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Losses in Viable Cells of Salmonellae upon Inoculation into Dry Animal Feeds of Various Types1
890
RESEARCH NOTES
when fed by themselves. However, when 0.5% cottonseed lipids were incorporated into the diets, a higher amount of discoloration was produced. REFERENCES Kemmerer, A. R., B. W. Heywang and M. G. Vavich, 1961. Effect of Sterculia foetida oil on gossypol discoloration in cold storage eggs and the mechanism of gossypol discoloration. Poultry Sci. 40: 1045-1048. Kemmerer, A. R., B. W. Heywang, H. E. Nordby and R. A. Phelps, 1962. Effect of cottonseed oil on discoloration of cold storage eggs. Poultry Sci. 4 1 : 1101-1103.
LOSSES IN VIABLE CELLS OF SALMONELLAE UPON INOCULATION INTO DRY ANIMAL FEEDS OF VARIOUS TYPES 1 D. A. A. MOSSEL AND M. J. KOOPMAN Department of Bacteriology, Central Institute for Nutrition and Food Research T.N.O. Zeist, The Netherlands (Received for publication January 15, 1965)
The difficulties encountered by Rasmussen et al. (1964) in interpreting the discrepancy between the heat resistance of Salmonellae in artifically versus naturally contaminated meals prompts us to report our data on the losses of viable cells observed upon inoculating several Salmonella serotypes into dry animal feeds. Our procedure was similar to that of Rasmussen et al. (1964). Two hundred gramme portions of fish meal, meat and bone meal, etc. were first of all freed from all viable Enterobacteriaceae by a procedure which we found very effective without unduly changing the gross composition of animal feeds, i.e. overnight incubation in a closed flask at 55 ± 2°C. Five gram1 The results reported in this paper are part of an investigation carried out under contract nr. 199/RB with the International Atomic Energy Agency, Vienna, Austria.
mes of this pre-pasteurized product were then placed in a sterile mortar of c. 15 cm. diameter and mixed, as thoroughly as possible, with one to five ml. of brain heart infusion broth suspensions of various Salmonella serotypes containing the order 1010-1012 viable cells per ml. This primary mixture was finally triturated with successive portions of c. 10 grammes of meal until the 200 grammes were exhausted. Immediately after inoculation the numbers of viable cells of Salmonella per gramme of meal were determined by plating one ml of suitable dilutions in violet red bile glucose agar (Mossel et al. 1962). After layering the inoculated agar with some 3 ml. of sterile agar, the plates were incubated for 20 to 48 hours at 36 ±1°C. When this procedure was followed without any precaution, losses in viable cells of the order 10~5 were generally observed. By pre-chilling the inoculum and washing it
Downloaded from http://ps.oxfordjournals.org/ at University of Leeds on May 17, 2015
contained less than 0.1% extractable lipids, 0.02 % free gossypol and approximately 1 % bound gossypol. For a positive control crystalline gossypol acetate was incorporated into the basal at levels given in Table 1. Cottonseed lipids (gossypol removed) were incorporated into one-half of the diets at a level of 0.5%. The discoloration in the eggs was determined by procedures previously reported (Kemmerer et al., 1961). Table 1 summarizes the results obtained. It is apparent that neither of the cottonseed meals caused appreciable egg discolor
891
RESEARCH NOTES TABLE 1.—Reduction in numbers of viable cells per gramme of fish meal occurring upon inoculation of fish meal with three different serotypes of Salmonella Series 2
Series 1 Serotype Montevideo senftenberg typhimurium
0.2X106 0.3X108 0.4X10*
Mode of inoculation
calculated count as inoculation determined 0.3X108 0.3X108 0.1X108
TABLE 3.—Reduction in numbers of viable cells per gramme of fish meal occurring upon various ways of inoculation with Salmonellae
calc.
determ.
o.sxio" o.ixios 11 0.2X10 0.7X109
0.9X10? 0.4X10?
TABLE 2.—Spontaneous reduction in numbers of viable cells of Salmonella senftenberg per one gramme of various substrata of the same Ow (c. 0.46) Calculated inoculum c. 10" cells/g. Period of storage (days) at 14+1 °C.
Fish meal
Meat meal
Casein
0 1 2 5
0.3X10« 0.6X10 6 0.4X10 6 0.2X10 5
0.3X10' 0.1X10' 0.6X10" 0.3X10 6
0.2X10 6 0.1X105 0.9X10 4 0.6X10*
lyophilized cells
binza newington senftenberg
chilled washed cells
biw.a newington senftenberg typhimurium
c.
Calculated
Found
0.6X10? 0.5X10« 0.6X10?
0.9X105 0.8X10* 0.7X106
0.8X108 0.1X109 0.7X10" 0.8X109
0.1X10? 0.4X10« 0.4X10? 0.2X108
cells to an environment of different aw, mostly called osmotic shock (Dunklin and Puck, 1948; Webb, 1964). In order to verify this hypothesis, the same experiments as described above were carried out once more, using fish meal as the substrate, but inoculating this material in parallel with a moist pellet of Salmonella cells, versus lyophilized preparations of the same serotypes. The results obtained in these tests are shown in Table 3. Apparently any gross change in aw whether in the positive or in the negative direction, effects serious losses in viable cells of these enteric bacteria. A further reduction in numbers of viable cells was observed when meals inoculated in this way were stored at about 14°C. as indicated in Table 2. Such an effect has also been established by earlier investigators (Brown and Gibbons, 1950; van der Schaaf et al., 1962). We do not at all doubt that bacterial cells imbedded in protein and protected by lipids, as they may occur in naturally contaminated feeds, may be more protected from a lethal heat treatment than cells brought into such products artifically. Yet we think that the spontaneous decay of viable cells of Salmonellae upon inoculation, and perhaps also during some period of storage before heating, as described in this note has led to an overestimation of the initial counts of the artificially inoculated meals and that this may very well have been the main cause of the discrep-
Downloaded from http://ps.oxfordjournals.org/ at University of Leeds on May 17, 2015
with refrigerated saline in combination with inoculating prechilled meals only, these reductions in viable cells were decreased to a significantly lower order. Table 1 gives a general impression of the type of figures obtained when fish meal is inoculated in this way with various Salmonella serotypes. In Table 2 the behaviour of one serotype, viz. senftenberg, in various dry meals is presented. In these tests the meals had all been conditioned to the same water activity viz. aw = 0.46. This had been carried out by exposing them in a vacuum desiccator to a reference sample of fish meal of which the aw value had been determined at 0.46 by the use of the LiCl cell, described by Mossel and van Kuijk (19SS). The data in Table 2 show that the chemical composition of the dry product seems to play an important role in the degree of killing of the enteric bacteria used: fish meal and casein are the most, meat meal the least hostile to Salmonellae at a given aw. Nevertheless the lethal effect seems to be mainly due to exposing the
Inoculation
Serotype of Salmonella
892
RESEARCH
ancy between the D-values for Salmonellae in artificially versus naturally contaminated meals, as observed by Rasmussen et al. (1964). REFERENCES
determination of the equilibrium relative humidity of foods. Food Research, 20: 415-423. Mossel, D. A. A., W. H. J. Mengerink and H. H. Schots, 1962. Use of a modified MacConkey agar medium for the selective growth and enumeration of Enterobacteriaceae. J. Bacteriol. 84: 381. Rasmussen, O. G., R. Hansen, N. J. Jacobs and O. H. M. Wilder, 1964. Dry heat resistance of Salmonellae in rendered animal by-products. Poultry Sci. 43 : 1151-1157. Schaaf, A. van der, H. J. M. van Zijl and F. M. Hagens, 1962. Diermeel en salmonellosis. Tijdschr. Diergeneesk. Utrecht, 87: 211-221. Webb, S. J., 1964. Bound water, metabolites and genetic continuity. Nature, 203: 374-377.
THE EFFECT OF DEBEAKING ON THE MATING ABILITY OF MEAT TYPE MALES L. D.
Poultry Science Department,
TINDEIX1
University of Georgia, Athens
(Received for publication January 25, 1965)
The debeaking of breeder cockerels is not a common practice due to the belief that the male's ability to mate will be impaired and result in poor fertility. Evidence indicating that debeaking does not adversely affect fertility has been presented by Sherwood and Milby (1956), Bauermann (1959), and Siegel and Martin (1960). Tindell (1964) indicated that debeaking was one means of reducing mortality among males held in confinement from 8-38 weeks of age, but did not present any data on subsequent mating ability or fertility. The data reported in this paper were obtained in conjunction with another study utilizing F 3 generation males selected in divergent directions for mating ability. A random bred control line was also measured. All males were debeaked (one-half of the upper beak was removed) at 15 weeks of age, with the exception of an additional 1
Present Address: Southern Regional PoultryGenetics Laboratory, ARS, USDA, Athens, Georgia.
group of low line males which were not debeaked. Mating behavior observations were initiated for the high, low and randombred male lines (debeaked) when they were 35 weeks of age. The late hatch of low line males (not debeaked) was also measured for mating behavior at this time, but were 30 weeks of age. All males were observed for four trials of eight minutes each for a total of 32 minutes observation per male. A male was tested only one time on a particular day, with each trial being at a different hour of the afternoon (1:00-5:00 p.m.). After being tested the male was returned to the individual cage within the pen in which he was tested. A random sample of each line was represented in each of the four pens (34-35 females per pen). Upon completion of the mating behavior trials, all males were subjectively scored according to their beak ratio (length of the upper beak as compared with the length of the lower beak) from .4 to 1.0 (Table 1). A score of 1.0 indicated that the beak was
Downloaded from http://ps.oxfordjournals.org/ at University of Leeds on May 17, 2015
Brown, H. J., and N. E. Gibbons, 1950. Enterococci as an index of fecal contamination in egg products. Canad. J. Research, 28F: 107-117. Dunklin, E. W., and T. T. Puck, 1948. The lethal effect of relative humidity on air-borne bacteria. J. Exper. Med. 87 : 87-101. Mossel, D. A. A., and H. J. L. van Kuijk, 1955. A new and simple technique for the direct
NOTES
RESEARCH NOTES
when fed by themselves. However, when 0.5% cottonseed lipids were incorporated into the diets, a higher amount of discoloration was produced. REFERENCES Kemmerer, A. R., B. W. Heywang and M. G. Vavich, 1961. Effect of Sterculia foetida oil on gossypol discoloration in cold storage eggs and the mechanism of gossypol discoloration. Poultry Sci. 40: 1045-1048. Kemmerer, A. R., B. W. Heywang, H. E. Nordby and R. A. Phelps, 1962. Effect of cottonseed oil on discoloration of cold storage eggs. Poultry Sci. 4 1 : 1101-1103.
LOSSES IN VIABLE CELLS OF SALMONELLAE UPON INOCULATION INTO DRY ANIMAL FEEDS OF VARIOUS TYPES 1 D. A. A. MOSSEL AND M. J. KOOPMAN Department of Bacteriology, Central Institute for Nutrition and Food Research T.N.O. Zeist, The Netherlands (Received for publication January 15, 1965)
The difficulties encountered by Rasmussen et al. (1964) in interpreting the discrepancy between the heat resistance of Salmonellae in artifically versus naturally contaminated meals prompts us to report our data on the losses of viable cells observed upon inoculating several Salmonella serotypes into dry animal feeds. Our procedure was similar to that of Rasmussen et al. (1964). Two hundred gramme portions of fish meal, meat and bone meal, etc. were first of all freed from all viable Enterobacteriaceae by a procedure which we found very effective without unduly changing the gross composition of animal feeds, i.e. overnight incubation in a closed flask at 55 ± 2°C. Five gram1 The results reported in this paper are part of an investigation carried out under contract nr. 199/RB with the International Atomic Energy Agency, Vienna, Austria.
mes of this pre-pasteurized product were then placed in a sterile mortar of c. 15 cm. diameter and mixed, as thoroughly as possible, with one to five ml. of brain heart infusion broth suspensions of various Salmonella serotypes containing the order 1010-1012 viable cells per ml. This primary mixture was finally triturated with successive portions of c. 10 grammes of meal until the 200 grammes were exhausted. Immediately after inoculation the numbers of viable cells of Salmonella per gramme of meal were determined by plating one ml of suitable dilutions in violet red bile glucose agar (Mossel et al. 1962). After layering the inoculated agar with some 3 ml. of sterile agar, the plates were incubated for 20 to 48 hours at 36 ±1°C. When this procedure was followed without any precaution, losses in viable cells of the order 10~5 were generally observed. By pre-chilling the inoculum and washing it
Downloaded from http://ps.oxfordjournals.org/ at University of Leeds on May 17, 2015
contained less than 0.1% extractable lipids, 0.02 % free gossypol and approximately 1 % bound gossypol. For a positive control crystalline gossypol acetate was incorporated into the basal at levels given in Table 1. Cottonseed lipids (gossypol removed) were incorporated into one-half of the diets at a level of 0.5%. The discoloration in the eggs was determined by procedures previously reported (Kemmerer et al., 1961). Table 1 summarizes the results obtained. It is apparent that neither of the cottonseed meals caused appreciable egg discolor
891
RESEARCH NOTES TABLE 1.—Reduction in numbers of viable cells per gramme of fish meal occurring upon inoculation of fish meal with three different serotypes of Salmonella Series 2
Series 1 Serotype Montevideo senftenberg typhimurium
0.2X106 0.3X108 0.4X10*
Mode of inoculation
calculated count as inoculation determined 0.3X108 0.3X108 0.1X108
TABLE 3.—Reduction in numbers of viable cells per gramme of fish meal occurring upon various ways of inoculation with Salmonellae
calc.
determ.
o.sxio" o.ixios 11 0.2X10 0.7X109
0.9X10? 0.4X10?
TABLE 2.—Spontaneous reduction in numbers of viable cells of Salmonella senftenberg per one gramme of various substrata of the same Ow (c. 0.46) Calculated inoculum c. 10" cells/g. Period of storage (days) at 14+1 °C.
Fish meal
Meat meal
Casein
0 1 2 5
0.3X10« 0.6X10 6 0.4X10 6 0.2X10 5
0.3X10' 0.1X10' 0.6X10" 0.3X10 6
0.2X10 6 0.1X105 0.9X10 4 0.6X10*
lyophilized cells
binza newington senftenberg
chilled washed cells
biw.a newington senftenberg typhimurium
c.
Calculated
Found
0.6X10? 0.5X10« 0.6X10?
0.9X105 0.8X10* 0.7X106
0.8X108 0.1X109 0.7X10" 0.8X109
0.1X10? 0.4X10« 0.4X10? 0.2X108
cells to an environment of different aw, mostly called osmotic shock (Dunklin and Puck, 1948; Webb, 1964). In order to verify this hypothesis, the same experiments as described above were carried out once more, using fish meal as the substrate, but inoculating this material in parallel with a moist pellet of Salmonella cells, versus lyophilized preparations of the same serotypes. The results obtained in these tests are shown in Table 3. Apparently any gross change in aw whether in the positive or in the negative direction, effects serious losses in viable cells of these enteric bacteria. A further reduction in numbers of viable cells was observed when meals inoculated in this way were stored at about 14°C. as indicated in Table 2. Such an effect has also been established by earlier investigators (Brown and Gibbons, 1950; van der Schaaf et al., 1962). We do not at all doubt that bacterial cells imbedded in protein and protected by lipids, as they may occur in naturally contaminated feeds, may be more protected from a lethal heat treatment than cells brought into such products artifically. Yet we think that the spontaneous decay of viable cells of Salmonellae upon inoculation, and perhaps also during some period of storage before heating, as described in this note has led to an overestimation of the initial counts of the artificially inoculated meals and that this may very well have been the main cause of the discrep-
Downloaded from http://ps.oxfordjournals.org/ at University of Leeds on May 17, 2015
with refrigerated saline in combination with inoculating prechilled meals only, these reductions in viable cells were decreased to a significantly lower order. Table 1 gives a general impression of the type of figures obtained when fish meal is inoculated in this way with various Salmonella serotypes. In Table 2 the behaviour of one serotype, viz. senftenberg, in various dry meals is presented. In these tests the meals had all been conditioned to the same water activity viz. aw = 0.46. This had been carried out by exposing them in a vacuum desiccator to a reference sample of fish meal of which the aw value had been determined at 0.46 by the use of the LiCl cell, described by Mossel and van Kuijk (19SS). The data in Table 2 show that the chemical composition of the dry product seems to play an important role in the degree of killing of the enteric bacteria used: fish meal and casein are the most, meat meal the least hostile to Salmonellae at a given aw. Nevertheless the lethal effect seems to be mainly due to exposing the
Inoculation
Serotype of Salmonella
892
RESEARCH
ancy between the D-values for Salmonellae in artificially versus naturally contaminated meals, as observed by Rasmussen et al. (1964). REFERENCES
determination of the equilibrium relative humidity of foods. Food Research, 20: 415-423. Mossel, D. A. A., W. H. J. Mengerink and H. H. Schots, 1962. Use of a modified MacConkey agar medium for the selective growth and enumeration of Enterobacteriaceae. J. Bacteriol. 84: 381. Rasmussen, O. G., R. Hansen, N. J. Jacobs and O. H. M. Wilder, 1964. Dry heat resistance of Salmonellae in rendered animal by-products. Poultry Sci. 43 : 1151-1157. Schaaf, A. van der, H. J. M. van Zijl and F. M. Hagens, 1962. Diermeel en salmonellosis. Tijdschr. Diergeneesk. Utrecht, 87: 211-221. Webb, S. J., 1964. Bound water, metabolites and genetic continuity. Nature, 203: 374-377.
THE EFFECT OF DEBEAKING ON THE MATING ABILITY OF MEAT TYPE MALES L. D.
Poultry Science Department,
TINDEIX1
University of Georgia, Athens
(Received for publication January 25, 1965)
The debeaking of breeder cockerels is not a common practice due to the belief that the male's ability to mate will be impaired and result in poor fertility. Evidence indicating that debeaking does not adversely affect fertility has been presented by Sherwood and Milby (1956), Bauermann (1959), and Siegel and Martin (1960). Tindell (1964) indicated that debeaking was one means of reducing mortality among males held in confinement from 8-38 weeks of age, but did not present any data on subsequent mating ability or fertility. The data reported in this paper were obtained in conjunction with another study utilizing F 3 generation males selected in divergent directions for mating ability. A random bred control line was also measured. All males were debeaked (one-half of the upper beak was removed) at 15 weeks of age, with the exception of an additional 1
Present Address: Southern Regional PoultryGenetics Laboratory, ARS, USDA, Athens, Georgia.
group of low line males which were not debeaked. Mating behavior observations were initiated for the high, low and randombred male lines (debeaked) when they were 35 weeks of age. The late hatch of low line males (not debeaked) was also measured for mating behavior at this time, but were 30 weeks of age. All males were observed for four trials of eight minutes each for a total of 32 minutes observation per male. A male was tested only one time on a particular day, with each trial being at a different hour of the afternoon (1:00-5:00 p.m.). After being tested the male was returned to the individual cage within the pen in which he was tested. A random sample of each line was represented in each of the four pens (34-35 females per pen). Upon completion of the mating behavior trials, all males were subjectively scored according to their beak ratio (length of the upper beak as compared with the length of the lower beak) from .4 to 1.0 (Table 1). A score of 1.0 indicated that the beak was
Downloaded from http://ps.oxfordjournals.org/ at University of Leeds on May 17, 2015
Brown, H. J., and N. E. Gibbons, 1950. Enterococci as an index of fecal contamination in egg products. Canad. J. Research, 28F: 107-117. Dunklin, E. W., and T. T. Puck, 1948. The lethal effect of relative humidity on air-borne bacteria. J. Exper. Med. 87 : 87-101. Mossel, D. A. A., and H. J. L. van Kuijk, 1955. A new and simple technique for the direct
NOTES