Bull Vet Inst Pulawy 53, 439-444, 2009
OCCURRENCE OF FOUR MAJOR FOOD-BORNE PATHOGENS
IN CATTLE SLAUGHTERED IN POLAND
KINGA WIECZOREK, EDYTA DENIS, AND JACEK OSEK
Department of Hygiene of Food of Animal Origin,
National Veterinary Research Institute,
24-100 Pulawy, Poland
[email protected]
Received for publication May 08, 2009
Abstract
A survey was conducted in order to determine the incidence of Campylobacter sp., Salmonella sp., L. monocytogenes, and
VTEC in slaughtered cattle in Poland. Overall, 276 swab samples of cattle hides and 276 samples from the corresponding carcasses
were analysed. Additionally, 238 beef meat samples, purchased at retail level, were tested. The hides were more frequently
contaminated with Campylobacter sp. (24.6%) than with any other pathogens. On the other hand, the carcasses had the highest
contamination with VTEC (10.9%). The occurrence of food-borne pathogens in minced beef was between 1.3% (VTEC) and 24.3%
(L. monocytogenes). The findings reinforce the importance of controlling zoonotic pathogens in meat through a complete, continuous
farm-to-fork test system.
Key words: cattle, beef, food-borne pathogens, meat inspection.
The contamination of beef during slaughter and
the processing of the carcasses is a major risk of
subsequent food-borne pathogen infection in humans. A
high proportion of these illnesses originate from the
direct consumption of contaminated meat. Cattle may
carry several pathogens in their gastro-intestinal tract,
which are then excreted in the faeces. The animals
shedding the pathogens in their faeces may contaminate
many other animals in the herd. Microorganisms can be
also transferred to the carcass from the hide during the
slaughter and dressing processes. Red meat animals can
be infected with, or carry a wide range of,
microorganisms, which are potentially pathogenic for
man. The most important of these are zoonotic bacteria,
principally Campylobacter sp., Salmonella sp., Listeria
monocytogens, and verocytotoxigenic Escherichia coli
(VTEC). According to the EFSA report, the two most
frequently reported zoonotic diseases in humans in the
EU in 2007 were Campylobacter and Salmonella
infections, with incidences of 45.2 and 31.1 per 100,000
people, respectively (14). The infection with
Campylobacter sp., especially C. jejuni, is one of the
leading causes of bacterial diarrhoea world-wide, and
the most common antecedent to the neuropathies such as
Guillian-Barré syndrome (GBS). Most S. enterica
serotypes cause self-limiting gastroenteritis, which is
very often associated with diarrhoea, stomach cramps, or
vomiting and fever. The third pathogen mentioned
above - VTEC - is characterised by the production of
Shiga toxins. It can cause infection, which is one of the
more serious forms of food-illness, as it can lead to
serious, sometimes fatal, complications. Cattle are
known to be the most important reservoir for VTEC, but
in most cases they are asymptomatic carriers (9). L.
monocytogenes is also widely distributed in the
environment. Infections caused by this microorganism
may result in a wide range of clinical symptoms. Such
infections are usually rare, but the mortality rate may be
as high as 20%. The outbreaks have been associated
with a variety of contaminated food, including beef (14).
The aim of this study was to evaluate the
potential risk of hide-to-cross-contamination during the
slaughter of cattle and at the retail level, by determining
the prevalence of the major food-borne pathogens.
Material and Methods
Sample collection. The two hundred and
seventy-six cattle slaughtered in the eastern part of
Poland used in this study were collected between
November 2007 and December 2008. Three different
slaughterhouses were visited, usually once a week, and
each time samples from ten cattle were taken. The
number of samples collected from each plant is shown in
Table 1.
440
Table 1
The number of samples taken from slaughterhouses
Source
Number of samples
Slaughterhouse No. 1
Slaughterhouse No. 2
Slaughterhouse No. 3
Total
38
14
224
276
Percentage of
samples
13.8%
5.1%
81.1%
100%
Table 2
Occurrence of Campylobacter sp., L. monocytogenes, Salmonella sp., and VTEC in slaughtered cattle and minced beef
Samples positive for:
Source
Campylobacter sp.
L. monocytogenes
Salmonella sp.
VTEC
Hide
28/276
6/276
54/276*
68/276(24.6%)
sample
(10.1%)
(2.2%)
(19.6%)
Carcass
8/276
7/276
5/276
30/276*
sample
(2.9%)
(2.5%)
(1.8%)
(10.9%)
23/276*
1/276
0/276
0/276
Cross contamination
(8.3%)
(0.36%)
5/238
58/238
6/238
3/238*
Minced beef sample
(2.1%)
(24.3%)
(2.5%)
(1.3%)
*No. of samples confirmed as VTEC by PCR
Pathogen
Salmonella sp.
Campylobacter sp.
L. monocytogenes
VTEC
Age of
cattle
(years)
Sample
origin
Hide
1-3
Carcass
Hide
4-6
Carcass
Hide
>6
Carcass
Hide
Unknown
Carcass
Table 3
Number of strains isolated from different sources
The source of strains
Hide
Carcass
Meat
4
3
6
68
8
5
28
7
56
11
9
4
Together
13
81
91
24
Table 4
Occurrence of pathogens in slaughtered cattle according to their age
Samples positive for:
Number of
samples
Campylobacter sp.
L. monocytogenes
Salmonella sp.
tested
16/73
3/52
1/52
52
(30.8%)
(5.8%)
(1.9%)
2/52
0/52
0/52
52
(3.8%)
8/30
5/30
1/30
30
(26.7%)
(16.7%)
(3.3%)
1/30
1/30
30
0/30
(3.3%)
(3.3%)
41/174
16/174
4/174
174
(23.6%)
(9.2%)
(2.3%)
5/174
6/174
3/174
174
(2.9%)
(3.4%)
(1.7%)
3/20
4/20
20
0/20
(15.0%)
(20.0%)
1/20
1/20
20
0/20
(5.0%)
(5.0%)
VTEC
1/52
(1.9%)
1/52
(1.9%)
7/30
(23.3%)
2/30
(6.7%)
39/174
(22.4%)
27/174
(15.5%)
7/20
(35.0%)
0/20
441
The samples were collected from the hides
before removal of the hide after exsanguination, with
sterile swabs wiped across the 400 cm2 area of brisket.
The same method was used for the carcasses after
evisceration, where the 400 cm2 brisket area was wiped
with four sterile swabs. Then all the samples were
placed in an ice-pack container, taken to the laboratory
and analysed within 24 h. A total of 238 minced beef
samples purchased from local supermarkets were also
tested. The minced beef was analysed between
November 2007 and December 2008.
Microbiological analyses. In the laboratory,
200 ml of Maximum Recovery Dilution (MRD, Oxiod,
United Kingdom) was added to each swab and
stomached for 3 min. After centrifugation at 15,000 g
for 10 min, the samples were analysed for the presence
of Campylobacter sp., L. monocytogenes, Salmonella
sp., and VTEC. The ISO standard methods, with some
modifications as described below, were used. Regarding
Campylobacter sp. the suspected colonies were
suspended in 1 ml of sterile water and centrifuged at
13,000 g for 3 min. Afterwards, the DNA was extracted
using the Genomic-Mini kit (A&A Biotechnology,
Poland) according to the manufacturer’s instruction. The
Campylobacter sp. was identified using multiplex PCR
(m-PCR) as described by Wieczorek and Osek (17). All
suspected L. monocytogenes and Salmonella sp. colonies
were subjected to biochemical analysis using Api®
Listeria and ID 32 E test (bioMérieux, France),
respectively. The detection and identification of VTEC
were performed using the ISO 16654 standard and PCR
protocol (11). All the samples were cultured by
incubating them in Tryptone Soya Broth (TSB, Merck,
Germany) for 24 h at 41.5°C. Then, 1 ml of subsamples
was centrifuged at 15,000 g for 2 min. The supernatant
was centrifuged again at 13,000 g for 3 min, and the
pellet was suspended in 1 ml of PBS (Biochrom, United
Kingdom) and centrifuged at 13,000 g for 3 min. After
that, the bacterial pellet was suspended in 1 ml of sterile
water, heated at 99°C for 5 min (Thermomixer,
Eppendorf, Germany), and centrifuged at 13,000 g for 1
min. The supernatant (5 µl) was subsequently used as a
source of DNA.
Results
During the study period, a total of 276 hides
and the 276 corresponding carcasses, as well as 238
meat samples were analysed for the presence of
Campylobacter sp., L. monocytogenes, Salmonella sp.,
and VTEC. The occurrence of at least one pathogen was
found in 95 (34.4%) of the 276 hides, 14 (5.1%) of the
carcasses and 33 (12.0%) of both hides and the
corresponding carcasses. In the meat samples, the
presence of at least one pathogen was found in 67
(28.2%) of the 238 samples (Table 2). The numbers of
strains isolated from the samples tested are shown in
Table 3.
In the case of the 276 hides analysed, the most
prevalent bacteria were Campylobacter sp. (24.6%)
followed by VTEC (19.6%). Most of the Campylobacter
isolates (34 isolates) were identified as C. jejuni. L.
monocytogenes was found in 28 (10.1%) samples. Only
five (1.8%) out of the 276 hides were contaminated with
Salmonella sp. On the other hand, the bovine carcasses
were mostly contaminated with VTEC (10.9%). The
remaining bacterial pathogens were found in only a few
carcasses. The rate of the transfer from hide to the
corresponding carcasses was low, except in the case of
VTEC (where 10.9% of cross-contaminations were
found) (Table 2).
It was noted that some hides were contaminated
with more than one pathogen, e.g. Campylobacter and
VTEC were found in 12 samples, and Campylobacter
and L. monocytogenes in seven animals. On the other
hand, only three carcasses were contaminated with more
than one pathogen – VTEC and L. monocytogenes were
found in two carcasses, and Campylobacter and VTEC
in one sample (Fig. 1).
12
10
8
6
4
Hide
Carcass
2
0
ono
L. m
o+V
lm.
+Sa
n
.mo
EC
+VT
p.+L
Ca m
ono
L. m
no
T EC
.mo
p+V
Ca m
p+L
Ca m
TEC
Fig. 1. Number of animals positive for multiple pathogens.
442
Table 4 shows the prevalence of four bacterial
pathogens detected in the hides and carcasses according
to the age of the slaughtered animals. Most of the
animals involved in the study were over 6-year-old (174
cattle; 63.0%). The next group were the animals below
three years of life (52; 18.8%), followed by cattle
between four and six years (30; 10.9%), whereas the age
of the remaining 20 animals (7.2 %) was not specified
during the slaughter process. It was found that the cattle
hides were contaminated in all the age groups of
animals, especially with Campylobacter sp. (for animals
aged 1-3 years it was 30.8%), and VTEC (animals of
unknown age – 35%). On the other hand, the most
contaminated bovine carcasses were from animals >6year-old, in which VTEC were found in 15.5% of the
samples tested. It was also noted that the carcasses of
some groups of cattle were free of Campylobacter sp.
(animals 4-6 years of age), L. monocytogens (cattle of 13 years and of unknown age), Salmonella sp. (animals of
1-3 years) and VTEC (cattle of unknown age) (Table 4).
In the case of the minced beef, a total of 238
samples were tested. Many of them - 58 (24.3%) - were
contaminated with L. monocytogenes. Other pathogens
were found in a few of the samples analysed: five
(2.1%) were positive for Campylobacter sp. (C. jejuni –
two samples, and C. coli – three samples), six (2.5%)
with Salmonella sp., and VTEC was found in three
(1.3%) samples. The results obtained are shown in Table
2. Only five meat samples were contaminated with more
than one pathogen; L. monocytogenes and Salmonella
were found in four samples, whereas Campylobacter
and L. monocytogenes were found in one sample.
Discussion
The observed prevalence of pathogens in the
beef chain varies considerably in the surveys. It is
important to recognise that the data from any
surveillance programme will be strongly influenced by
the techniques used for the identification of the
microorganisms. This is mainly due to the differences in
the sampling strategies and the analytical methods
applied. Sometimes, different parts of the food chain are
also examined in different ways. The variation between
the surveys makes the data difficult to compare. For all
these reasons, when interpreting the data from the food
and the animals it is important to note that the
information from the different investigations are not
directly comparable, especially between countries.
Variations related to the abattoirs. In this
study, the prevalence of pathogens on the animal from
the exsanguinations and the point at which the viscera
are removed, but before any further cutting, trimming,
and washing, was examined. The testing of 276 hides
and the same number of corresponding carcasses
showed that Campylobacter sp. was the most common
pathogen present in bovine hides. The prevalence of
Campylobacter in cattle in many countries is well
documented. In 2007, examinations were performed in
14 European countries, and the occurrence was below
25% in most reported cases. However, a high prevalence
was reported in Denmark - 70.5% (14). In contrast, none
of the 100 carcasses sampled in Northern Ireland carried
Campylobacter sp. (7). Our examinations are at the
mean European level, and yielded 24.6% and 2.9%
positive hides and corresponding carcasses. This fact
indicates the importance of cattle as a Campylobacter
reservoir or carrier in Poland. Moreover, we classified
38 (50.0%) Campylobacter isolates as C. jejuni, and 35
(46.1%) as C. coli. In comparison, on English farms
where Campylobacter was isolated from 27.7% of the
study population, the vast majority of the isolates
(95.2%) were C. jejuni (2).
The prevalence of verotoxigenic E. coli during
the abattoir processing is very diverse, and it strongly
depends on the sampling point. Additionally, the most
widely used analytical method only detects E. coli
O157, and only a few investigations have been
conducted with methods aimed at identifying other
serotypes of VTEC. The majority of the data from the
cattle was obtained by investigating faecal samples from
single animals. The research conducted on the preevisceration carcasses showed that VTEC contaminated
from 7% to 43.4% of the samples, but the postevisceration prevalence was distinctly lower (0.3%3.2%) (3, 9, 13). In our study, VTEC was identified in
54 (19.6%) and 30 (10.9%) of the 276 hides and the
corresponding 276 carcasses. These results seem to be
significantly higher than the data mentioned above.
However, according to the EFSA report, the occurrence
of VTEC in European countries ranged from 0% to
22.1%.
Data on the third tested pathogen - L.
monocytogenes - are considerably less available,
particularly at the production point at the abattoir
processing stages. The occurrence of L. monocytogenes
in cattle in 2007 in the EU, where the examinations were
performed in 13 countries, ranged from 0% to 11.8%
(14). Rivera-Betancourt et al. (13), in a survey of
animals at slaughter, investigated the L. monoctogenes
contamination of the hides. The prevalence observed
was 9.9% of the 1,033 animals examined, and the rate
was considerably lower than those observed for
Salmonella and E. coli O157. In our study, most of the
positive samples were obtained from the hides (10.1%),
compared to the corresponding carcasses (2.5%).
The number of Salmonella positive samples
found in our study was six (2.2%) and five (1.8%) for
hides and the corresponding carcasses, respectively.
This is a little bit higher than the other observations
concerning, especially, the carcass samples. The EFSA
reported that in countries with monitoring programmes
based on swab samples, the prevalence of Salmonella on
carcasses was lower than 1% in most of the Member
States, but the point at which the sampling was carried
out was not specified. The highest level was found in
Estonia and Spain - 1.8% and 6.7% of positive samples,
respectively. On the other hand, some authors reported a
higher frequency of Salmonella in the animals analysed.
According to Barkocy-Gallagher et al. (4), these bacteria
were detected in 12.7% of 1,060 pre-evisceration
carcasses, and 0.1% of post-intervention carcasses.
Salmonella was also isolated in 4.7% of the 531 faecal
443
and environmental samples collected in cattle herds in
Virginia, USA, and in 7.6% of beef carcass samples in
Irish abattoirs (8, 16).
Variations related to retail level. Minced beef
may be produced at the primary processing plant, where
the animals are slaughtered, or at a second premises. The
occurrence of zoonotic pathogens in raw meat are
variable, although most often are between 1% and 10%
depending on the organisms, geographical factors,
and/or meat production practices (10).
According to our results, L. monocytogenes was
the most prevalent pathogen in the meat samples. The
summarised data from several European countries
showed that the L. monocytogenes prevalence in red,
mixed, or unspecified meat ranged from 0% to 4.0%,
with a mean of 2.5% (EFSA). A high prevalence of L.
monocytogenes (52%) was observed in a survey of 100
retail samples of minced beef in Canada (5).
Furthermore, in our study of raw products such as
minced beef, these bacteria are more frequently isolated
than the other pathogens (24.3% positive samples).
Many of the monitoring programmes of on
bovine meat and products thereof are based on sampling
at the slaughterhouse and meat cutting plants. The
proportion of Salmonella positive units in minced meat,
meat preparations, and meat products of beef origin
varied from 0.2% to 4.0% in 2007 (EFSA). The results
of our research showed that Salmonella was present in
2.5% samples.
In 2007, only Italy out of the four countries
reporting MSs found samples of fresh bovine meat
positive for Campylobacter at the retail level (2.4%).
These results obtained were similar to those in the
present study (2.1% positive samples).
Bovine meat is commonly perceived to be a
major source of food-borne VTEC infections for
humans. A prevalence of 0.76% and 2.8% of VTEC was
reported in the UK and Ireland, respectively, which is
also a typical range for other European countries (14).
The occurrence of VTEC obtained in our study (1.3%)
was at a similar level. However, as shown by some
authors, beef contamination with VTEC varied widely,
and ranged from 0.01% to 54% for O157 and 1.7% to
62.5% for non-O157 VTEC (6).
Implications of the results on meat safety.
The risk factors of human Campylobacter sp.,
Salmonella, VTEC, or Listeria sp. infections include the
consumption of contaminated meat, and the handling of
the contaminated raw meat, as well as crosscontamination with other ready-to-eat products. It is
very important that a not too-simplistic view is taken
regarding the results of our investigation and of others’
observations (12, 15). However, the results indicate that
the brisket area of bovine carcasses carries food-borne
pathogens relatively frequently. The prevalence on hides
is generally much higher, and declines throughout the
abattoir process. All the aforementioned pathogens
follow a similar trend. On the other hand, there was no
correlation between the age group of the animals and the
presence of food-borne pathogens. Minced beef can also
be contaminated with different microbial pathogens,
especially L. monocytogenes, but also with
Campylobacter, Salmonella, and VTEC. The results of
this study showed that the prevalence rates of the
pathogens examined varied widely, from 1.4% (VTEC)
to 23.8% (L. monoctygenes). The data as reported in this
survey, to the authors’ knowledge, is the first piece of
information reporting the incidence of major food-borne
pathogens in cattle slaughtered in Poland, and provides
same useful information for future research.
The findings reinforce the importance of the
adequate cooking of meat and good hygiene to avoid
cross-contamination. However, there is no doubt that
infection with food-borne pathogens in red-meat animals
represents a substantial risk to human health (1, 12).
Acknowledgments: This work was
financially supported by the EU FP6 Integrated Project
ProSafeBeef, contract No. FOOD-CT-2006-36241.
References
1.
Adak G.K., Meakins S.M., Yip H., Lopman B.A.,
O'Brien S.J.: Disease risks from foods, England and
Wales, 1996-2000. Emerg Infect Dis 2005, 11, 365-372.
2. Akopayanz N., Bukanov N.O. Westblom T.U., Kresovich
S., Berg D.E.: DNA diversity among clinical isolates of
Helicobacter pylori detected by PCR-based RAPD
fingerprinting. Nucleic Acids Res 1992, 20, 5137-5142.
3. Arthur T.M., Bosilevac J.M., Nou X., Shackelford S.D.,
Wheeler T.L., Kent M.P., Jaroni D., Pauling B., Allen
D.M., Koohmaraie M.: Escherichia coli O157 prevalence
and enumeration of aerobic bacteria, Enterobacteriaceae,
and Escherichia coli O157 at various steps in commercial
beef processing plants. J Food Prot 2004, 67, 658-665.
4. Barkocy-Gallagher G.A., Arthur T.M., RiveraBetancourt M., Nou X., Shackelford S.D., Wheeler T.L.,
Koohmaraie M.: Seasonal prevalence of Shiga toxinproducing Escherichia coli, including O157:H7 and nonO157 serotypes, and Salmonella in commercial beef
processing plants. J Food Prot 2003, 66, 1978-1986.
5. Bohaychuk V.M., Gensler G.E., King R.K., Manninen
K.I., Sorensen O., Wu J.T., Stiles M.E., McMullen L.M.:
Occurrence of pathogens in raw and ready-to-eat meat
and poultry products collected from the retail
marketplace in Edmonton, Alberta, Canada. J Food Prot
2006, 69, 2176-2182.
6. Hussein H.S, Bollinger L.M.: Prevalence of Shiga toxinproducing Escherichia coli in beef cattle. J Food Prot
2005, 68, 2224-2241.
7. Madden R.H., Espie W.E., Moran L., McBride J., Scates
P.: Occurrence of Escherichia coli O157:H7, Listeria
monocytogenes, Salmonella and Campylobacter spp. on
beef carcasses in Northern Ireland. Meat Sci 2001, 58,
343-346.
8. McEvoy J.M., Doherty A.M., Sheridan J.J., Blair I.S.,
McDowell D.A.: The prevalence of Salmonella spp. in
bovine faecal, rumen and carcass samples at a
commercial abattoir. J Appl Microbiol 2003, 94, 693700.
9. McEvoy J.M., Doherty A.M., Sheridan J.J., ThomsonCarter F.M., Garvey P., McGuire L., Blair I.S.,
McDowell D.A.: The prevalence and spread of
Escherichia coli O157:H7 at a commercial beef abattoir.
J Appl Microbiol 2003, 95, 256-266.
10. Nørrung B., Buncic S.: Microbial safety of meat in the
European Union. Meat Sci 2008, 78, 14-24.
444
11. Osek J.: Identifying shigatoxigenic Escherichia coli in
bovine carcasses by the PCR method. Vet Med 2008, 64,
179-182.
12. Rhoades J., Koutsoumanis K. Duffy G.: Prevalence and
concentration of verocytotoxigenic Escherichia coli,
Salmonella enterica, and Listeria monocytogenes in the
beef production chain. Food Microbiol 2009, 26, 357376.
13. Rivera-Betancourt M., Shackelford S.D., Arthur T.M.,
Westmoreland K.E., Bellinger G., Rossman M., Reagan
J.O., Koohmaraie M.: Prevalence of Escherichia coli
O157:H7, Listeria monocytogenes, and Salmonella in
two geographically distant commercial beef processing
plants in the United States. J Food Prot 2004, 67, 295302.
14. The Community Summary Report on Trends and Sources
of Zoonoses and Zoonotic Agents in the European Union
in 2007. http://www.efsa.europa.eu.
15. Humphrey T., Jorgensen F.: Pathogens on meat and
infection in animals: Establishing a relationship using
Campylobacter and Salmonella as examples. Meat Sci
2006, 74, 89-97.
16. Warnick L.D., Crofton L.M., Pelzer K.D., Hawkins M.J.:
Risk factors for clinical salmonellosis in Virginia, USA
cattle herds. Prev Vet Med 2001, 49, 259-275.
17. Wieczorek K., Osek J.: Multiplex PCR assays for
simultaneous identification of Campylobacter jejuni and
Campylobacter coli. Vet Med 2005, 61, 797-799.