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Veterinary Quarterly Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tveq20
Salmonella contamination of poultry flocks in the Netherlands a
a
a
A. W. v.d. Giessen , R. Peters , P.A.T.A. Berkers , b
W. H. Jansen & S. H. W. Notermans
a
a
Laboratory for Water and Food Microbiology , National Institute of Public Health and Environmental Hygiene , P.O. box 1, Bilthoven, 3720 BA, The Netherlands b
Laboratory for Bacteriology , National Institute of Public Health and Environmental Hygiene , P.O. Box 1, Bilthoven, 3720 BA, The Netherlands Published online: 01 Nov 2011.
To cite this article: A. W. v.d. Giessen , R. Peters , P.A.T.A. Berkers , W. H. Jansen & S. H. W. Notermans (1991) Salmonella contamination of poultry flocks in the Netherlands, Veterinary Quarterly, 13:1, 41-46, DOI: 10.1080/01652176.1991.9694283 To link to this article: http://dx.doi.org/10.1080/01652176.1991.9694283
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sublicensing, systematic supply, or distribution in any form to anyone is expressly
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Salmonella contamination of poultry flocks in the Netherlands A. W. v.d. Giessen', R. Peters', P.A.T.A. Berkersi, W. H. Jansen2, and S. H. W. Notermansl
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SUMMARY The contamination of poultry in the Netherlands with Salmonella enteritidis was tested. For this, different methods (detection ofS. enteritidis in faecal samples of 25 g; detection ofS. enteritidis in cloaca! swabs; detection ofS. enteritidis by serological testing of antibodies in serum) were compared for their efficiency to detect S. enteritidis in flocks of poultry. Testing ?ffaecal samples In which 14 flocksclearly yielded the best results. This method was used in a transmission study, descending from a contaminated primary mother flock were screened for the presence of S. enteritidis. The method was also used for screening 49 flocks of laying hens and52 flocks of broiler chickens throughout the Netherlands. From the transmission study it became clear that S. enteritidis, phage type 2 (Dutch phage set) was isolated both from the mother flock and from five of the descendent flocks. Screening of poultry flocks for the presence of salmonella revealed that salmonella was present in 47% of the layer flocks and in 94% of the broiler flocks. S. enteritidis was isolated from 15% of the flocks screened. INTRODUCTION
In the Netherlands exact information the occurrence of salmonellosis is not available. However, based on sentinel on studies it can be estimated that there are approximately 200,000 cases of human diarrhoea caused by salmonella yearly (1). In a number of countries S. enteritidis has emerged as a major serotype causing salMonellosis (2). Particularly in the northeastern United States and in Great Britain, there has been an epidemic increase in S. enteritidis infections during the past five years (3, 4). On the basis of surveillance studies the number of cases of salnionellosis estimated at in the Netherlands caused by this serotype in 1987 and 1988 was 7,000 and 15,000, respectively. For 1989 a total of 50,000 cases is xPected (results to be published). More and more, outbreaks ofS. enteritidis have been associated significantly with consumption of raw or poorly cooked poultry products such as poultry meat, eggs and egg products in which raw eggs are used as ingredients (5, 6, 7, 8). Evidence has been provided that transovarian transmission of S. enteritidis does occur in poultry (9, 10, 11). This transovarian transmission
is the cause of internal contamination of the In order to find ?ut more about the situation in the Netherlands, different egg. methods were tested ttor their efficiency in detecting S. enteritidis in flocks of poultry. Furthermore, .he potential for S. enteritidis transmission from a contaminated primary flock to descendent flocks was tested. Finally, the degree of contamination with salmonella of poultry in the Netherlands in the summer of 1989 was estimated. MATERIALS
AND METHODS
Comparison study of detection methods for salmonella in poultry Three
different methods were compared for their efficiency in detecting S. enteritidis in flock of poultry that had shown evidence of natural S. enteritidis infection. From this Hock 68 faecal samples, 100 cloacal swabs and 100 serum samples were taken at random. a,
I
National Institute of Public Health and Environmental Hygiene, Laboratory for Water and Food Microbiology, P.O. box I, 3720 BA Bilthoven, The Netherlands. National Box I, Institute of Public Health and Environmental Hygiene, Laboratory for Bacteriology, P.O. 3720 BA Bilthoven, The Netherlands.
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No 1,
JANUARY 1991
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Each faecal sample consisted of fresh faecal material obtained from at least 10 different places and was mixed thoroughly. Detection of S. enteritidis in faecal 1) was carried out by using a draft standard method described by ISO/DISsamples (method 6579. According to this method a 25 g portion of faeces was added to 225 ml of pre-enrichment medium. After incubation for 18 h, 0.1 ml of the culture obtained was transferred into 10 ml of selective RV medium. After incubation for 24 h and 48 h the culture in the RV medium was plated out on a selective agar medium. After incubation of the plates for 24 h colonies suspected of salmonella were confirmed biochemically and serologically. Detection of S. enteritidis in cloacal swabs (method 2) was done by using the same isolation procedure. Serum samples were tested for the presence of antibodies against by using a slide-agglutination test (method 3). This test is based on detection S. enteritidis against the 0-antigens 9, 12 and the H-antigens g, m. Antibodies against S. of antibodies enteritidis are considered to be present if agglutination occurs with S. enteritidis (0:9,12; H:g,m) and if this reaction is positive with S. pullorum (0:9,12; H:nm) and with S. godesberg (0:30; H:g,m). Additionally, three variations of method I were compared for their efficiency to detect S. enteritidis in 27 flocks of poultry. In the first place, 20 faecal samples were obtained from each flock and examined individually by using method 1 (method 1A). Secondly, those 20 faecal samples
were mixed into two pool samples, which were examined 1 (method 1B). Finally, the cultures obtained by non-selective enrichment by using method of those 20 faecal samples were mixed into two wet-pool samples, which were examined by using method 1 (method IC). All isolated strains of salmonella were sero- and phagetyped at the National Salmonella Centre (W. J. van Leeuwen and W. H. Jansen)., Transmission of S. enteritidis from a primary flock to descendent flocks A primary mother flock, which was known to be contaminated with S. enteritidis, as well as 14 descendent flocks were screened for the presence of S. enteritidis. Each faecal sample tested consisted of fresh faecal material obtained from at least five different The faecal sample was mixed and tested for the presence of salmonella by usingplaces. the abovedescribed method IA. Contamination of poultry with salmonella During the first period of 1989, 49 flocks of laying hens and 52 flocks of broiler
chickens were selected at random and screened for the presence of salmonella (especially S.
enteritidis). Each faecal sample tested consisted of fresh faecal material obtained least 10 different places. After mixing the samples were tested for the presence of from at salmonella by using the above-described method 1A. RESULTS
Methods for detection of salmonella Table IA shows the comparison between the results of, the different methods for
detection of S. enteritidis in a flock of poultry. This flock was known to be
contaminated with S. enteritidis during the breeding period. No clinical disorders were observed in this flock. In none of the 100 cloacal swabs tested (method 2) was S. enteritidis detected. However, in 26 out of 68 samples of faeces the presence of S. enteritidis was demonstrated (method 1). Of 100 serum samples tested (method 3), three gave a positive reaction. Autopsy of these three birds did not show clinical disorders, and by culturing assay (isolation according to method 1) no salmonella was isolated from any of the organs of these birds. Also, S. enteritidis was not found to be present in the caecal contents. Little difference was found between the results of the three variations of method 1 (Table 1B). S. enteritidis was isolated by at least one of the methods from four of the 27 flocks of poultry. S. enteritidis was demonstrated three times by IA and IC and twice by method 1B. Although salmonella was detected methods in all 27 flocks of poultry by at least one of the methods, one flock was found to be negative by method 1A, four flocks were found negative by method 1B, and two flocks 42
THE VETERINARY QUARTERLY. VOL. 13, No. I, JANUARY 1991
Table I.
Comparison of methods I for detection of S. enteritidis in flocks of poultry. IA: Samples obtained from one single flock number of samples
method 1 method 2 method 3
number of samples with S. enteridtidis
68
26
100
0
100
3
IB: Samples obtained from different flocks number of flocks with salmonella
with S. enteritidis
27
26
3
27
23
2
27
25
3
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number of flocks
method IA method 1B method IC I
number of flocks
method 1: detection in faecal samples of 25 g by using the draft standard method ISO/DIS 6579. method 2: detection in cloacal swabs by using the draft standard method ISO/DIS 6579. method 3: detection of antibodies in serum by using a slide-agglutination test. method IA: 20 faecal samples obtained from one flock were examined individually according to method 1.
method 1B: the 20 faecal samples from method IA were mixed into 2 pool samples, which were examined according to method I. method IC: the cultures obtained by non-selective enrichment of the 20 faecal samples of method IA were mixed into 2 wet-pool samples, which were examined according to method 1.
were found to be negative by method IC. As expected, more different serotypes were isolated from each flock by using method IA than by using methods 1B or IC. Transmission of S. enteritidis
Table 2 shows the results of the transmission study. S. enteritidis was detected in six of the 14 tested flocks descending from the mother flock. Phage type 2 (Dutch phage set) was found both in the mother flock and in five of the descendent flocks.
Contamination of poultry with salmonella
Table 3 shows the results of the screening study. Salmonella was found to be present in 23 of the 49 flocks of laying hens (47%) and in 49 of the 52 flocks of broiler chickens (94%). S. enteritidis was isolated from nine flocks of laying hens (18%) and from six flocks of broiler chickens (12%). An overview of the most frequently isolated serotypes and S. enteritidis phage types is shown in Table 4. S. infantis, S. virchow, S. typhimurium, S. enteritidis and S. hadar were isolated most frequently. Of the serotype S. enteritidis, phage type 1 was demonstrated in eight flocks and phage type 2 in three flocks. Phage types 1 and 2, according to the Dutch phage set, appear to be identical to types 4 and 8 of the Colindale system (England) (12). THE VETERINARY QUARTERLY, VOI..
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Table 2.
Occurrence' of S. enteritidis in a primary mother flock and in 14 number of flocks
primary mother flock descendent flocks
descendent flocks.
number of flocks with S. enteritidis
number of flocks with S. enteritildis phage type 22
1
1
14
6
5
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I For detection of S. enterititdis method IA was used. 2 Dutch phage set.
Table 3. Occurrence' of salmonella in flocks of laying hens and flocks of broiler-chickens on 59 poultry farms in the Netherlands.
Laying hens Broiler chickens Total
number of flocks screened
number of flocks with salmonella
number of flocks with S. enteritidis
49
23
9
52
49
6
101
71
15
' For detection of salmonella, method IA was used.
Table 4. Overview of the most frequently isolated serotypes (A) and S. enteritidis phage types' (B) in 101 flocks of poultry. 4A serotypc
number of flocks in which present
S. infantis S. virchow S. typhimurium S. enteritidis S. hadar
21 21 17
percentage of salmonella-positive flocks
15
30.9 30.9 25.0 -20.6
12
17.6
4.B
phage type S. enteritidis phage type 1 S. enteritidis phage type 2 S. enteritidis unknown types
number of flocks in which present
percentage of
8 3
53.3 20.0
7
46.6
S. enteritidis-positive flocks
'Dutch phage set.
44
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DISCUSSION
The results of the screening study make clear that a large part of the poultry in the Netherlands is contaminated with salmonella. This organism was detected in 47% of the layer flocks screened, while,94% of the broiler flocks screened were found to be salmonella-positive. S. enteritidis was isolated from 15% of the flocks tested. The actual degree of contamination with S. enteritidis in the Netherlands in the summer of 1989 can be compared with the situation in England in 1988 (12). The serotypes isolated most frequently in this study are the same as those isolated most frequently from poultry in the Netherlands in 1988, according to data of the National Salmonella Centre. It is of great importance that S. enteritidis has been emerging as a major serotype in poultry since 1988. Because of this
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tncrease the chance of being infected with S. enteritidis by consumption of improperly prepared poultry products has increased. Poorly prepared poultry meat as well as the consumption of raw or soft-boiled eggs and foods containing such ingredients may well be a hazard to public health (3, 8). In all probability the increase in the contamination of poultry with S. enteritidis is the cause of the considerable increase in human cases of salmonellosis caused by this organism during 1988 and 1989.
The results of the comparison study for detection of salmonella make clear that isolation of salmonella from faecal samples of 25 g is the best method for detection of this organism in flocks of poultry. Although there was little difference between the results of the three variations of method 1, more flocks were found to be 'false% negative by using the pool sampling method and the wet-pool sampling method.
Taking into account the extra information that is obtained by isolation of more serotypes from each flock, the examination of several samples per flock must be considered to be the most reliable method. The negative results obtained by using cloacal swabs might be due to the small amount of material that is obtained for examination in comparison with the amount of faeces obtained by using method I. The slide-agglutination test for detection of circulating antibodies against S. enteritidis was found te be unsuitable for detection of this organism in birds. Furthermore, the absence of salmonella in seropositive birds indicates that the presence of antibodies in serum may not be indicative of actual infection, as antibodies against S. enteritidis may be produced in an early stage of life. However, more sensitive immuno-assays, such as the enzyme-linked immunosorbent assay
and latex-agglutination, should be tested for their suitability as pre-screening methods. A positive test may indicate the presence of S. enteritidis or a history of S. enteritidis in the flock (13, 14).
As poultry products especially may lead to salmonellosis, efforts to reduce S. enteritidis infections in humans should focus on decreasing the contamination of poultry with this organism. Salmonella can be introduced into a flock of poultry
by different routes of infection. Newly hatched chicks as well as feed and environmental factors have been implicated in the infection of flocks (15). According to St. Louis et al. (3) both S. enteritidis and S. typhimurium are suspected of having the potential for transovarian transmission of infection. The results of our transmission study clearly demonstrate transmission of S. enteritidis from an apparently healthy primary flock to descendent flocks. However, such a transmission is not essentially transovarian. Eggs contaminated externally by faeces containing S. enteritidis contribute to the transmission of this organism as well. It is clear that supply of eggs from an infected mother flock could lead to a pyramidal increase in the degree of contamination of progeny. Measures to reduce contamination of poultry with S. enteritidis should include clearance of contaminated flocks, a proper cleaning and disinfection of contaminated poultryTHE VETERINARY QUARTERLY. Vol.. 13, No. I, JANUARY 1991
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houses and prevention of environmental contamination. Food should be free of salmonella and attention should be paid to the processing of poultry litter. The question is, however, whether these preventive measures can be effectuated adequately. Taking into account the persistence of salmonella in the environment (the organism is not host specific and multiplication occurs outside animals) (16, 17), succes by those measures is not to be expected within short time. Therefore, an additional decontamination of poultry meat by pasteurisation or irradiation should be considered to increase the safety of the product. Eggs for consumption should only be used when orginating from flocks free of S. enteritidis and S. typhimurium. ACKNOWLEDGEMENTS
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This work was carried out on behalf of the Veterinary Health Chief Inspectorate (RIVM project nr. 148122). Thanks are due to M. Hout for his practical assistance and to W. Edel for his constructive criticism. REFERENCES 1.
Hoogenboom-Verdegaal AMM, During M, Leentvaar-Kuypers A, Peerbooms PGH, Kooij WCM, Vlerken R van, and Sobczak H. Epidemiologisch en microbiologisch onderzoek met betrekking tot gastro-enteritis bij de mens in de regio's Amsterdam en Helmond, in 1987. RIVM
report no. 148612001. RIVM, The Netherlands, 1989. Report of WHO on epidemiological emergency in poultry and egg salmonellosis. Report no. WHO/CDS/VPH/89.82. WHO, 1989. 3. St. Louis ME, Morse DL, Potter ME, DeMelfi TM, Guzewich JJ, Tauxe RV, and Blake PA. The emergence of grade A eggs as a major source of Salmonella enteritidis infections. J Am Med Assoc 2.
1988; 259: 2103-7.
4. Anonymous. Salmonella enteritidis phage type 4: chicken and egg's. Lancet 1988; 2: 720-2, 5. Rowe B, Coyle EF, Ribeiro CD, Howard AJ, Palmer SR, Jones HI, and Ward L. Salmonella enteritidis phage type 4 infection: association with hen's eggs. Lancet 1988; 2: 720-2. 6. Rowe B, Mawer SL, and Spain GE. Salmonella enteritidis phage type 4 and hen's eggs. Lancet 1989; 1: 280-1.
7.
Veterinary Investigation Service. Unprecedented increase in Salmonella enteritidis food poisoning. Vet Rec 1988; 123-45.
8. Cowden JM, Lynch D, Joseph CA, O'Mahony M, Mawer SL, Rowe B, and Bartlett CLR. Case control study of infections with Salmonella enteritidis phage type 4 in England. Br Med J 1989; 299: 771-3.
9. O'Brien JDP. Salmonella enteritidis infection in broiler chickens. Vet Rec 1988; 122: 214. 10. Lister SA. Salmonella enteritidis in broilers and broiler breeders. Vet Rec 1988; 123: 350. I 1. Hopper SA and Mawer SL. Salmonella enteritidis in a commercial layer flock. Vet Rec 1988; 123: 351.
12. Rowe B. Central Public Health Laboratory, London, UK, personal communication, 1989. 13. Cooper GL, Nicholas RA, and Bracewell CD. Serological and bActeriological investigations of chickens from flocks naturally infected with Salmonella enteritidis. Vet Rec 1989; 125: 567-72. 14. Barrow P, Hassan JO, Mocket APA, and McLeod S. Detection of Salmonella infection by ELISA.
Vet Rec 1989; 125: 586. d'Aoust JY. Salmonella. In: Doyle MP (Ed), Foodborne Bacterial Pathogens, 1989; Chapt 5, 328447, M Dekker, New York, Basel. 16. Edel W, Guinee PAM, Schothorst M van, and Kampelmacher EH. Salmonella cycles in foods with special reference to the effects of environmental factors, including feeds. Proc Symp Microbiol, Food Borne Infections and Intoxications, Ottawa, 1973. 17. Edel W, Schothorst M van, and Kampelmacher EH. Epidemiologisch Salmonella onderzoek in een bepaald gebied. Tijdschr Diergeneeskd 1975; 100: 1304-11. 15.
46
THE VETERINARY QUARTERLY. VOL.
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JANUARY 1991
Veterinary Quarterly Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tveq20
Salmonella contamination of poultry flocks in the Netherlands a
a
a
A. W. v.d. Giessen , R. Peters , P.A.T.A. Berkers , b
W. H. Jansen & S. H. W. Notermans
a
a
Laboratory for Water and Food Microbiology , National Institute of Public Health and Environmental Hygiene , P.O. box 1, Bilthoven, 3720 BA, The Netherlands b
Laboratory for Bacteriology , National Institute of Public Health and Environmental Hygiene , P.O. Box 1, Bilthoven, 3720 BA, The Netherlands Published online: 01 Nov 2011.
To cite this article: A. W. v.d. Giessen , R. Peters , P.A.T.A. Berkers , W. H. Jansen & S. H. W. Notermans (1991) Salmonella contamination of poultry flocks in the Netherlands, Veterinary Quarterly, 13:1, 41-46, DOI: 10.1080/01652176.1991.9694283 To link to this article: http://dx.doi.org/10.1080/01652176.1991.9694283
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sublicensing, systematic supply, or distribution in any form to anyone is expressly
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Salmonella contamination of poultry flocks in the Netherlands A. W. v.d. Giessen', R. Peters', P.A.T.A. Berkersi, W. H. Jansen2, and S. H. W. Notermansl
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SUMMARY The contamination of poultry in the Netherlands with Salmonella enteritidis was tested. For this, different methods (detection ofS. enteritidis in faecal samples of 25 g; detection ofS. enteritidis in cloaca! swabs; detection ofS. enteritidis by serological testing of antibodies in serum) were compared for their efficiency to detect S. enteritidis in flocks of poultry. Testing ?ffaecal samples In which 14 flocksclearly yielded the best results. This method was used in a transmission study, descending from a contaminated primary mother flock were screened for the presence of S. enteritidis. The method was also used for screening 49 flocks of laying hens and52 flocks of broiler chickens throughout the Netherlands. From the transmission study it became clear that S. enteritidis, phage type 2 (Dutch phage set) was isolated both from the mother flock and from five of the descendent flocks. Screening of poultry flocks for the presence of salmonella revealed that salmonella was present in 47% of the layer flocks and in 94% of the broiler flocks. S. enteritidis was isolated from 15% of the flocks screened. INTRODUCTION
In the Netherlands exact information the occurrence of salmonellosis is not available. However, based on sentinel on studies it can be estimated that there are approximately 200,000 cases of human diarrhoea caused by salmonella yearly (1). In a number of countries S. enteritidis has emerged as a major serotype causing salMonellosis (2). Particularly in the northeastern United States and in Great Britain, there has been an epidemic increase in S. enteritidis infections during the past five years (3, 4). On the basis of surveillance studies the number of cases of salnionellosis estimated at in the Netherlands caused by this serotype in 1987 and 1988 was 7,000 and 15,000, respectively. For 1989 a total of 50,000 cases is xPected (results to be published). More and more, outbreaks ofS. enteritidis have been associated significantly with consumption of raw or poorly cooked poultry products such as poultry meat, eggs and egg products in which raw eggs are used as ingredients (5, 6, 7, 8). Evidence has been provided that transovarian transmission of S. enteritidis does occur in poultry (9, 10, 11). This transovarian transmission
is the cause of internal contamination of the In order to find ?ut more about the situation in the Netherlands, different egg. methods were tested ttor their efficiency in detecting S. enteritidis in flocks of poultry. Furthermore, .he potential for S. enteritidis transmission from a contaminated primary flock to descendent flocks was tested. Finally, the degree of contamination with salmonella of poultry in the Netherlands in the summer of 1989 was estimated. MATERIALS
AND METHODS
Comparison study of detection methods for salmonella in poultry Three
different methods were compared for their efficiency in detecting S. enteritidis in flock of poultry that had shown evidence of natural S. enteritidis infection. From this Hock 68 faecal samples, 100 cloacal swabs and 100 serum samples were taken at random. a,
I
National Institute of Public Health and Environmental Hygiene, Laboratory for Water and Food Microbiology, P.O. box I, 3720 BA Bilthoven, The Netherlands. National Box I, Institute of Public Health and Environmental Hygiene, Laboratory for Bacteriology, P.O. 3720 BA Bilthoven, The Netherlands.
THE VETERINARY
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No 1,
JANUARY 1991
41
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Each faecal sample consisted of fresh faecal material obtained from at least 10 different places and was mixed thoroughly. Detection of S. enteritidis in faecal 1) was carried out by using a draft standard method described by ISO/DISsamples (method 6579. According to this method a 25 g portion of faeces was added to 225 ml of pre-enrichment medium. After incubation for 18 h, 0.1 ml of the culture obtained was transferred into 10 ml of selective RV medium. After incubation for 24 h and 48 h the culture in the RV medium was plated out on a selective agar medium. After incubation of the plates for 24 h colonies suspected of salmonella were confirmed biochemically and serologically. Detection of S. enteritidis in cloacal swabs (method 2) was done by using the same isolation procedure. Serum samples were tested for the presence of antibodies against by using a slide-agglutination test (method 3). This test is based on detection S. enteritidis against the 0-antigens 9, 12 and the H-antigens g, m. Antibodies against S. of antibodies enteritidis are considered to be present if agglutination occurs with S. enteritidis (0:9,12; H:g,m) and if this reaction is positive with S. pullorum (0:9,12; H:nm) and with S. godesberg (0:30; H:g,m). Additionally, three variations of method I were compared for their efficiency to detect S. enteritidis in 27 flocks of poultry. In the first place, 20 faecal samples were obtained from each flock and examined individually by using method 1 (method 1A). Secondly, those 20 faecal samples
were mixed into two pool samples, which were examined 1 (method 1B). Finally, the cultures obtained by non-selective enrichment by using method of those 20 faecal samples were mixed into two wet-pool samples, which were examined by using method 1 (method IC). All isolated strains of salmonella were sero- and phagetyped at the National Salmonella Centre (W. J. van Leeuwen and W. H. Jansen)., Transmission of S. enteritidis from a primary flock to descendent flocks A primary mother flock, which was known to be contaminated with S. enteritidis, as well as 14 descendent flocks were screened for the presence of S. enteritidis. Each faecal sample tested consisted of fresh faecal material obtained from at least five different The faecal sample was mixed and tested for the presence of salmonella by usingplaces. the abovedescribed method IA. Contamination of poultry with salmonella During the first period of 1989, 49 flocks of laying hens and 52 flocks of broiler
chickens were selected at random and screened for the presence of salmonella (especially S.
enteritidis). Each faecal sample tested consisted of fresh faecal material obtained least 10 different places. After mixing the samples were tested for the presence of from at salmonella by using the above-described method 1A. RESULTS
Methods for detection of salmonella Table IA shows the comparison between the results of, the different methods for
detection of S. enteritidis in a flock of poultry. This flock was known to be
contaminated with S. enteritidis during the breeding period. No clinical disorders were observed in this flock. In none of the 100 cloacal swabs tested (method 2) was S. enteritidis detected. However, in 26 out of 68 samples of faeces the presence of S. enteritidis was demonstrated (method 1). Of 100 serum samples tested (method 3), three gave a positive reaction. Autopsy of these three birds did not show clinical disorders, and by culturing assay (isolation according to method 1) no salmonella was isolated from any of the organs of these birds. Also, S. enteritidis was not found to be present in the caecal contents. Little difference was found between the results of the three variations of method 1 (Table 1B). S. enteritidis was isolated by at least one of the methods from four of the 27 flocks of poultry. S. enteritidis was demonstrated three times by IA and IC and twice by method 1B. Although salmonella was detected methods in all 27 flocks of poultry by at least one of the methods, one flock was found to be negative by method 1A, four flocks were found negative by method 1B, and two flocks 42
THE VETERINARY QUARTERLY. VOL. 13, No. I, JANUARY 1991
Table I.
Comparison of methods I for detection of S. enteritidis in flocks of poultry. IA: Samples obtained from one single flock number of samples
method 1 method 2 method 3
number of samples with S. enteridtidis
68
26
100
0
100
3
IB: Samples obtained from different flocks number of flocks with salmonella
with S. enteritidis
27
26
3
27
23
2
27
25
3
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number of flocks
method IA method 1B method IC I
number of flocks
method 1: detection in faecal samples of 25 g by using the draft standard method ISO/DIS 6579. method 2: detection in cloacal swabs by using the draft standard method ISO/DIS 6579. method 3: detection of antibodies in serum by using a slide-agglutination test. method IA: 20 faecal samples obtained from one flock were examined individually according to method 1.
method 1B: the 20 faecal samples from method IA were mixed into 2 pool samples, which were examined according to method I. method IC: the cultures obtained by non-selective enrichment of the 20 faecal samples of method IA were mixed into 2 wet-pool samples, which were examined according to method 1.
were found to be negative by method IC. As expected, more different serotypes were isolated from each flock by using method IA than by using methods 1B or IC. Transmission of S. enteritidis
Table 2 shows the results of the transmission study. S. enteritidis was detected in six of the 14 tested flocks descending from the mother flock. Phage type 2 (Dutch phage set) was found both in the mother flock and in five of the descendent flocks.
Contamination of poultry with salmonella
Table 3 shows the results of the screening study. Salmonella was found to be present in 23 of the 49 flocks of laying hens (47%) and in 49 of the 52 flocks of broiler chickens (94%). S. enteritidis was isolated from nine flocks of laying hens (18%) and from six flocks of broiler chickens (12%). An overview of the most frequently isolated serotypes and S. enteritidis phage types is shown in Table 4. S. infantis, S. virchow, S. typhimurium, S. enteritidis and S. hadar were isolated most frequently. Of the serotype S. enteritidis, phage type 1 was demonstrated in eight flocks and phage type 2 in three flocks. Phage types 1 and 2, according to the Dutch phage set, appear to be identical to types 4 and 8 of the Colindale system (England) (12). THE VETERINARY QUARTERLY, VOI..
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Table 2.
Occurrence' of S. enteritidis in a primary mother flock and in 14 number of flocks
primary mother flock descendent flocks
descendent flocks.
number of flocks with S. enteritidis
number of flocks with S. enteritildis phage type 22
1
1
14
6
5
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I For detection of S. enterititdis method IA was used. 2 Dutch phage set.
Table 3. Occurrence' of salmonella in flocks of laying hens and flocks of broiler-chickens on 59 poultry farms in the Netherlands.
Laying hens Broiler chickens Total
number of flocks screened
number of flocks with salmonella
number of flocks with S. enteritidis
49
23
9
52
49
6
101
71
15
' For detection of salmonella, method IA was used.
Table 4. Overview of the most frequently isolated serotypes (A) and S. enteritidis phage types' (B) in 101 flocks of poultry. 4A serotypc
number of flocks in which present
S. infantis S. virchow S. typhimurium S. enteritidis S. hadar
21 21 17
percentage of salmonella-positive flocks
15
30.9 30.9 25.0 -20.6
12
17.6
4.B
phage type S. enteritidis phage type 1 S. enteritidis phage type 2 S. enteritidis unknown types
number of flocks in which present
percentage of
8 3
53.3 20.0
7
46.6
S. enteritidis-positive flocks
'Dutch phage set.
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DISCUSSION
The results of the screening study make clear that a large part of the poultry in the Netherlands is contaminated with salmonella. This organism was detected in 47% of the layer flocks screened, while,94% of the broiler flocks screened were found to be salmonella-positive. S. enteritidis was isolated from 15% of the flocks tested. The actual degree of contamination with S. enteritidis in the Netherlands in the summer of 1989 can be compared with the situation in England in 1988 (12). The serotypes isolated most frequently in this study are the same as those isolated most frequently from poultry in the Netherlands in 1988, according to data of the National Salmonella Centre. It is of great importance that S. enteritidis has been emerging as a major serotype in poultry since 1988. Because of this
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tncrease the chance of being infected with S. enteritidis by consumption of improperly prepared poultry products has increased. Poorly prepared poultry meat as well as the consumption of raw or soft-boiled eggs and foods containing such ingredients may well be a hazard to public health (3, 8). In all probability the increase in the contamination of poultry with S. enteritidis is the cause of the considerable increase in human cases of salmonellosis caused by this organism during 1988 and 1989.
The results of the comparison study for detection of salmonella make clear that isolation of salmonella from faecal samples of 25 g is the best method for detection of this organism in flocks of poultry. Although there was little difference between the results of the three variations of method 1, more flocks were found to be 'false% negative by using the pool sampling method and the wet-pool sampling method.
Taking into account the extra information that is obtained by isolation of more serotypes from each flock, the examination of several samples per flock must be considered to be the most reliable method. The negative results obtained by using cloacal swabs might be due to the small amount of material that is obtained for examination in comparison with the amount of faeces obtained by using method I. The slide-agglutination test for detection of circulating antibodies against S. enteritidis was found te be unsuitable for detection of this organism in birds. Furthermore, the absence of salmonella in seropositive birds indicates that the presence of antibodies in serum may not be indicative of actual infection, as antibodies against S. enteritidis may be produced in an early stage of life. However, more sensitive immuno-assays, such as the enzyme-linked immunosorbent assay
and latex-agglutination, should be tested for their suitability as pre-screening methods. A positive test may indicate the presence of S. enteritidis or a history of S. enteritidis in the flock (13, 14).
As poultry products especially may lead to salmonellosis, efforts to reduce S. enteritidis infections in humans should focus on decreasing the contamination of poultry with this organism. Salmonella can be introduced into a flock of poultry
by different routes of infection. Newly hatched chicks as well as feed and environmental factors have been implicated in the infection of flocks (15). According to St. Louis et al. (3) both S. enteritidis and S. typhimurium are suspected of having the potential for transovarian transmission of infection. The results of our transmission study clearly demonstrate transmission of S. enteritidis from an apparently healthy primary flock to descendent flocks. However, such a transmission is not essentially transovarian. Eggs contaminated externally by faeces containing S. enteritidis contribute to the transmission of this organism as well. It is clear that supply of eggs from an infected mother flock could lead to a pyramidal increase in the degree of contamination of progeny. Measures to reduce contamination of poultry with S. enteritidis should include clearance of contaminated flocks, a proper cleaning and disinfection of contaminated poultryTHE VETERINARY QUARTERLY. Vol.. 13, No. I, JANUARY 1991
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houses and prevention of environmental contamination. Food should be free of salmonella and attention should be paid to the processing of poultry litter. The question is, however, whether these preventive measures can be effectuated adequately. Taking into account the persistence of salmonella in the environment (the organism is not host specific and multiplication occurs outside animals) (16, 17), succes by those measures is not to be expected within short time. Therefore, an additional decontamination of poultry meat by pasteurisation or irradiation should be considered to increase the safety of the product. Eggs for consumption should only be used when orginating from flocks free of S. enteritidis and S. typhimurium. ACKNOWLEDGEMENTS
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This work was carried out on behalf of the Veterinary Health Chief Inspectorate (RIVM project nr. 148122). Thanks are due to M. Hout for his practical assistance and to W. Edel for his constructive criticism. REFERENCES 1.
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report no. 148612001. RIVM, The Netherlands, 1989. Report of WHO on epidemiological emergency in poultry and egg salmonellosis. Report no. WHO/CDS/VPH/89.82. WHO, 1989. 3. St. Louis ME, Morse DL, Potter ME, DeMelfi TM, Guzewich JJ, Tauxe RV, and Blake PA. The emergence of grade A eggs as a major source of Salmonella enteritidis infections. J Am Med Assoc 2.
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Vet Rec 1989; 125: 586. d'Aoust JY. Salmonella. In: Doyle MP (Ed), Foodborne Bacterial Pathogens, 1989; Chapt 5, 328447, M Dekker, New York, Basel. 16. Edel W, Guinee PAM, Schothorst M van, and Kampelmacher EH. Salmonella cycles in foods with special reference to the effects of environmental factors, including feeds. Proc Symp Microbiol, Food Borne Infections and Intoxications, Ottawa, 1973. 17. Edel W, Schothorst M van, and Kampelmacher EH. Epidemiologisch Salmonella onderzoek in een bepaald gebied. Tijdschr Diergeneeskd 1975; 100: 1304-11. 15.
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