Salmonella radicidation of poultry carcasses

R.W.A.W. Mulder

    Research output: Thesisexternal PhD, WU

    Abstract

    <u>Validity of methods</u><p/>Experiments were carried out In which it was assessed which <u>Salmonella</u> isolation method is the most productive one In the examination of broiler carcasses. Refrigerated, refrigerated and radiated (2.50 kGy), frozen and frozen and radiated (2.50 kGy) samples of broilers were examined. After evaluation of all results It was concluded that the following method was the most productive one:<br/>1. pre-enrichment in buffered peptone water at 37 °C for 20 hours<br/>2. enrichment in selenite cystine broth at 37 °C and at 43 °C for 24 hours<br/>3. selective plating on brilliant green sulphapyridine agar and XLD- agar at 37 °C for 24 hours<p/>From this study It could not be concluded that other methods were not sufficiently reliable to be applied in other situations.<p/>However, it became clear that when sublethally damaged salmonellae are expected to be present, and when, as a result of the treatment, a different competitive microflora is present, adapted isolation methods should be used.<p/>The method chosen for <u>Salmonella</u> -isolation in the experiments described in this thesis was the ISO 3565/1975 reference method for isolation of salmonellae from meat and meat products. This method was ultimately preferred because of the experience obtained for many years in routine analyses.<p/>In two Instances other conditions than prescribed by the ISO-method were used; i.e. the samples were incubated at 37 °C or 43°C for 24 hours. The ISO-method as well as its slight modification proved to be reliable methods in the experiments reported.<p/><u>Escherichia coli versus Salmonella.</u><p/>The experiments have shown that the <u>Escherichia coli</u> K12 NDA strain tested can not be used as index microorganism for <u>Salmonella</u> : in this case <u>Salmonella niloese</u> and <u>Salmonella panama</u> on broilers, though in liquid and solid culture media the D <sub><font size="-1">10</font></sub> -values of these three microorganisms were similar.<p/>The experiments with pure cultures and with artificially contaminated carcasses showed that the following factors influenced D <sub><font size="-1">10</font></sub> -values:<br/>1. the culture media used before, during and after irradiation;<br/>2. the temperature during irradiation;<br/>3. the physiological age of the bacteria.<p/>No influence on D <sub><font size="-1">10</font></sub> -values could be observed from<br/>1. the initial cell concentration;<br/>2. the oxygen partial pressure.<p/>The results of the experiments showed that after a radiation treatment with 2.50 kGy <u>Salmonella</u> -positive samples were found; In those cases the numbers of cfu's of <u>Enterobacteriaceae</u> were below the detection limit. This indicates that no other microorganism or groups of microorganisms can consistently serve as the sole index microorganism(s) for <u>Salmonella.</u><p/><u>Effect of 2.50 kGy on salmonellae and Enterobacteriaceae.</u><p/>Poultry carcasses were found frequently contaminated with salmonellae. Per carcass a maximum number of 1,400 cfu per 1,000 cm <sup><font size="-1">2</font></SUP>skin were estimated by a MPN method. However, 90% of the total of the examined carcasses in this specific experiment contained less than 100 cfu's per 1,000 cm <sup><font size="-1">2</font></SUP>. Similar data are given in literature (161,200,260,306).<p/>A radicidation treatment with a dose of 2.50 kGy markedly reduced the total number of <u>Salmonella</u> -positive carcasses, as well as the number of salmonellae per carcass.<p/>This dose, however, could not guarantee a <u>Salmonella</u> -"free" product. The number of <u>Salmonella</u> contaminated carcasses was reduced tenfold (table 38). After irradiation at +5°C with a dose of 2.50 kGy two series of pooled samples of broiler skin were found <u>Salmonella</u> -positive by a MPN method. The serotypes isolated were agona and infantis.<p/>Before the radiation treatment these samples proved to be very sparsely contaminated with salmonellae (numbers of 2 cfu per 1,000 cm <sup><font size="-1">2</font></SUP>).<p/>It was shown in previous experiments that the D <sub><font size="-1">10</font></sub> -values of these two serotypes were comparable to other serotypes, so the two did not belong to a radio-resistant group.<p/><u>Salmonella</u> -positive samples which were observed in experiments in which they were expected to be absent, according to their estimated D <sub><font size="-1">10</font></sub> -values, were also found in :<br/>1. experiments in which the resulting microflora after certain radiation treatments was identified;<br/>2. experiments in which the effect on Salmonella of freezing and storage of radiated carcasses was studied.<p/>In some experiments artificially contaminated carcasses were used. In the case <u>of Salmonella</u> niloese and <u>Salmonella panama</u> a protective action was afforded by the carcass skin. This resulted in D <sub><font size="-1">10</font></sub> -values of 1.07 and 1.29 kGy respectively, when irradiation was carried out at -18 °C and in D <sub><font size="-1">10</font></sub> -values of 0.62 and 0.67 kGy at +5 °C.<p/>The D <sub><font size="-1">10</font></sub> -values of <u>Salmonella</u><u>panama</u> estimated in skin samples were found higher (P&lt;0.01) than the values which were estimated under optimal conditions In liquid culture media, when the D <sub><font size="-1">10</font></sub> -values was 0.52 kGy. Different factors as discussed earlier exert an influence on D <sub><font size="-1">10</font></sub> -values and therefore on the lethality of the radiation treatment. Only speculations can be made on the mechanism of the protective action of skin. Perhaps salmonellae meet less competition from the naturally present microflora than <u>Escherichia coli</u> , resulting in a stronger attachment to the skin. It is possible that this protection is provided by skin components or by extracellular polysaccharides which are produced by attached bacteria (14,216).<p/>A model was designed to assess the probability of survival after irradiation. Following the model assumptions it was shown that the detection level of the <u>Salmonella</u> -isolation methods employed was very important in the finding of <u>Salmonella</u> -positive samples after irradiation, and hence the estimated lethality of a given radiation treatment. Several bacteria, such as species of <u>Alcaligenes</u> , <u>Escherichia</u> , <u>Pseudomonas</u> and <u>Salmonella</u> were found in radiation treated products in which they were not expected according to their D <sub><font size="-1">10</font></sub> -values.<p/>The application of low doses of ionizing radiation followed by freezing and storage of the products during a certain period had more success In reaching the final goal : a <u>Salmonella</u> -"free" end product.<p/>To demonstrate a correlation between the number of <u>Enterobacteriaceae</u> and the number of salmonellae present per carcass, also <u>Enterobacteriaceae</u> cfu counts were assessed.<p/>Surprising was the presence of <u>Salmonella</u> cfu in samples of broiler skin on which the number of <u>Enterobacteriaceae</u> cfu's were below 10 per 10 cm <sup><font size="-1">2</font></SUP>of skin. This effect was not found in samples of thaw water.<p/>A difference in lethal effect on salmonellae was not found between irradiation at +5°C and at -18°C. In literature (207) proof of differences in lethal effects of irradiation carried out at different temperatures have been reported.<p/>The results obtained with these experiments point to a preference for the application of the radiation treatment at +5 °C but the statistical superiority over the other results was not proven.<p/>By means of estimation of the number of cfu's of <u>Enterobacteriaceae</u> no answer to this question could be obtained. All counts of cfu's proved less than 10 per 10 cm <sup><font size="-1">2</font></SUP>or per ml. After a three months' storage period at -18°C. both groups of radiation treated carcasses were <u>Salmonel1a</u> -negative. This was not the case with untreated carcasses, from which <u>Salmonella</u> could be isolated during the whole storage period.<p/>In summary, it can be said that irradiation of broiler carcasses with a dose of 2.50 kGy results in a decrease of the total number of <u>Salmonella</u> contaminated carcasses as well as of the number of salmonellae per carcass. As the experiments showed very promising results with these combined treatments, further investigations should be made in the field of the application of the combined use of freezing and irradiation. <u></u><p/><u>Surviving microflora.</u><p/>The microflora surviving after irradiation with 2.50 kGy will not cause unexpected problems as demonstrated by the nature and the composition of the microflora. The residual "total" count of 100 - 1000 cells per 10 cm2 or per m] consists of bacilli, micrococci, streptococci, yeasts and moulds. Sporadically an <u>Enterobacteriaceae</u> was also isolated.<p/>No stimulating effect as suggested in literature was observed in the enumeration of various microorganisms, especially <u>Staphylococcus aureus</u> and <u>Salmonella</u> , when catalase was added to the surface of the selective agar media. The mechanism of the beneficial effect of catalase on the enumeration of yeasts and moulds and of the inhibitive effect on cfu's of <u>Bacillus cereus</u> should be studied in more detail in future. Solid medium repair methods used in the enumeration of stressed microorganisms did not result in better recoveries of the microorganisms investigated.<p/>The presence of <u>Salmonella</u> in the end product after irradiation with 2.50 kGY shows that this dose does not guarantee a <u>Salmonella</u> -"free" end product. The question arose which dose is needed to guarantee this. The broiler skin and irradiation in the frozen condition provided a protection of <u>Salmonella</u> against irradiation. The highest D <sub><font size="-1">10</font></sub> -value found was approx. 1.29 kGy. If the 7 D <sub><font size="-1">10</font></sub> -concept is applied a dose of approx. 9.00 kGy at -18 °C is needed for thaw water.<p/>This dose is rather high, but nowadays doses up to 10.00 kGy are accepted by international organizations. In case of poultry products it is expected that at this dose quality defects will occur.<p/>If "absence" of <u>Salmonella</u> in radiated skin samples is required a dose of 3.90 kGy is needed with irradiation at -18°C and a dose of 1.95 kGy at +50 °C. The data presented, however, show that such high doses are not necessary. Experiments have shown that the number of salmonellae per carcass will be lower than 100 cfu. An additional storage period for 1 to 3 months at -18 °C leads to a decrease in the number of salmonellae per carcass. In literature (259) it was shown that freezing resulted in an 0.5 log cycle reduction of the number of salmonellae cfu. The application of a radiation dose of 2.50 kGy does not guarantee a <u>Salmonella</u> -"free" end product. The microbiological quality, with respect to total counts and to the presence of potentially pathogenic microorganisms, is Improved In comparison with the untreated slaughtered product. <u></u><p/><u>Practical consequences.</u><p/>The application of a radiation dose of 2.50 kGy was not sufficient to destroy all salmonellae present per carcass.<p/>The surviving microflora of the carcasses consisted of bacilli. micrococcl, streptococci, yeasts and moulds. The final counts on the radiation treated carcasses were so low that no dangerous effect can be expected.<p/><u>Salmonella</u> -contaminated broiler carcasses contained on an average 100 cfu's per carcass (minimum : 2, maximum : 19400).<p/>After irradiation only 2 series of pooled skin samples contained 2 cfu. D <sub><font size="-1">10</font></sub> -values for <u>Salmonella panama</u> after irradiation at +5°C and at -18 °C were 0.67 kGy and 1.29 kGy respectively.<p/>As can be seen from these D <sub><font size="-1">10</font></sub> -values and from the number of <u>Salmonella</u> cfu's per carcass, generally, the carcasses will be <u>Salmonella</u> -negative after irradiation with 2.50 kGy. An extra effect on the number of <u>Salmonella</u> -positive carcasses as well as on the number of salmonellae per carcass can be obtained by freezing and additional storage for several months' at -18 °C<p/>An extrapolation to the whole Dutch broiler production of the lethal effect of the radiation treatment as measured in the experiments described may not be made, because of uncertainty with respect to the variance in average numbers of <u>Salmonella</u> cfu's per carcass, the variance in D <sub><font size="-1">10</font></sub> -values and the <u>Salmonella</u> detection level obtained by the <u>Salmonella</u> -isolation method used.<p/>Nevertheless the results presented here indicate that in case the whole Dutch broiler production (approx. 3.0 <strong>.</strong> 10 <sup><font size="-1">7</font></SUP>carcasses) is treated with a dose of 2.50 kGy and assuming a D <sub><font size="-1">10</font></sub> -value of 0.80 kGy, the maximal result would be 1 positive carcass in 55 carcasses, which means that the number of <u>Salmonella</u> contaminated carcasses is decreased 14 times.<p/>Additional storage for several months' at -18 °C affords an extra beneficial effect to this respect.<p/>A combined treatment of freezing and Irradiation can be recommended to reduce the dissemination of <u>Salmonella.</u>
    Original languageEnglish
    QualificationDoctor of Philosophy
    Awarding Institution
    Supervisors/Advisors
    • Kampelmacher, E.H., Promotor, External person
    • Mossel, D.A.A., Co-promotor, External person
    Award date19 May 1982
    Place of PublicationWageningen
    Publisher
    Publication statusPublished - 1982

    Fingerprint

    poultry carcasses
    Salmonella
    irradiation
    microorganisms
    dosage
    Enterobacteriaceae
    broiler chickens
    isolation techniques
    Salmonella Panama
    freezing
    sampling

    Keywords

    • poultry meat
    • chicken meat
    • duck meat
    • animal products
    • bacteriology
    • bacterial count
    • adulteration
    • contamination
    • aging
    • defects
    • deterioration
    • netherlands

    Cite this

    Mulder, R. W. A. W. (1982). Salmonella radicidation of poultry carcasses. Wageningen: Mulder.
    Mulder, R.W.A.W.. / Salmonella radicidation of poultry carcasses. Wageningen : Mulder, 1982. 158 p.
    @phdthesis{159755ba405f4214a68a03d3bc6358da,
    title = "Salmonella radicidation of poultry carcasses",
    abstract = "Validity of methodsExperiments were carried out In which it was assessed which Salmonella isolation method is the most productive one In the examination of broiler carcasses. Refrigerated, refrigerated and radiated (2.50 kGy), frozen and frozen and radiated (2.50 kGy) samples of broilers were examined. After evaluation of all results It was concluded that the following method was the most productive one:1. pre-enrichment in buffered peptone water at 37 °C for 20 hours2. enrichment in selenite cystine broth at 37 °C and at 43 °C for 24 hours3. selective plating on brilliant green sulphapyridine agar and XLD- agar at 37 °C for 24 hoursFrom this study It could not be concluded that other methods were not sufficiently reliable to be applied in other situations.However, it became clear that when sublethally damaged salmonellae are expected to be present, and when, as a result of the treatment, a different competitive microflora is present, adapted isolation methods should be used.The method chosen for Salmonella -isolation in the experiments described in this thesis was the ISO 3565/1975 reference method for isolation of salmonellae from meat and meat products. This method was ultimately preferred because of the experience obtained for many years in routine analyses.In two Instances other conditions than prescribed by the ISO-method were used; i.e. the samples were incubated at 37 °C or 43°C for 24 hours. The ISO-method as well as its slight modification proved to be reliable methods in the experiments reported.Escherichia coli versus Salmonella.The experiments have shown that the Escherichia coli K12 NDA strain tested can not be used as index microorganism for Salmonella : in this case Salmonella niloese and Salmonella panama on broilers, though in liquid and solid culture media the D 10 -values of these three microorganisms were similar.The experiments with pure cultures and with artificially contaminated carcasses showed that the following factors influenced D 10 -values:1. the culture media used before, during and after irradiation;2. the temperature during irradiation;3. the physiological age of the bacteria.No influence on D 10 -values could be observed from1. the initial cell concentration;2. the oxygen partial pressure.The results of the experiments showed that after a radiation treatment with 2.50 kGy Salmonella -positive samples were found; In those cases the numbers of cfu's of Enterobacteriaceae were below the detection limit. This indicates that no other microorganism or groups of microorganisms can consistently serve as the sole index microorganism(s) for Salmonella.Effect of 2.50 kGy on salmonellae and Enterobacteriaceae.Poultry carcasses were found frequently contaminated with salmonellae. Per carcass a maximum number of 1,400 cfu per 1,000 cm 2skin were estimated by a MPN method. However, 90{\%} of the total of the examined carcasses in this specific experiment contained less than 100 cfu's per 1,000 cm 2. Similar data are given in literature (161,200,260,306).A radicidation treatment with a dose of 2.50 kGy markedly reduced the total number of Salmonella -positive carcasses, as well as the number of salmonellae per carcass.This dose, however, could not guarantee a Salmonella -{"}free{"} product. The number of Salmonella contaminated carcasses was reduced tenfold (table 38). After irradiation at +5°C with a dose of 2.50 kGy two series of pooled samples of broiler skin were found Salmonella -positive by a MPN method. The serotypes isolated were agona and infantis.Before the radiation treatment these samples proved to be very sparsely contaminated with salmonellae (numbers of 2 cfu per 1,000 cm 2).It was shown in previous experiments that the D 10 -values of these two serotypes were comparable to other serotypes, so the two did not belong to a radio-resistant group.Salmonella -positive samples which were observed in experiments in which they were expected to be absent, according to their estimated D 10 -values, were also found in :1. experiments in which the resulting microflora after certain radiation treatments was identified;2. experiments in which the effect on Salmonella of freezing and storage of radiated carcasses was studied.In some experiments artificially contaminated carcasses were used. In the case of Salmonella niloese and Salmonella panama a protective action was afforded by the carcass skin. This resulted in D 10 -values of 1.07 and 1.29 kGy respectively, when irradiation was carried out at -18 °C and in D 10 -values of 0.62 and 0.67 kGy at +5 °C.The D 10 -values of Salmonellapanama estimated in skin samples were found higher (P<0.01) than the values which were estimated under optimal conditions In liquid culture media, when the D 10 -values was 0.52 kGy. Different factors as discussed earlier exert an influence on D 10 -values and therefore on the lethality of the radiation treatment. Only speculations can be made on the mechanism of the protective action of skin. Perhaps salmonellae meet less competition from the naturally present microflora than Escherichia coli , resulting in a stronger attachment to the skin. It is possible that this protection is provided by skin components or by extracellular polysaccharides which are produced by attached bacteria (14,216).A model was designed to assess the probability of survival after irradiation. Following the model assumptions it was shown that the detection level of the Salmonella -isolation methods employed was very important in the finding of Salmonella -positive samples after irradiation, and hence the estimated lethality of a given radiation treatment. Several bacteria, such as species of Alcaligenes , Escherichia , Pseudomonas and Salmonella were found in radiation treated products in which they were not expected according to their D 10 -values.The application of low doses of ionizing radiation followed by freezing and storage of the products during a certain period had more success In reaching the final goal : a Salmonella -{"}free{"} end product.To demonstrate a correlation between the number of Enterobacteriaceae and the number of salmonellae present per carcass, also Enterobacteriaceae cfu counts were assessed.Surprising was the presence of Salmonella cfu in samples of broiler skin on which the number of Enterobacteriaceae cfu's were below 10 per 10 cm 2of skin. This effect was not found in samples of thaw water.A difference in lethal effect on salmonellae was not found between irradiation at +5°C and at -18°C. In literature (207) proof of differences in lethal effects of irradiation carried out at different temperatures have been reported.The results obtained with these experiments point to a preference for the application of the radiation treatment at +5 °C but the statistical superiority over the other results was not proven.By means of estimation of the number of cfu's of Enterobacteriaceae no answer to this question could be obtained. All counts of cfu's proved less than 10 per 10 cm 2or per ml. After a three months' storage period at -18°C. both groups of radiation treated carcasses were Salmonel1a -negative. This was not the case with untreated carcasses, from which Salmonella could be isolated during the whole storage period.In summary, it can be said that irradiation of broiler carcasses with a dose of 2.50 kGy results in a decrease of the total number of Salmonella contaminated carcasses as well as of the number of salmonellae per carcass. As the experiments showed very promising results with these combined treatments, further investigations should be made in the field of the application of the combined use of freezing and irradiation. Surviving microflora.The microflora surviving after irradiation with 2.50 kGy will not cause unexpected problems as demonstrated by the nature and the composition of the microflora. The residual {"}total{"} count of 100 - 1000 cells per 10 cm2 or per m] consists of bacilli, micrococci, streptococci, yeasts and moulds. Sporadically an Enterobacteriaceae was also isolated.No stimulating effect as suggested in literature was observed in the enumeration of various microorganisms, especially Staphylococcus aureus and Salmonella , when catalase was added to the surface of the selective agar media. The mechanism of the beneficial effect of catalase on the enumeration of yeasts and moulds and of the inhibitive effect on cfu's of Bacillus cereus should be studied in more detail in future. Solid medium repair methods used in the enumeration of stressed microorganisms did not result in better recoveries of the microorganisms investigated.The presence of Salmonella in the end product after irradiation with 2.50 kGY shows that this dose does not guarantee a Salmonella -{"}free{"} end product. The question arose which dose is needed to guarantee this. The broiler skin and irradiation in the frozen condition provided a protection of Salmonella against irradiation. The highest D 10 -value found was approx. 1.29 kGy. If the 7 D 10 -concept is applied a dose of approx. 9.00 kGy at -18 °C is needed for thaw water.This dose is rather high, but nowadays doses up to 10.00 kGy are accepted by international organizations. In case of poultry products it is expected that at this dose quality defects will occur.If {"}absence{"} of Salmonella in radiated skin samples is required a dose of 3.90 kGy is needed with irradiation at -18°C and a dose of 1.95 kGy at +50 °C. The data presented, however, show that such high doses are not necessary. Experiments have shown that the number of salmonellae per carcass will be lower than 100 cfu. An additional storage period for 1 to 3 months at -18 °C leads to a decrease in the number of salmonellae per carcass. In literature (259) it was shown that freezing resulted in an 0.5 log cycle reduction of the number of salmonellae cfu. The application of a radiation dose of 2.50 kGy does not guarantee a Salmonella -{"}free{"} end product. The microbiological quality, with respect to total counts and to the presence of potentially pathogenic microorganisms, is Improved In comparison with the untreated slaughtered product. Practical consequences.The application of a radiation dose of 2.50 kGy was not sufficient to destroy all salmonellae present per carcass.The surviving microflora of the carcasses consisted of bacilli. micrococcl, streptococci, yeasts and moulds. The final counts on the radiation treated carcasses were so low that no dangerous effect can be expected.Salmonella -contaminated broiler carcasses contained on an average 100 cfu's per carcass (minimum : 2, maximum : 19400).After irradiation only 2 series of pooled skin samples contained 2 cfu. D 10 -values for Salmonella panama after irradiation at +5°C and at -18 °C were 0.67 kGy and 1.29 kGy respectively.As can be seen from these D 10 -values and from the number of Salmonella cfu's per carcass, generally, the carcasses will be Salmonella -negative after irradiation with 2.50 kGy. An extra effect on the number of Salmonella -positive carcasses as well as on the number of salmonellae per carcass can be obtained by freezing and additional storage for several months' at -18 °CAn extrapolation to the whole Dutch broiler production of the lethal effect of the radiation treatment as measured in the experiments described may not be made, because of uncertainty with respect to the variance in average numbers of Salmonella cfu's per carcass, the variance in D 10 -values and the Salmonella detection level obtained by the Salmonella -isolation method used.Nevertheless the results presented here indicate that in case the whole Dutch broiler production (approx. 3.0 . 10 7carcasses) is treated with a dose of 2.50 kGy and assuming a D 10 -value of 0.80 kGy, the maximal result would be 1 positive carcass in 55 carcasses, which means that the number of Salmonella contaminated carcasses is decreased 14 times.Additional storage for several months' at -18 °C affords an extra beneficial effect to this respect.A combined treatment of freezing and Irradiation can be recommended to reduce the dissemination of Salmonella.",
    keywords = "pluimveevlees, kippenvlees, eendenvlees, dierlijke producten, bacteriologie, kiemgetal, vervalsing, besmetting, verouderen, gebreken, achteruitgang (deterioration), nederland, poultry meat, chicken meat, duck meat, animal products, bacteriology, bacterial count, adulteration, contamination, aging, defects, deterioration, netherlands",
    author = "R.W.A.W. Mulder",
    note = "WU thesis 895 Proefschrift Wageningen",
    year = "1982",
    language = "English",
    publisher = "Mulder",

    }

    Mulder, RWAW 1982, 'Salmonella radicidation of poultry carcasses', Doctor of Philosophy, Wageningen.

    Salmonella radicidation of poultry carcasses. / Mulder, R.W.A.W.

    Wageningen : Mulder, 1982. 158 p.

    Research output: Thesisexternal PhD, WU

    TY - THES

    T1 - Salmonella radicidation of poultry carcasses

    AU - Mulder, R.W.A.W.

    N1 - WU thesis 895 Proefschrift Wageningen

    PY - 1982

    Y1 - 1982

    N2 - Validity of methodsExperiments were carried out In which it was assessed which Salmonella isolation method is the most productive one In the examination of broiler carcasses. Refrigerated, refrigerated and radiated (2.50 kGy), frozen and frozen and radiated (2.50 kGy) samples of broilers were examined. After evaluation of all results It was concluded that the following method was the most productive one:1. pre-enrichment in buffered peptone water at 37 °C for 20 hours2. enrichment in selenite cystine broth at 37 °C and at 43 °C for 24 hours3. selective plating on brilliant green sulphapyridine agar and XLD- agar at 37 °C for 24 hoursFrom this study It could not be concluded that other methods were not sufficiently reliable to be applied in other situations.However, it became clear that when sublethally damaged salmonellae are expected to be present, and when, as a result of the treatment, a different competitive microflora is present, adapted isolation methods should be used.The method chosen for Salmonella -isolation in the experiments described in this thesis was the ISO 3565/1975 reference method for isolation of salmonellae from meat and meat products. This method was ultimately preferred because of the experience obtained for many years in routine analyses.In two Instances other conditions than prescribed by the ISO-method were used; i.e. the samples were incubated at 37 °C or 43°C for 24 hours. The ISO-method as well as its slight modification proved to be reliable methods in the experiments reported.Escherichia coli versus Salmonella.The experiments have shown that the Escherichia coli K12 NDA strain tested can not be used as index microorganism for Salmonella : in this case Salmonella niloese and Salmonella panama on broilers, though in liquid and solid culture media the D 10 -values of these three microorganisms were similar.The experiments with pure cultures and with artificially contaminated carcasses showed that the following factors influenced D 10 -values:1. the culture media used before, during and after irradiation;2. the temperature during irradiation;3. the physiological age of the bacteria.No influence on D 10 -values could be observed from1. the initial cell concentration;2. the oxygen partial pressure.The results of the experiments showed that after a radiation treatment with 2.50 kGy Salmonella -positive samples were found; In those cases the numbers of cfu's of Enterobacteriaceae were below the detection limit. This indicates that no other microorganism or groups of microorganisms can consistently serve as the sole index microorganism(s) for Salmonella.Effect of 2.50 kGy on salmonellae and Enterobacteriaceae.Poultry carcasses were found frequently contaminated with salmonellae. Per carcass a maximum number of 1,400 cfu per 1,000 cm 2skin were estimated by a MPN method. However, 90% of the total of the examined carcasses in this specific experiment contained less than 100 cfu's per 1,000 cm 2. Similar data are given in literature (161,200,260,306).A radicidation treatment with a dose of 2.50 kGy markedly reduced the total number of Salmonella -positive carcasses, as well as the number of salmonellae per carcass.This dose, however, could not guarantee a Salmonella -"free" product. The number of Salmonella contaminated carcasses was reduced tenfold (table 38). After irradiation at +5°C with a dose of 2.50 kGy two series of pooled samples of broiler skin were found Salmonella -positive by a MPN method. The serotypes isolated were agona and infantis.Before the radiation treatment these samples proved to be very sparsely contaminated with salmonellae (numbers of 2 cfu per 1,000 cm 2).It was shown in previous experiments that the D 10 -values of these two serotypes were comparable to other serotypes, so the two did not belong to a radio-resistant group.Salmonella -positive samples which were observed in experiments in which they were expected to be absent, according to their estimated D 10 -values, were also found in :1. experiments in which the resulting microflora after certain radiation treatments was identified;2. experiments in which the effect on Salmonella of freezing and storage of radiated carcasses was studied.In some experiments artificially contaminated carcasses were used. In the case of Salmonella niloese and Salmonella panama a protective action was afforded by the carcass skin. This resulted in D 10 -values of 1.07 and 1.29 kGy respectively, when irradiation was carried out at -18 °C and in D 10 -values of 0.62 and 0.67 kGy at +5 °C.The D 10 -values of Salmonellapanama estimated in skin samples were found higher (P<0.01) than the values which were estimated under optimal conditions In liquid culture media, when the D 10 -values was 0.52 kGy. Different factors as discussed earlier exert an influence on D 10 -values and therefore on the lethality of the radiation treatment. Only speculations can be made on the mechanism of the protective action of skin. Perhaps salmonellae meet less competition from the naturally present microflora than Escherichia coli , resulting in a stronger attachment to the skin. It is possible that this protection is provided by skin components or by extracellular polysaccharides which are produced by attached bacteria (14,216).A model was designed to assess the probability of survival after irradiation. Following the model assumptions it was shown that the detection level of the Salmonella -isolation methods employed was very important in the finding of Salmonella -positive samples after irradiation, and hence the estimated lethality of a given radiation treatment. Several bacteria, such as species of Alcaligenes , Escherichia , Pseudomonas and Salmonella were found in radiation treated products in which they were not expected according to their D 10 -values.The application of low doses of ionizing radiation followed by freezing and storage of the products during a certain period had more success In reaching the final goal : a Salmonella -"free" end product.To demonstrate a correlation between the number of Enterobacteriaceae and the number of salmonellae present per carcass, also Enterobacteriaceae cfu counts were assessed.Surprising was the presence of Salmonella cfu in samples of broiler skin on which the number of Enterobacteriaceae cfu's were below 10 per 10 cm 2of skin. This effect was not found in samples of thaw water.A difference in lethal effect on salmonellae was not found between irradiation at +5°C and at -18°C. In literature (207) proof of differences in lethal effects of irradiation carried out at different temperatures have been reported.The results obtained with these experiments point to a preference for the application of the radiation treatment at +5 °C but the statistical superiority over the other results was not proven.By means of estimation of the number of cfu's of Enterobacteriaceae no answer to this question could be obtained. All counts of cfu's proved less than 10 per 10 cm 2or per ml. After a three months' storage period at -18°C. both groups of radiation treated carcasses were Salmonel1a -negative. This was not the case with untreated carcasses, from which Salmonella could be isolated during the whole storage period.In summary, it can be said that irradiation of broiler carcasses with a dose of 2.50 kGy results in a decrease of the total number of Salmonella contaminated carcasses as well as of the number of salmonellae per carcass. As the experiments showed very promising results with these combined treatments, further investigations should be made in the field of the application of the combined use of freezing and irradiation. Surviving microflora.The microflora surviving after irradiation with 2.50 kGy will not cause unexpected problems as demonstrated by the nature and the composition of the microflora. The residual "total" count of 100 - 1000 cells per 10 cm2 or per m] consists of bacilli, micrococci, streptococci, yeasts and moulds. Sporadically an Enterobacteriaceae was also isolated.No stimulating effect as suggested in literature was observed in the enumeration of various microorganisms, especially Staphylococcus aureus and Salmonella , when catalase was added to the surface of the selective agar media. The mechanism of the beneficial effect of catalase on the enumeration of yeasts and moulds and of the inhibitive effect on cfu's of Bacillus cereus should be studied in more detail in future. Solid medium repair methods used in the enumeration of stressed microorganisms did not result in better recoveries of the microorganisms investigated.The presence of Salmonella in the end product after irradiation with 2.50 kGY shows that this dose does not guarantee a Salmonella -"free" end product. The question arose which dose is needed to guarantee this. The broiler skin and irradiation in the frozen condition provided a protection of Salmonella against irradiation. The highest D 10 -value found was approx. 1.29 kGy. If the 7 D 10 -concept is applied a dose of approx. 9.00 kGy at -18 °C is needed for thaw water.This dose is rather high, but nowadays doses up to 10.00 kGy are accepted by international organizations. In case of poultry products it is expected that at this dose quality defects will occur.If "absence" of Salmonella in radiated skin samples is required a dose of 3.90 kGy is needed with irradiation at -18°C and a dose of 1.95 kGy at +50 °C. The data presented, however, show that such high doses are not necessary. Experiments have shown that the number of salmonellae per carcass will be lower than 100 cfu. An additional storage period for 1 to 3 months at -18 °C leads to a decrease in the number of salmonellae per carcass. In literature (259) it was shown that freezing resulted in an 0.5 log cycle reduction of the number of salmonellae cfu. The application of a radiation dose of 2.50 kGy does not guarantee a Salmonella -"free" end product. The microbiological quality, with respect to total counts and to the presence of potentially pathogenic microorganisms, is Improved In comparison with the untreated slaughtered product. Practical consequences.The application of a radiation dose of 2.50 kGy was not sufficient to destroy all salmonellae present per carcass.The surviving microflora of the carcasses consisted of bacilli. micrococcl, streptococci, yeasts and moulds. The final counts on the radiation treated carcasses were so low that no dangerous effect can be expected.Salmonella -contaminated broiler carcasses contained on an average 100 cfu's per carcass (minimum : 2, maximum : 19400).After irradiation only 2 series of pooled skin samples contained 2 cfu. D 10 -values for Salmonella panama after irradiation at +5°C and at -18 °C were 0.67 kGy and 1.29 kGy respectively.As can be seen from these D 10 -values and from the number of Salmonella cfu's per carcass, generally, the carcasses will be Salmonella -negative after irradiation with 2.50 kGy. An extra effect on the number of Salmonella -positive carcasses as well as on the number of salmonellae per carcass can be obtained by freezing and additional storage for several months' at -18 °CAn extrapolation to the whole Dutch broiler production of the lethal effect of the radiation treatment as measured in the experiments described may not be made, because of uncertainty with respect to the variance in average numbers of Salmonella cfu's per carcass, the variance in D 10 -values and the Salmonella detection level obtained by the Salmonella -isolation method used.Nevertheless the results presented here indicate that in case the whole Dutch broiler production (approx. 3.0 . 10 7carcasses) is treated with a dose of 2.50 kGy and assuming a D 10 -value of 0.80 kGy, the maximal result would be 1 positive carcass in 55 carcasses, which means that the number of Salmonella contaminated carcasses is decreased 14 times.Additional storage for several months' at -18 °C affords an extra beneficial effect to this respect.A combined treatment of freezing and Irradiation can be recommended to reduce the dissemination of Salmonella.

    AB - Validity of methodsExperiments were carried out In which it was assessed which Salmonella isolation method is the most productive one In the examination of broiler carcasses. Refrigerated, refrigerated and radiated (2.50 kGy), frozen and frozen and radiated (2.50 kGy) samples of broilers were examined. After evaluation of all results It was concluded that the following method was the most productive one:1. pre-enrichment in buffered peptone water at 37 °C for 20 hours2. enrichment in selenite cystine broth at 37 °C and at 43 °C for 24 hours3. selective plating on brilliant green sulphapyridine agar and XLD- agar at 37 °C for 24 hoursFrom this study It could not be concluded that other methods were not sufficiently reliable to be applied in other situations.However, it became clear that when sublethally damaged salmonellae are expected to be present, and when, as a result of the treatment, a different competitive microflora is present, adapted isolation methods should be used.The method chosen for Salmonella -isolation in the experiments described in this thesis was the ISO 3565/1975 reference method for isolation of salmonellae from meat and meat products. This method was ultimately preferred because of the experience obtained for many years in routine analyses.In two Instances other conditions than prescribed by the ISO-method were used; i.e. the samples were incubated at 37 °C or 43°C for 24 hours. The ISO-method as well as its slight modification proved to be reliable methods in the experiments reported.Escherichia coli versus Salmonella.The experiments have shown that the Escherichia coli K12 NDA strain tested can not be used as index microorganism for Salmonella : in this case Salmonella niloese and Salmonella panama on broilers, though in liquid and solid culture media the D 10 -values of these three microorganisms were similar.The experiments with pure cultures and with artificially contaminated carcasses showed that the following factors influenced D 10 -values:1. the culture media used before, during and after irradiation;2. the temperature during irradiation;3. the physiological age of the bacteria.No influence on D 10 -values could be observed from1. the initial cell concentration;2. the oxygen partial pressure.The results of the experiments showed that after a radiation treatment with 2.50 kGy Salmonella -positive samples were found; In those cases the numbers of cfu's of Enterobacteriaceae were below the detection limit. This indicates that no other microorganism or groups of microorganisms can consistently serve as the sole index microorganism(s) for Salmonella.Effect of 2.50 kGy on salmonellae and Enterobacteriaceae.Poultry carcasses were found frequently contaminated with salmonellae. Per carcass a maximum number of 1,400 cfu per 1,000 cm 2skin were estimated by a MPN method. However, 90% of the total of the examined carcasses in this specific experiment contained less than 100 cfu's per 1,000 cm 2. Similar data are given in literature (161,200,260,306).A radicidation treatment with a dose of 2.50 kGy markedly reduced the total number of Salmonella -positive carcasses, as well as the number of salmonellae per carcass.This dose, however, could not guarantee a Salmonella -"free" product. The number of Salmonella contaminated carcasses was reduced tenfold (table 38). After irradiation at +5°C with a dose of 2.50 kGy two series of pooled samples of broiler skin were found Salmonella -positive by a MPN method. The serotypes isolated were agona and infantis.Before the radiation treatment these samples proved to be very sparsely contaminated with salmonellae (numbers of 2 cfu per 1,000 cm 2).It was shown in previous experiments that the D 10 -values of these two serotypes were comparable to other serotypes, so the two did not belong to a radio-resistant group.Salmonella -positive samples which were observed in experiments in which they were expected to be absent, according to their estimated D 10 -values, were also found in :1. experiments in which the resulting microflora after certain radiation treatments was identified;2. experiments in which the effect on Salmonella of freezing and storage of radiated carcasses was studied.In some experiments artificially contaminated carcasses were used. In the case of Salmonella niloese and Salmonella panama a protective action was afforded by the carcass skin. This resulted in D 10 -values of 1.07 and 1.29 kGy respectively, when irradiation was carried out at -18 °C and in D 10 -values of 0.62 and 0.67 kGy at +5 °C.The D 10 -values of Salmonellapanama estimated in skin samples were found higher (P<0.01) than the values which were estimated under optimal conditions In liquid culture media, when the D 10 -values was 0.52 kGy. Different factors as discussed earlier exert an influence on D 10 -values and therefore on the lethality of the radiation treatment. Only speculations can be made on the mechanism of the protective action of skin. Perhaps salmonellae meet less competition from the naturally present microflora than Escherichia coli , resulting in a stronger attachment to the skin. It is possible that this protection is provided by skin components or by extracellular polysaccharides which are produced by attached bacteria (14,216).A model was designed to assess the probability of survival after irradiation. Following the model assumptions it was shown that the detection level of the Salmonella -isolation methods employed was very important in the finding of Salmonella -positive samples after irradiation, and hence the estimated lethality of a given radiation treatment. Several bacteria, such as species of Alcaligenes , Escherichia , Pseudomonas and Salmonella were found in radiation treated products in which they were not expected according to their D 10 -values.The application of low doses of ionizing radiation followed by freezing and storage of the products during a certain period had more success In reaching the final goal : a Salmonella -"free" end product.To demonstrate a correlation between the number of Enterobacteriaceae and the number of salmonellae present per carcass, also Enterobacteriaceae cfu counts were assessed.Surprising was the presence of Salmonella cfu in samples of broiler skin on which the number of Enterobacteriaceae cfu's were below 10 per 10 cm 2of skin. This effect was not found in samples of thaw water.A difference in lethal effect on salmonellae was not found between irradiation at +5°C and at -18°C. In literature (207) proof of differences in lethal effects of irradiation carried out at different temperatures have been reported.The results obtained with these experiments point to a preference for the application of the radiation treatment at +5 °C but the statistical superiority over the other results was not proven.By means of estimation of the number of cfu's of Enterobacteriaceae no answer to this question could be obtained. All counts of cfu's proved less than 10 per 10 cm 2or per ml. After a three months' storage period at -18°C. both groups of radiation treated carcasses were Salmonel1a -negative. This was not the case with untreated carcasses, from which Salmonella could be isolated during the whole storage period.In summary, it can be said that irradiation of broiler carcasses with a dose of 2.50 kGy results in a decrease of the total number of Salmonella contaminated carcasses as well as of the number of salmonellae per carcass. As the experiments showed very promising results with these combined treatments, further investigations should be made in the field of the application of the combined use of freezing and irradiation. Surviving microflora.The microflora surviving after irradiation with 2.50 kGy will not cause unexpected problems as demonstrated by the nature and the composition of the microflora. The residual "total" count of 100 - 1000 cells per 10 cm2 or per m] consists of bacilli, micrococci, streptococci, yeasts and moulds. Sporadically an Enterobacteriaceae was also isolated.No stimulating effect as suggested in literature was observed in the enumeration of various microorganisms, especially Staphylococcus aureus and Salmonella , when catalase was added to the surface of the selective agar media. The mechanism of the beneficial effect of catalase on the enumeration of yeasts and moulds and of the inhibitive effect on cfu's of Bacillus cereus should be studied in more detail in future. Solid medium repair methods used in the enumeration of stressed microorganisms did not result in better recoveries of the microorganisms investigated.The presence of Salmonella in the end product after irradiation with 2.50 kGY shows that this dose does not guarantee a Salmonella -"free" end product. The question arose which dose is needed to guarantee this. The broiler skin and irradiation in the frozen condition provided a protection of Salmonella against irradiation. The highest D 10 -value found was approx. 1.29 kGy. If the 7 D 10 -concept is applied a dose of approx. 9.00 kGy at -18 °C is needed for thaw water.This dose is rather high, but nowadays doses up to 10.00 kGy are accepted by international organizations. In case of poultry products it is expected that at this dose quality defects will occur.If "absence" of Salmonella in radiated skin samples is required a dose of 3.90 kGy is needed with irradiation at -18°C and a dose of 1.95 kGy at +50 °C. The data presented, however, show that such high doses are not necessary. Experiments have shown that the number of salmonellae per carcass will be lower than 100 cfu. An additional storage period for 1 to 3 months at -18 °C leads to a decrease in the number of salmonellae per carcass. In literature (259) it was shown that freezing resulted in an 0.5 log cycle reduction of the number of salmonellae cfu. The application of a radiation dose of 2.50 kGy does not guarantee a Salmonella -"free" end product. The microbiological quality, with respect to total counts and to the presence of potentially pathogenic microorganisms, is Improved In comparison with the untreated slaughtered product. Practical consequences.The application of a radiation dose of 2.50 kGy was not sufficient to destroy all salmonellae present per carcass.The surviving microflora of the carcasses consisted of bacilli. micrococcl, streptococci, yeasts and moulds. The final counts on the radiation treated carcasses were so low that no dangerous effect can be expected.Salmonella -contaminated broiler carcasses contained on an average 100 cfu's per carcass (minimum : 2, maximum : 19400).After irradiation only 2 series of pooled skin samples contained 2 cfu. D 10 -values for Salmonella panama after irradiation at +5°C and at -18 °C were 0.67 kGy and 1.29 kGy respectively.As can be seen from these D 10 -values and from the number of Salmonella cfu's per carcass, generally, the carcasses will be Salmonella -negative after irradiation with 2.50 kGy. An extra effect on the number of Salmonella -positive carcasses as well as on the number of salmonellae per carcass can be obtained by freezing and additional storage for several months' at -18 °CAn extrapolation to the whole Dutch broiler production of the lethal effect of the radiation treatment as measured in the experiments described may not be made, because of uncertainty with respect to the variance in average numbers of Salmonella cfu's per carcass, the variance in D 10 -values and the Salmonella detection level obtained by the Salmonella -isolation method used.Nevertheless the results presented here indicate that in case the whole Dutch broiler production (approx. 3.0 . 10 7carcasses) is treated with a dose of 2.50 kGy and assuming a D 10 -value of 0.80 kGy, the maximal result would be 1 positive carcass in 55 carcasses, which means that the number of Salmonella contaminated carcasses is decreased 14 times.Additional storage for several months' at -18 °C affords an extra beneficial effect to this respect.A combined treatment of freezing and Irradiation can be recommended to reduce the dissemination of Salmonella.

    KW - pluimveevlees

    KW - kippenvlees

    KW - eendenvlees

    KW - dierlijke producten

    KW - bacteriologie

    KW - kiemgetal

    KW - vervalsing

    KW - besmetting

    KW - verouderen

    KW - gebreken

    KW - achteruitgang (deterioration)

    KW - nederland

    KW - poultry meat

    KW - chicken meat

    KW - duck meat

    KW - animal products

    KW - bacteriology

    KW - bacterial count

    KW - adulteration

    KW - contamination

    KW - aging

    KW - defects

    KW - deterioration

    KW - netherlands

    M3 - external PhD, WU

    PB - Mulder

    CY - Wageningen

    ER -

    Mulder RWAW. Salmonella radicidation of poultry carcasses. Wageningen: Mulder, 1982. 158 p.