Effect of Moisture on Salmonella Populations in Animal Feeds

INDUCED EGG PRODUCTION CYCLES 1942. Game bird investigations. Missouri Agri. Expt. Sta. Bull. 435. Rowan, W., 1929. Experiments in bird migration. I. Manipulation of the reproductive cycle: Seasonal histological changes in the gonads. Proc. Boston Soc. Nat. Hist. 39: 151-208. Sunde, M. L., and H. R. Bird, 1959. The effect of pullet maturity on fertility and hatchability of eggs. Poultry Sci. 38: 272-279. Wolfson, A., 1954. Production of repeated gonadal, fat and molt cycles within one year in the

717

junco and White-crowned Sparrow by manipulation of day length. J. Exp. Zool. 125: 353376. Wilson, W. O., H. Abplanalp and L. Arrington, 1962a. Sexual development of coturnix as affected by changes in photoperiods. Poultry Sci. 4 1 : 17-22. Wilson, W. O., F. X. Ogasawara and V. S. Asmundson, 1962b. Artificial control of egg production in turkeys by photoperiods. Poultry Sci. 4 1 : 1169-1175.

VIRGINIA L. CARLSON AND G. H. SNOEYENBOS Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, Massachusetts 01002 (Received for publication November 28, 1969)

I

T IS well documented that microorganisms require chemically unbound water for growth. Water activity (aw) is defined as "the relative humidity of an atmosphere with which a product is in equilibrium" (Scott, 1953) and is used as a measurement of the available water. Relatively little information is available on the relationship between salmonella microbial contaminants and the water activity of animal feedstuffs. Scott and Christian (19S3) reported that salmonellae grew in liquid media having aw's between 0.945 and 0.995, while in foods the aw necessary for growth was slightly higher. Liu et al. (1969) noted that a Salmonella senftenberg 775W population would multiply in meat and bone meal having aw's of 0.950 and higher at holding temperatures of 25° and 37°C. This investigation was undertaken to determine the effect of water activity on the dynamics of four salmonella species in a variety of animal by-products and finished feeds.

MATERIALS AND METHODS

Moisture level adjustment. Animal feeds used were meat and bone meal, fish meal, feather meal, poultry by-product meal, and a poultry mash. Feed moisture levels were adjusted so as to range from 5-40% in 5% increments as follows: 100 gm. samples were sterilized in mason jars for 2 hr. at 15 psig. Two-gm. portions of each 100 gm. sample were dried in a covered dish using a heated vacuum desiccator at 100 mm. Hg for 4 hr. A stream of air, dried by passage through flasks of H 2 S0 4 and CaCl2, flowed through the desiccator during drying. Dried samples were cooled to ambient temperature in a desiccator containing a drying agent prior to weighing to avoid moisture uptake. The loss in weight was used to compute the percent dry weight. Predetermined moisture levels were then prepared by adding a calculated amount of sterile distilled water to each 100 gm. sample, and held at 4°C. for 5 days to allow for moisture stabilization. Simulated-natural contamination. A

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Effect of Moisture on Salmonella Populations in Animal Feeds

718

V. L. CARLSON AND G. H. SNOEYENBOS

RESULTS

Figures 1-5 show changes for S. typhimurium populations in the several sterile feedstuffs at the various moisture levels. Population increase was obtained in 30% moisture fish meal, feather meal, and meat

and bone meal. Multiplication first occurred in 40% moisture poultry by-product meal. At 5% moisture, the contaminating salmonella population remained relatively stable throughout the holding period at all moisture levels in all feeds used. As the moisture levels were increased from 5% the rate of salmonella die-off tended to increase in all the by-products tested until a water activity was reached which allowed multiplication. Since salmonella multiplication occurred in sterile feedstuffs at high moisture levels (30 and 40%), the non-sterile by-products were contaminated with S. typhimurium to determine if the natural microflora would effect the growth of salmonellae. Results clearly indicated that the salmonella growth curve was essentially identical to that in the sterile by-product feedstuffs during the first four days of incubation. However, after four days of incubation at 37°C. the abundance of mold prevented accurate cell counts. Table 1 lists the water activities of the several feeds at the various moisture levels. An aw level of approximately 0.96 (Liu et al., 1969) appears to be required for salmonella increase in these products. Poultry mash having an aw of 0.97 (40% moisture) failed to allow multiplication of the test populations; similar lack of multiplication occurred in a mash slurry. Data obtained for S. enteriditis, S. heidelberg, and S. tennessee were similar to S. typhimurium (Figs. 1-5) and therefore are not reproduced. DISCUSSION

Results indicate that populations of the four salmonella species tested behave similarly in all by-products used at the different moisture levels. Low moisture levels allow the contaminating salmonella population to remain relatively stable for extended periods of time. There appears to be

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simulated-natural method of contamination was used in an attempt to duplicate salmonella contamination occurring in the field. One ml. of 24 hr. broth cultures of Salmonella typhimurium, S. tennessee, S. enteriditis, and S. heidelberg, were each inoculated into separate 100 gm. samples of 40% moisture meat and bone meal, mixed on a paint conditioner, and incubated at 37°C. for 48 hr. Each sample was then dried to a moisture level of approximately S % by the previously described drying procedure. Resulting cell populations of these inoculums were approximately 1 X 10' cells/ gm. and were relatively stable during the 9 month testing period. A 1 gm. portion of each inoculum was then mixed with 99 gm. of each sterile feedstuff. Cell counts were taken immediately after introduction into the test feed of the several moisture levels, and at specified intervals during a 21 day holding period at 37°C. The salmonella populations were determined by transferring 3-1 gm. portions of each sample into dilution bottles containing 99 ml. of a 1% peptone phosphate buffer solution, making appropriate dilutions, and preparing three brilliant green agar pour plates (BGA; Difco) from each dilution. Colony counts were made after 48 hr. plate incubation at 37°C. Water activity measurements of the test feedstuffs were obtained by transferring moisture stabilized feeds to a 150 X 20 mm. petri dish and holding in a closed chamber with a wide range sensing element connected to an electric hygrometer indicator that measured humidity within the chamber.

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MOISTURE AND SALMONELLA I N FEEDS

6 -

MEAT

BONE

MEAL

AT

37 C

-O..

5 X>' ~

a

.&' • —40%

-a..

4-

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12

3 4 DAYS

FIG. 1. Effect of various moisture levels on a S. typhimurium population in meat and bone meal at 37°C. for 21 days.

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V. L. CARLSON AND G. H. SNOEYENBOS

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FIG. 2. Effect of various moisture levels on a S. typhimurium population in fish meal at 37°C. for 21 days.

an inverse relationship between the viable cell count and moisture level; as the moisture level increases, the viable cell count

decreases, except at moisture levels that allow multiplication. Failure of poultry mash to support multi-

721

MOISTURE AND SALMONELLA I N FEEDS

FEATHER MEAL AT 3 7 ° C

-0—40% *X> — 3 0 %

-*—-10%

\ ^ — 2 0 % ^—25% I I I ! 12 3 4

1 7

1 14

•-—15%

1 21

DAYS FIG. 3. Effect of various moisture levels on a S. typhimurium population in feather meal at 37°C. for 21 days.

plication of salmonella even at aw levels higher than 0.970 was unexpected and cannot be explained at the present time. Several lots of starter type mash without known

bacterial inhibitors were tested without significantly different results. Additional work is necessary to confirm and explain these unexpected results. However, sal-

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—_ _

722

V. L. CARLSON AND G. H. SNOEYENBOS

POULTRY BY-PRODUCT MEAL AT 37°C

DAYS FIG. 4. Effect of various moisture levels on a S. typhimurium population in poultry by-product meal at 37°C. for 21 days.

monella can remain stable in poultry mash having low moisture levels as Fig. 5 shows, S. typhimurium is generally regarded as one of the most frequent serotypes infect-

ing animals (Moran et al., 1964), but has been found only infrequently in animal byproducts (Allred et al., 1967; Moran et al., 1964; Snoeyenbos et al., 1967). The failure

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— 40 %

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MOISTURE AND SALMONELLA IN FEEDS

POULTRY AAASH AT 37°C

rrn 12

3 4

DAYS

FIG. 5. Effect of various moisture levels on a S. typhimurium population in poultry mash at 37°C. for 21 days.

to find important differences in growth and stability characteristics in animal by-products among the serotypes tested suggests

that the low frequency of S. typhimurium in animal by-products cannot be explained in this manner. It is possible that S. typhi-

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^^-•—10%

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V. L.

CARLSON AND G.

H.

SNOEYENBOS

TABLE 1. Water activity of several feeds of different moisture levels

PERCENT TEST

MOISTURE

FEED 5

JO

15

20

25

0.405

0.650 0.820 0.910

FISH MEAL

0.410

0.710

FEATHER MEAL

0.405

0.700 0.860 0.935

POULTRY MEAL

0.415

0.670 0.810

0.865 0.910

POULTRY MASH

0.410

0.640

murium only appears to be relatively more common than other serotypes in animals because of greater likelihood of isolation due to generally greater virulence, higher excretion rate or related characteristics. It has been demonstrated that molds grow at aw levels substantially lower than those necessary for salmonella multiplication (Pitt and Christian, 1968). Storage of feedstuffs at aw levels sufficiently low to prevent mold growth would preclude growth of salmonella. However, local condensation of moisture on walls of containers used for storing low moisture feeds could allow development of dangerously large salmonella populations.

0.950 0.970

0.860 0.920 0.950 0.970

0.805

0.960

40 0.990 0.980

0.980

0.990

0.950

0.970

0.870 0.930 0.950

0.970

(aw's approximately 0.96 or higher). Multiplication was not demonstrated in starter mash even of moisture levels as high as 40% (a w 0.97). Growth patterns were not significantly different among the four test strains in the feedstuffs used. Natural feed microflora did not prevent the salmonella contaminants from multiplying at moisture levels that allowed growth in non-sterile feeds. However, mold was a severe problem in nonsterile feeds at high moisture levels after four days incubation. ACKNOWLEDGEMENT

SUMMARY

This work was supported in part by the American Feed Manufacturers Association, Chicago, Illinois.

Population dynamics of four salmonella species in a variety of animal by-products were found to be related to the aw (water activity) of the feedstuff. Test feeds having low aw's (0.40S-0.41S) permitted the salmonella contaminants to remain relatively stable during the 21-day holding period at 37°C. Population dieoff was directly proportional to elevation in moisture level, except at moisture levels that allowed growth

REFERENCES Alfred, J. N., J. W. Walker, V. C. Beal, Jr. and F. W. Germaine, 1967 A survey to determine the Salmonella contamination rate in livestock and poultry feeds. J. Am. Vet. Med. Assoc. 151: 1857-1860. Christian, J. D. B., and W. J. Scott, 1953. Water relations of salmonellae at 30 C. Australian J. Biol. Sci. 6: 565-573. Liu, R., G. H. Snoeyenbos and V. L. Carlson, 1969. Effect of moisture and storage tempera-

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MEAT a BONE MEAL

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MOISTURE AND SALMONELLA IN FEEDS ture on a Salmonella senftenberg 775W population in meat and bone meal. Poultry Sci. 48: 1628-1633. Moran, A B., C. D. Van Houweling and E. M. Ellis, 1964. The results of typing salmonella from animal sources in the United States. Proc. National Conf. Salmonellosis: 33-35. Morehouse, L. C , and E. E. Wedman, 1961. Salmonella and other disease producing organisms in animal by-products—a survey. J. Am. Vet. Med. Assoc. 139: 989-995.

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Pitt, J. I., and J. H. B. Christian, 1968. Water relations of Xerophilic fungi isolated from prunes. Appl. Microbiol. 16: 1853-1858. Scott, W. J., 1953. Water relations of Staphylococcus aureus at 30 C. Australian J. Biol. Sci. 6: 549-564. Snoeyenbos, G. H., V. L. Carlson, B. A. McKie and C. F. Smyser, 1967. An epidemiological study of salmonellosis of chickens. Avian Dis. 11: 653-667.

E. T. MORAN, JR. AND H. L. ORR Department of Poultry Science, University of Guelph, Guelph, Ontario, Canada (Received for publication November 29. 1969)

A

N EARLIER report on the percentage • of various carcass parts indicated that differences due to age and sex with one commercial source broiler chicken between 6 and 10 weeks were not particularly outstanding (Moran and Orr, 1969). Confirming the data shown by Swanson et al. (1964) the female was observed to have a larger proportion of breast but smaller percentage of drumstick than the male. With regard to age there appeared small but noticeable increases in the relative amount of breast at 8 and 9 weeks with the male and female, respectively. To investigate the degree of variation which might be encountered with the multitude of commercial crosses yet avoid actual experimentation with each, Moran et al. (1970) determined the differences which existed between two of the most commonly used broiler breeds and their reciprocal crosses. The breeds studied were pure but not inbred lines of Cornish and White Rocks. Contrary to expectation, alterations in the relative yields of the various commercial parts due to breed and cross though somewhat variable were not evident.

Because there were only small and/or infrequent differences ever noted in any of the parts with age, sex or between primary component breeds, it was our opinion that there would be no variation of any commercial consequence with the many available crosses. This opinion met with some objection by personnel of a broiler parent breeding operation. Reliable sources had reported that this particular strain source ultimately yielded carcasses with a larger breast than those of some competitors. Upon considering the implications as they could affect advertising and sales their concern was warranted. While these aforementioned field reports could well be true, we felt any differences involved were more probably related to alterations in the degree of fleshing than true relative yield change. As the data of Moran and Orr (1969) illustrate, fleshing grade on the breast is significantly related to its percentage cooked meat yield. Though an increase in the degree of fleshing is not likely to be localized but proportionately spread over the entire bird with muscle mass, one might expect the heavily fleshed parts to benefit with any improvements more so

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Influence of Strain on the Yield of Commercial Parts from the Chicken Broiler Carcass