by a
multidrug-resistant strain of S. typhimurium were detected. The index case was
admitted on 26 August 1999. Fifteen (45%) cases occurred between April and June
24
2000. PFGE results suggested that this strain of S. typhimurium might have been
introduced into the hospital environment by a foal presenting with diarrhea. The
hospital was closed on June 13, and intensive environmental cleaning and
disinfection were completed. Enforcement of infectious disease control protocols in
hospitals and environmental and patient surveillance is needed to prevent outbreaks
of salmonellosis. &COPY; 2005 Elsevier B.V. All rights reserved.
6DUMHDQW.& :LOOLDPV6. +LQWRQ$ 7KH HIIHFW RI HOHFWURQ EHDP LUUDGLDWLRQ RQ WKH
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7KH HIIHFW RI KLJKHQHUJ\ HOHFWURQ EHDP LUUDGLDWLRQ RQ WKH VXUYLYDO RI
Salmonella enterica serovar Typhimurium and psychrotrophic bacteria on
commercial chicken breast meat was evaluated. Fresh chicken breast meat was
purchased from a local poultry processor, inoculated with 8 log(10) cfu/mL
Salmonella, packaged in Styrofoam trays and over wrapped with a polyvinyl chloride
film, and subjected to 0, 1, 2, or 3 kGy of irradiation. The packaged samples were
stored at 4&DEG; C and analyzed for Salmonella Typhimurium and psychrotrophic
organisms at 0, 2, 4, 6, 8,10,12, and 14 d of storage. Direct plating and enrichment
methods were used for S.Typhimurium analyses. The direct plating method revealed
a 4 log reduction in Salmonella for chicken breasts inoculated and treated with 1, 2,
or 3 kGy of irradiation. Psychrotrophic counts were conducted at 7&DEG; C for 10 d
and 25&DEG; C for 5 d to determine the effect of incubation methods on the
recovery of psychrotrophic organisms. The enrichment method resulted in the repair
of injured Salmonella cells and an elevated Salmonella Typhimurium count for all
irradiation dosages when compared with data reported for the direct plating method.
In general, psychrotrophic counts increased as storage time increased. However,
psychrotrophic counts decreased (P &LT; 0.05) as the irradiation dosage increased.
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GLOXWLRQ UDWHV -2851$/2)(19,5210(17$/6&,(1&($1'+($/7+3$57%
3(67,&,'(6)22'&217$0,1$176$1'$*5,&8/785$/:$67(6 The objective of the study was to determine the frequency of spontaneous acquisition
of resistance to select antibiotics by Salmonella Typhimurium (ST) when grown in
glucose amended continuous flow culture at slow (D = 0.025 h(-1)) or fast (D = 0.27
h-1) dilution rates. The bacterium was grown in LB minimal medium (pH 6.25)
containing no antibiotics. Upon achieving steady state, samples were plated to tryptic
soy agar (TSA) alone or supplemented (per ml) with 2 and 16 &mu; g
oxytetracycline, 4 and 16 &mu; g tetracycline, 2 and 64 &mu; g kanamycin, and 0.25
and 2 &mu; g enrofloxacin. Regardless of growth rate, CFU of resistant ST from the
TSA containing antibiotics was less than 2 x 10(1) except for 2 &mu; g kanamycin
and 0.25 &mu; g enrofloxacin treatments (higher than 1 x 10(9) and 4 x 10(7) CFU
of resistant ST for trials 1 and 2, respectively). Frequency of recovering resistant ST
from the TSA containing the higher antibiotic concentrations was less than 1 in 10(9)
25
for all antibiotics, but was higher on the media containing 2 &mu; g kanamycin and
0.25 &mu; g enrofloxacin at both slow and fast growth rates. In general, minimal
susceptibility differences were detected for isolates from slow and fast dilution rates.
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Nontyphoidal salmonellae are among the leading causes of food-borne disease in the
United States. Because of the importance of Salmonella enterica in food-borne
disease, numerous typing methodologies have been developed. Among the several
molecular typing methods, pulsed-field gel electrophoresis (PFGE) is currently
considered the "gold standard" technique in typing Salmonella. The aim of this study
was to compare the discriminatory power of PFGE to multilocus sequence typing
(MLST) in typing Salmonella enterica serovar Typhimurium clinical isolates. A total
of 85 Salmonella Typhimurium clinical isolates from cattle were used in this study.
PFGE using Xbal was performed on the 85 isolates by the Centers for Disease
Control and Prevention method, and data were analyzed using the BioNumerics
software package. Fifty PFGE profiles were observed among the isolates, and these
grouped into three major clusters. For the MLST analysis, the manB, pduF, glnA, and
spaM genes were amplified by PCR from the same 85 isolates. DNA sequencing of
these four genes, manB, pduF, glnA, and spaM, showed no genetic diversity among
the isolates tested, with a 100% identity in nucleotide sequence. Moreover, the DNA
sequences of the aforementioned genes showed 100% identity to the sequence
reported in GenBank for the S. enterica serovar Typhimurium LT2 strain. Therefore,
MLST, using these genes, lacks the discriminatory power of PFGE for typing
Salmonella enterica serovar Typhimurium.
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The increase in AmpC-mediated resistance in salmonellae constitutes a serious
public health concern, since these enzymes confer resistance to a wide range of
&beta;-lactams. One hundred six isolates were selected from 278,308 Salmonella
isolates based on resistance to ampicillin and cephalosporins and were subjected to
further characterization. Nine isolates had a cefoxitin inhibition diameter :517 mm
and were proven to be AmpC positive by multiplex PCR. Sequence analysis revealed
the presence of bla(DHA-1), bla(CMY-2), and bla(CMY-4) genes. All nine isolates
presented different pulsed-field gel electrophoresis restriction profiles. The AmpC
genetic determinants were present in transferable plasmids of around 11, 42, 70, 98,
and 99 MDa. A combination of size and restriction fragment length polymorphism
(RFLP) analysis showed that all the bla(CMY) plasmids investigated in our study
were different, which suggests that bla(CMY) may be located in different plasmid
environments. Some United Kingdom isolates linked to foreign travel showed RFLP
plasmid patterns consistent with plasmids previously seen in the United States, which
suggests that bla(CMY-2) has also been disseminated through plasmid transfer. The
26
fact that two of the domestically acquired United Kingdom isolates presented
previously unseen RFLP plasmid patterns could indicate that these strains have
followed routes different from those prevalent in North America or other parts of the
world. This study represents the first report of bla(CMY) genes in Salmonella
isolates in the United Kingdom and the first report of CMY-4 in Salmonella enterica
serotype Senftenberg worldwide.
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Contaminated feed is a source of infection with Salmonella for livestock, including
pigs. Because pigs rarely show clinical signs of salmonellosis, undetected carriers can
enter the food production chain. In a "Farm to Fork" food safety concept, safe feed is
the first step for ensuring safe food. Heat treatment or adding organic acids are
process steps for reducing or eliminating a contamination with Salmonella. The aims
of this study were (I) to estimate the probability and the level of Salmonella
contamination in batches of feed for finishing pigs in Swiss mills and (II) to assess
the efficacy of specific process steps for reducing the level of contamination with
Salmonella. A quantitative release assessment was performed by gathering and
combining data on the various parameters having an influence on the final
contamination of feed. Fixed values and probability distributions attributed to these
parameters were used as input values for a Monte Carlo simulation. The simulation
showed that-depending on the production pathway-the probability that a batch of feed
for finishing pigs contains Salmonella ranged from 34% (for feed on which no
specific decontaminating step was applied) to 0% (for feed in which organic acids
were added and a heat treatment was implemented). If contamination occurred, the
level of contamination ranged from a few Salmonella kg(-1) feed to a maximum of
8E+04 Salmonella kg(-1) feed. Probability and levels of contamination were highest
when no production process able to reduce or eliminate the pathogen was
implemented. However, most of the Swiss production was shown to undergo some
kind of decontaminating step. A heat treatment, in combination with the use of
organic acids, was found as a solution of choice for the control of Salmonella in feed.
(c) 2004 Elsevier B. V. All rights reserved.
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An individual-based model (IbM) was developed to describe the growth and
migration of Salmonella enteritidis in hens’ eggs. The Bacteria Simulator (BacSim)
environment was used to implement the model; the bacteria are represented by
spheres that grow by nutrient uptake and divide in two daughter cells upon exceeding
a certain threshold volume. Motility of the Salmonella bacteria was described by a
run and tumble mechanism. For the sake of simplicity, the bacteria were assumed to
grow exponentially, an appropriate assumption for the initial phase of growth
relevant for shelf-life predictions. Both albumen and yolk were assumed to be
27
homogeneous. The impact of several model parameters (chemotaxis, growth rate,
initial contamination numbers and bacterial swimming speed) was assessed by a
sensitivity analysis. The results show that chemotaxis towards the yolk would have a
strong effect on the time needed to reach the vitelline membrane, an aspect that
future research should focus on. The contamination position had less impact on the
time to reach the vitelline membrane. The simulation results illustrate the need for
more detailed knowledge on the subject of bacterial migration in hens’ eggs. Our
model can easily incorporate this knowledge when it becomes available. (c) 2004
Elsevier B.V. All rights reserved.
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Salmonellosis is the major reason for foodborne infections in humans in
industrialised countries and is often associated with pork production. Hence, different
Salmonella monitoring programs have been developed. But high costs caused by the
elevated sampling numbers necessary make those efforts inefficient, especially in
countries with small production units. Lower Austria has many areas with small
production units and adoptation of a monitoring scheme based on existing programs
would riot be feasible financially. The development and application of a new
program has proven to be practicable and low in costs and hence a real alternative for
areas with variable development as to its agricultural infrastructure. A total of 2318
samples (meat juice) of slaughter pigs were examined for Salmonella antibodies with
a commercial test kit. Currently, the incidence of salmonellosis in slaughter pigs in
Lower Austria is low, with only 1.81% serological positives. The above screening
method costs mere 2 Euro cents per slaughter pig, hence makes this method suitable
for a yearly check up and it allows immediate regulatory intervention should this be
deemed appropriate.
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Passive laboratory-based surveillance data from Alberta Agriculture Food and Rural
Development were analyzed for common Salmonella serovars, prevalences, trends,
and for the presence of temporal clusters. There were 1767 isolates between October
1990 and December 2001 comprising 63 different serovars, including 961 isolates
from chickens, 418 from cattle, 108 from pigs, 102 from turkeys, and 178 from all
other species combined. Salmonella Typhimurium, Heidelberg, Hadar, Kentucky,
and Thompson were the 5 most frequently isolated serovars. Approximately 60% of
the S. Typhimurium were isolated from cattle, whereas over 90% of the S.
Heidelberg, Hadar, Kentucky, and Thompson were isolated from chickens.
Salmonella Enteritidis was rarely isolated. There was an increasing trend in isolates
from chickens, cattle, and pigs, and a decreasing trend in isolates from turkeys.
Temporal clusters were observed in 11 of 15 serovars examined in chickens (S.
Anatum, Heidelberg, Infantis, Kentucky, Mbandaka, Montevideo, Nienstedten,
28
Oranienburg, Thompson, Typhimurium, and Typhimurium var. Copenhagen), 5 of 5
serovars in cattle (S. Dublin, Montevideo, Muenster, Typhimurium, and
Typhimurium var. Copenhagen), and I of 3 serovars in pigs (S. Typhimurium). Shortduration clusters may imply point source infections, whereas long-duration clusters
may indicate an increase in the prevalence of the serovar, farm-to-farm transmission,
or a wide-spread common source. A higher concentration of clusters in the winter
months may reflect greater confinement, reduced ventilation, stressors, or increased
exposure to wildlife vectors that are sharing housing during the winter. Detection of
large clusters of Salmonella may have public health implications in addition to
animal health concerns.
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1. This study was to identify the risk factors for Salmonella spp. contamination of
Senegalese chicken carcases during slaughtering. One hundred and twenty traditional
slaughterhouses were studied from January 2000 to December 2002 in and around
Dakar. A questionnaire was answered by the slaughterers, and samples of breast skin
were taken to assess the Salmonella status of chicken carcases.
2. Results showed that 43.3% of the batches were contaminated with Salmonella,
indicating Salmonella Hadar and Salmonella Brancaster as the two main serovars.
3. Salmonella contamination of the carcases after slaughtering was related to
contamination of the live birds. Feed withdrawal before slaughtering and thorough
cleaning and disinfection procedures decreased the risk of contamination.
4. One individual worker for each slaughtering stage was also associated with a
decreasing risk of contamination. Using scalding water for plucking the chicken
carcases increased contamination risk.
5. These results will help slaughters to produce safer products for local consumers.
/R\QDFKDQ$7 +DUULV'/ 'RVH GHWHUPLQDWLRQ IRU DFXWH 6DOPRQHOOD LQIHFWLRQ LQ SLJV
$33/,('$1'(19,5210(17$/0,&52%,2/2*< 0$< Pigs were exposed to various levels of Salmonella enterica subsp. enterica serovar
Typhimurium by either intranasal inoculation or by subjecting them to a
contaminated environment. More than 10(3) salmonellae were required to induce
acute Salmonella infection. These results indicate that intervention against acute
Salmonella infection in lairage may be more readily achieved than previously
thought.
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-2851$/2)$17,0,&52%,$/&+(027+(5$3< 0$< Objectives: Two multiresistant Salmonella enterica subsp. enterica serovar Agona
isolates from pig carcasses were investigated for antimicrobial resistance genes and
their location with particular reference to the detection of class 1 integrons.
29
Methods: The two S. Agona isolates were investigated for their in vitro susceptibility
to antimicrobial agents and their plasmid content. The resistance genes and class 1
amplicons were identified by PCR assays. Amplicons of class 1 integrons were
cloned and sequenced. Transferability of resistance plasmids was confirmed by
conjugation.
Results: Both S. Agona isolates carried conjugative plasmids of approximately 150
kb which harboured all resistance genes detected in the respective isolates. S. Agona
231 was resistant to chloramphenicol by catA1, to tetracycline and minocycline by
tet(B), and to sulphonamides by sul1. In addition, it harboured a streptomycin
resistance gene strA and a class 1 integron with a new aadA variant designated
aadA23, which mediates resistance to streptomycin and spectinomycin. S. Agona 242
also carried the genes catA1, tet(B), and sul1. Moreover, it harboured a second
sulphonamide resistance gene, sul2, and a class 1 integron with intact gene cassettes
carrying new variants of the trimethoprim resistance gene dfrA15b or the
chloramphenicol resistance gene cmlA4. The third gene cassette consisted of a
truncated aadA2 gene.
Conclusions: The results of this study show that large conjugative multiresistance
plasmids are present in S. Agona from pigs. Analysis of the class 1 integrons
revealed the presence of new variants of resistance genes so far not detected in
Salmonella isolates.
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FKLFNV ,1',$1-2851$/2)$1,0$/6&,(1&(6 $35 The effect of competitive exclusion (CE) in prevention of Salmonella Gallinarurn
colonization was compared with a live (rough) strain of the same organism in chicks.
A competitive exclusion (CE) product and the rough strain were administered to
different groups of one-day-old chicks, which were then challenged with the smooth
strain of S. Gallinarum on day 3. The colonization of Salmonella was significantly
reduced in both the treatment groups, as compared to the infected control group, up
to 21 days. However, CE had a better protection factor than the rough strain, thus
suggesting its better efficacy in reducing Salmonella colonization in the chicks.
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In this study the feasibility of 50- and 60-mer oligonucleotides in microarray analysis
for the detection and identification of antibiotic resistance genes in various
Salmonella strains was assessed. The specificity of the designed oligonucleotides was
evaluated, furthermore the optimal spotting concentration was determined. The
oligonucleotide microarray was used to screen two sets of Salmonella strains for the
presence of several antibiotic resistance genes. Set 1 consisted of strains with variant
Salmonella Genomic Island 1 (SGI1) multidrug resistance (MDR) regions of which
the antibiotic resistance profiles and genotypes were known. The second set
contained strains of which initially only phenotypic data were available. The
microarray results of the first set of Salmonella strains perfectly matched with the
30
phenotypic and genotypic information. The microarray data of the second set were
almost completely in concordance with the available phenotypic data. It was
concluded that the microarray technique in combination with random primed
genomic labeling and 50- or 60-mer oligonucleotides is a powerful tool for the
detection of antibiotic resistance genes in bacteria. (c) 2005 Elsevier B.V. All rights
reserved.
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Proteins and genes involved in the recovery of heat-injured Salmonella Enteritidis
were investigated. Salmonella Enteritidis cells cultured overnight in tryptic soy broth
(TSB; nonselective medium) were suspended in citric acid-disodium hydrogen
phosphate buffer (pH 6). After heat treatment at 55&DEG; C for 15 min, the
culturable counts measured by tryptic soy agar (TSA; nonselective medium)
decreased from 108 to 107 CFU/ml. On the other hand, culturable counts measured
by desoxycholate-hydrogen sulfite-lactose (DHL) agar (selective medium) were
decreased from 108 to 101 CFU/ml by the same treatment. The results suggest that
99.9% of Salmonella Enteritidis detected on TSA were injured but recoverable.
When injured Salmonella Enteritidis was incubated in TSB, the culturable count
measured by TSA did not increase for 2 h, whereas that by DHL agar increased after
incubation for 30 min. After incubation for 2 h, the culturable count measured by
DHL agar reached a similar level with that by TSA, indicating that Salmonella
Enteritidis had recovered. The two-dimensional polyacrylamide gel electrophoresis
analysis revealed that elongation factor G (FusA) and pyrulvate kinase (PykF)
specifically increased in the cells just after heat treatment and in the recovery cells.
The levels of transcription of 86 stress-inducible genes were also investigated by
reverse transcription PCR. Nineteen heat-inducible (clpB, clpX, degP, dnaJ, fkpA,
ftsJ, gapA, hflB, hslJ, hslU, hslV, htpG, htrA, lon, mopA, mopB, mreB, rpoE, and
ppiD), and 12 oxidative-stress and DNA damage-inducible (ahpC, ahpF, fldB, fur,
grxA, dinF, katG, mutM, recA, soxR, trxC, and zwf) genes were transcribed
extensively during recovery in TSB. The results obtained in this study will be used to
develop the media or culture conditions that will promote recovery for the detection
of food poisoning bacteria, including injured cells from food products.
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To investigate the effects of sublethal stress on Salmonella thermal inactivation
kinetics, an eight-strain Salmonella cocktail was subjected to heat shock (30 min at
54&DEG; C), cold shock (2 h at 4&DEG; C, and starvation stress (10 days in
phosphate buffer at 4, harvested by centrifugation, and inoculated into irradiated
comminuted turkey. Immediately after stressing, the Salmonella cocktails contained
89.1% heat-injured, 44.7% cold-injured, and 67.7% starvation-injured cells, as
31
determined by plating on selective and nonselective media. D-60&DEG; C-values for
the heat-shocked cocktail (0.64 min on Trypticase soy agar containing 0.6% yeast
extract [TSAYE], 0.35 min on xylose lysine desoxycholate [XLD] agar) were higher
(P &LT; 0.05) than those for the unshocked control (0.41 min on TSAYE, 0.17 min
on XLD), whereas D-60&DEG; C-values for the cold-shocked cocktail (0.38 min on
TSAYE, 0.17 min on XLD) were not significantly different from those for the
control. Starved cells had the same D-60&DEG; C-value on TSAYE as did the
unshocked cocktail, but the D-60&DEG; C-value on XLD was significantly lower
(0.14 min). Although starvation and cold shock were not thermally protective, heat
shock increased thermal resistance, indicating that product history and the
physiological state of the Salmonella cells should be considered when developing
and validating thermal processes. D-60&DEG; C-values observed on selective media
were significantly lower than those observed on nonselective media for all stress
treatments and for the control. Therefore, nonselective culture media should be used
to assess the response of microorganisms to a thermal challenge when developing
performance standards for lethality.
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From March 2000 to September 2001, 608 samples of retail meat (136 pork, 70 beef,
202 chicken, and 200 ducks) and 110 samples of retail shrimp from six provinces of
the Mekong Delta in Vietnam were collected individually and examined for the
prevalence of Salmonella. Of the 718 samples examined, 243 (33.8%) were
Salmonella positive. Salmonella was isolated from 69.9% of the pork samples,
48.6% of the beef samples, 21.0% of the chicken meat samples, 22.3% of the duck
meat samples, and 24.5% of the shrimp samples. From 261 Salmonella isolates, 24
different serovars were identified. The predominant serovars of the isolates were
Salmonella ’Derby, Salmonella Weltevreden, and Salmonella London in pork;
Salmonella Weltevreden, Salmonella London, and Salmonella Dessau in beef;
Salmonella Emek, Salmonella Typhimurium, and Salmonella Dessau in chicken
meat; Salmonella Lexington, Salmonella Derby, and Salmonella Dessau in duck
meat; and Salmonella Weltevreden, Salmonella Tennessee, and Salmonella Dessau
in shrimps. Salmonella Bovismorbificans, Salmonella Derby, Salmonella Dessau,
and Salmonella Weltevreden were the most common serovars in all the samples
examined. These results indicate a high rate of contamination by Salmonella in retail
meats and shrimps in the Mekong Delta, Vietnam.
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From years 2000 to 2003, Salmonella was investigated from a total of 1785 samples
comprised of chicken intestinal samples, cloacal swabs, drag swabs, litter samples
and chick dust samples collected from 191 poultry breeding flocks belonging to 15
different chicken breeding stock companies in the Marmara region, Turkey by a
SYBR green-based real-time polymerase chain reaction (SGBRT-PCR), by a probespecific real-time polymerase chain reaction (PSRT-PCR) and by standardized
32
bacteriology as described in the manual of National Poultry Improvement Plan and
Auxillary Provisions, United States Department of Agriculture. Between January
2000 and July 2001, Salmonella was detected at the rates of 5.87% and 4.10% out of
a total of 1242 samples by SGBRT-PCR and bacteriology, respectively. From July
2001 until December 2003, Salmonella was found at rates of 11.42% and 5.52%
from a total of 543 samples by PSRT-PCR and bacteriology, respectively. The
dominant Salmonella serovar was determined as Salmonella enterica subsp. enterica
Serovar Enteritidis ( S. Enteritidis), while serogroup C1 and C2 in 2001 and
serogroup E1 in 2002 were isolated as additional serovars. As a conclusion, S.
Enteritidis seems to be the major problem in poultry breeding flocks in Turkey, and
both of the real-time polymerase chain reaction methods were found more sensitive
than standard bacteriology for the detection of Salmonella from poultry samples.
33
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34