Formalin Destruction of Salmonellae in Poultry Litter

Formalin Destruction of Salmonellae in Poultry Litter

J.E.WILLIAMS

USDA, Science and Education Administration, Agricultural Research, Southeast Poultry Research Laboratory, 934 College Station Road, Athens, Georgia 30605

(Received for publication March 10, 1980)

ABSTRACT Litter was sprayed with 2%, 4%, and 6% formalin solutions both with and without litter turnover after contamination either on the surface or by mixing well with feces containing salmonellae. Results showed that 4% and 6% formalin applied to the litter by a turnover, spray, turnover, spray, turnover, spray procedure completely destroyed the salmonellae. A 6% solution of formalin applied by a turnover, spray, turnover, spray procedure also completely destroyed the organisms. Direct surface spraying with either 4% or 6% formalin completely destroyed salmonellae deposited in feces on the litter surface; however, the organisms were not eliminated if the formalin was only surface sprayed after the organisms had been mixed into the litter before spraying. In the experiments with turnover and spray the organisms were not killed if there was a 2 day break between formalin applications. (Key words: Salmonella, Salmonella typhimurium, formalin, formaldehyde, salmonellacidal, bacteriocidal, poultry litter, nalidixic acid, poultry feces)

1980 Poultry Science 59:2717-2724

INTRODUCTION

High levels of salmonella organisms may build up in poultry litter where they tend to multiply; therefore, litter may serve as an important means by which salmonella infections are transmitted from infected to clean birds. Fanelli et al. (1970) have suggested that cycling of the organisms between the intestinal tracts of infected birds and the litter is important in maintaining the infection. Olesiuk et al. (1969), Snoeyenbos et al. (1970), Snoeyenbos (1971), and Bhatia et al. (1979) reported periodic cultures of litter samples to be a reasonably accurate and practical method of determining the salmonella status of chicken flocks and to be superior to the more direct methods of detecting flock infection. Poultry litter serves as an excellent carrier system for the perpetuation of salmonella infections under natural environmental conditions. Litter may also contaminate the surface of eggs with salmonellae from the contaminated feet of chickens or from direct contact with the surface of eggs laid on the floor.

New litter has been reported to favor the survival of salmonellae more than old litter in which the organisms tend to die out more rapidly (Botts et al, 1952; Snoeyenbos et al, 1967). Rigby and Pettit (1979) showed that Salmonella typhimurium can spread very rapidly in young chicks on new litter. Williams and Benson (1978) found that salmonellae will survive for at least 18 months in litter at room temperature. Turnbull and Snoeyenbos

(1973) reported that the salmonellacidal activity of chicken litter was the result of two principal factors: a water activity unfavorable to salmonella cell viability and high pH from ammonia dissolved in the available moisture of the litter. Duff et al. (1973) found that following cleaning, disinfecting, and fumigation, S. typhimurium could not be isolated from the litter in houses that had previously been infected with S. typhimurium.

In spite of the importance of litter in the spread of salmonella infections, little attention has been given to the destruction of salmonella organisms in it. Halbrook et al. (1951) used quicklime in built-up litter to reduce noticeably all classes of bacteria, yeasts, and molds. Best et al. (1970) found in two broiler units that litter naturally and artificially contaminated with S. thompson was disinfected within 15 hr by a combination of heating air and floors at 55 to 75 C. Only a combination of heated air and heated floor was effective for successful disinfection. Enos (1972) found the average levels of aflatoxin in litter of control pens ranged from 2.42 to 3.63 times greater than in pens in w'hich the litter was treated with propionic acid.

Woodward and Tudor (1973) added powdered sulfur and sodium acid sulfate to the litter of experimental pens of pigeons during studies of pox vaccines after an outbreak of S. typhimurium var. Copenhagen infection. They suggested that this procedure eliminated residual salmonellae in the pens because losses ceased within 2 weeks, and no further salmonella

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cases were observed. Veloso et al. (1974) reported that the bacterial count of poultry litter containing 3% formaldehyde flakes was reduced to about 10% of the control value for 3 weeks after which the count returned to control values. When the litter contained only 1% formaldehyde flakes, the bacterial count was reduced for 2 weeks and the extent of the reduction was not as great. The treatments had no significant effect on growth rate, feed efficiency, or mortality of broilers. Tudor (1978) recommended the use of sodium acid sulfate and wettable sulfur for salmonella destruction in poultry-raising operations by considerably lowering the pH.

Harry et al. (1973) found that exposure of finely sieved poultry house litter for 20 hr to methyl bromide gas at concentrations of 10 to 120 mg/liter resulted in a marked reduction in the number of 5. typhimurium. Tucker et al. (1975) found that Salmonella vircbow could not be eliminated from poultry litter by methyl bromide fumigation because the gas was not effective against the organisms in compacted feces present in the litter. Investigators reported (Anon, 1976) that British researchers recommended the addition of 21 g of a pelleted form of paraformaldehyde to the litter in each nest box to reduce bacterial contamination, including salmonellae. A mixed culture called Litter—Life has been added to litter for microbial destruction. Other preparations on the market to control microbial flora in litter are sorbic acid, Cu+ + , gentian violet, and sodium propionate.

Formalin was chosen for these studies because it has been extensively used with considerable success in fumigation and disinfection practices in the poultry industry, it is cheap and readily available, and it shows superior biocidal qualities toward salmonellae in in vitro tests (minimum inhibitory concentration). Formalin has been used by plant scientists for years to sanitize soil in preventing the spread of plant pathogens. Harry and Helmsley (1964) dispersed formalin in a deep litter house as a wet mist to reduce the coliform count of layers of dust within 1 day of treatment.

Tosh et al. (1966) treated soil samples with 3% and 5% formaldehyde solution. One week after application of formaldehyde, the fungus Histoplasma capsulatum could no longer be isolated from the soil. Robinson et al. (1970) showed that monthly sprayings of sheep yard debris containing salmonella bacteria with 5% formalin solution resulted in elimination of

these organisms after two sprayings. In the present study the efficiency of

formalin treatments in eliminating the salmonella populations in poultry litter was measured.

MATERIALS AND METHODS

Infecting Organisms. A 24-hr Veal Infusion broth culture of a strain of S. typhimurium resistant to nalidixic acid with a viable cell count of 53 x 107 organisms/ml was diluted 1:50 with sterile saline (.85%). Each of 150 1-day-old chicks was administered .3 ml of the diluted culture per os. Starting after 24 hr, all the feces from these chicks were collected on foil placed below the wire floors of each modified Horsfall unit (Drury et al., 1969) in which they were housed in groups of 10. Total fecal collections were continued each day for 1 week.

The collected feces were allowed to dry for 24 hr at room temperature and then placed in an unheated vacuum chamber for final drying. The dried feces were pulverized in a Waring blender under a hood, after which they were stored tightly closed at 4 C. The initial viable 5. typhimurium cell count of this powder was 11 X 10s cells per gram, which closely approximated all the counts subsequently made of the powder just before its use during the period of each of these experiments.

Experimental Test Units. Each experiment was conducted in a modified Horsfall unit, with a total area of .37 m 2 (4 ft2) . Enough litter, composed of new pinewood shavings, was placed into each unit in an amount to provide a depth of 7.6 to 10.2 cm (3 to 4 in).

Litter Contamination. The carrier system for the organisms used to contaminate the litter was a fresh fecal slurry prepared from salmonella-free feces collected from adult birds. For each experiment, this slurry was prepared by adding 100 ml of sterile distilled water to 100 g of fresh feces. To this was added 5 g of the fecal powder containing the salmonella. A fecal slurry was used for infection in an effort to closely simulate the fecal contamination of litter by carrier birds which occurs under natural conditions.

In each experimental unit 200 g of the salmonella-infected slurry were used to contaminate the litter. This contamination resulted in an S. typhimurium level of about 4,000 organisms per gram of litter. In Experiment 1, the contaminated slurry was evenly deposited


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directly on the surface of the litter with a sterile spatula to evenly distribute the individual aliquots. In Experiments 2, 3, and 4, the contaminated slurry was evenly mixed throughout the litter by hand; personnel wore rubber gloves for protection.

Formalin Treatments. In all experiments except Experiment 4, three concentrations of formalin were used: 2%, 4%, and 6%; in Experiment 4, only the 6% concentration was used. In Experiments 1 and 4, one application of 500 ml/.37 m2 (4 ft2) of the formalin solutions was sprayed evenly on the surface of the litter. In part 1 of Experiment 2, the formalin solutions were sprayed in one immediate application of 300 ml/.37 m2 (4 ft2) after litter turnover followed by a second immediate application of 300 ml/.37 m2 (4 ft2) after a second litter turnover.

In part 2 of Experiment 2, the formalin solutions were sprayed on the surface of the litter in one application of 300 ml/.37 m2 (4 ft2) after litter turnover; then the units were left closed for 2 days. The litter was then thoroughly turned over a second time, and the surface again sprayed with 300 ml/. 3 7 m2 (4 ft2) of formalin solution. In Experiment 3, one application of 200 ml/. 3 7 m2 (4 ft2) of formalin was applied to the surface of the freshly turned over litter; then the litter was turned over a second time and 200 ml/.37 m2 (4 ft2) of formalin was applied. The litter was then turned over a third time and 200 ml/. 3 7 m 2 (4 ft2) of formalin solution was applied.

The formalin solutions were sprayed into all the experimental units with a low pressure hand operated sprayer. Neither the particle size nor the flow rate of the sprayer was controlled. Tight gas masks were worn during the applications of the formalin solutions.

Controls. For each experimental unit treated with formalin, a control unit was contaminated in the same manner as the formalin-treated pens but received no treatment with any disinfectant.

Sentinel Chicks. At the end of 1 week after unit treatments with formalin, five one-day-old chicks were placed on the litter in each treated and control unit for salmonella detection.

Isolation from litter. The litter was collected on days 5, 7, 12, and 14 with paper cups and sterile plastic bags in 5-g amounts from each test and control unit and placed into 100 ml of Tetra-thionate Brilliant Green (TBG) broth containing 100 jug/ml of sodium nalidixate. This broth was incubated at 37 C for 24 hr and plated on

Brilliant Green (BG) agar containing 100 /xg/ml of sodium nalidixate. Typical salmonella colonies were picked from the plates and passed through Triple Sugar Iron (TSI) and Lysine Iron (LI) agars before biochemical and serological identification.

Isolation from Sentinel Chicks. At culture time each group of five chicks was placed in a plastic bag. Birds from the negative and'treated pens were cultured first and those from the positive control pens last. The hands were washed carefully after handling each group. Cloacal swabs were taken from the chicks on days 5, 7, 12, and 14, placed into tubes containing 9 ml of TBG broth with 100 /ul/ml of sodium nalidixate, and incubated at 37 C for 24 hr. The TBG broth was plated on BG agar containing 100 jul/ml of sodium nalidixate. Colonies were transferred from the plates to TSI and LI agars and identified biochemically and serologically as S. typhimurium.

After four culture tests of the litter and four of the cloacal swabs from the sentinel chicks, the experiment was ended.

RESULTS

Experiment 1. Results of Experiment 1 are given in Table 1. Surface spraying of surface contamination with 4% and 6% formalin concentrations completely eliminated detectable levels of salmonella organisms from the litter. A low level of salmonella contamination was noted in the litter treated with 2% formalin. None of the sentinel chicks in any of the pens treated with 2%, 4%, or 6% formalin revealed an S. typhimurium-posiw/e cloacal swab culture.

The untreated control pens all revealed positive litter samples at the time each culture was taken, and a high percentage of the cloacal swabs from the sentinel chicks in these pens were S. typhimurium positive. Negative controls remained negative during the entire experiment.

Experiment 2. Results of Experiment 2 are given in Table 2. Salmonellae were detected by both litter and sentinel bird culture in part 1 of Experiment 2, in which litter contaminated by mixing was turned over and treated with an application of 2% and 4% formalin, turned over again, and treated with a second application of 2% and 4% formalin. Thus, 2% and 4% formalin did not eliminate the salmonella organisms. An identical treatment with litter turnover and formalin application followed by litter turnover


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TABLE 1. Culture results of litter and sentinel chicks placed on litter surface sprayed with three levels of formalin after surface contamination (Experiment 1)

„ _ ,. . . , . „ a Days after sentinel chick placement Formalin concentration Littera i £. for surface treatment Chicks'5 5 7 12 14

2% Treated Litter 1/2 0/2 0/2 0/2 Chicks 0/10 0/10 0/10 0/10

Untreated controls Litter 2/2 2/2 2/2 2/2 Chicks 3/10 5/10 5/10 8/9





Negative controls Litter 0/1 0/1 0/1 0/1 Chicks 0/5 0/5 0/5 0/5

Number of S. typhimurium isolated/number of pens cultured.

Number of S. typhimurium isolated/number of chicks cultured.

and formalin application with 6% formalin revealed that detectable levels of salmonella contamination were totally eliminated. The higher concentration of formalin was apparently needed for complete elimination of the organisms.

In part 2 of Experiment 2, S. typhimurium remained at levels detectable by both litter and sentinel chick culture when the litter was sprayed with one application of either 2%, 4%, or 6% formalin after litter turnover followed by a second application after litter was again turned over after 2 days. Apparently during the 2 day interim period the salmonellae had not all been killed, and a bacterial buildup did not permit subsequent formalin applications to be effective in killing the organisms.

The untreated positive control units revealed a high level of contamination during the entire experiment. Negative controls remained negative during the entire experiment.

Experiment 3. Results of Experiment 3 are given in Table 3. When litter mixed during contamination was treated with 2% formalin, turned over, sprayed and mixed well with formalin three times, salmonellae were detectable by both litter and sentinel bird cultures. Thus, 2% formalin did not eliminate the organisms. The same treatment with 4% and 6%

formalin revealed that detectable levels of salmonella contamination were totally eliminated by turnover and spraying three times after contamination by mixing. The higher concentrations of formalin were apparently needed for complete elimination of the organisms.

In all the untreated positive control groups, salmonellae were isolated. Negative controls remained negative during the entire experiment.

Experiment 4. Results of Experiment 4 are given in Table 4. The level of S. typhimurium contamination remained very high for both the formalin-treated litter and untreated control litter for the entire period of this experiment. Sentinel chicks on both treated and untreated litter were also salmonella infected.

Although some salmonella inhibition was noted early in the formalin-treated groups, this inhibition tended to disappear with time, and all test groups became equally contaminated at a maximum level. Results of this experiment clearly show that the formalin was not able to penetrate into the litter after surface spraying and reach the organisms that had been mixed with the litter substance. These organisms apparently survived and multiplied in the litter.

Negative controls remained negative during the entire experiment.


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TABLE 2. Culture results of litter and sentinel chicks placed on litter containing well mixed contamination sprayed two times with litter turnover using three levels of formalin

with and without a time break (Experiment 2)

Formalin concentration and application time for mixed treatment

2% Treated (Part 1-2 immediate sprays)

2% Treated (Part 2-2 sprays with a 2-day break)

Untreated controls



Untreated controls



Untreated controls

Negative controls

Littera

Chicksb

Litter Chicks

Litter Chicks

Litter Chicks

Litter Chicks

Litter Chicks

Litter Chicks

Litter Chicks

Litter Chicks

Litter Chicks

Litter Chicks

5

0/2 0/10

2/2 10/10

2/2 8/10

0/2 3/10

2/2 10/10

2/2 9/10

0/2 0/10

1/2 8/10

2/2 9/10

0/1 0/5

Days after sentinel chick placement

7

0/2 10/10

2/2 10/10

2/2 9/9

0/2 7/10

2/2 10/10

2/2 9/9

0/2 0/10

1/2 9/10

2/2 9/10

0/1 0/5

12

1/2 10/10

2/2 10/10

2/2 9/9

0/2 9/10

2/2 10/10

2/2 9/9

0/2 0/10

2/2 10/10

2/2 9/10

0/1 0/5

14

2/2 10/10

2/2 10/10

2/2 8/8

1/2 10/10

2/2 10/10

2/2 8/8

0/2 0/10

2/2 10/10

2/2 9/9

0/1 0/5



DISCUSSION Salmonellae may readily reach high popula

tions in poultry litter after their introduction from feed or from other extraneous sources and their subsequent incubation under natural conditions in the poultry house. The findings obtained in these experiments show that 6%, but not 2% and not always 4%, aqueous formalin solution effectively eliminates salmonellae from poultry litter when litter is turned over and there is no time break. The formalin must come into direct contact with the contaminated surfaces to get the full effects of these treatments.

Because salmonella contamination is usually in the litter substance rather than just on the surface, litter turnover with formalin sprays will be the preferred method of application. Our research shows that after litter turnover, viable salmonellae can be eliminated from the litter by spraying with 6% formalin followed by a second litter turnover and a second spraying

with 6% formalin. For each of these two sprays, 300 ml/.37 m2 (4 ft2) of 6% formalin were used. Salmonellae can also be eliminated by spraying with 200 ml/.37 m2 (4ft2)of4%or6%formalin after litter turnover followed by a second litter turnover and a second spraying with 200 ml/.37 m2 (4 ft2) of 4% or 6% formalin followed by a third litter tur .over and third spraying with 200 ml/.37 m2 (4 ft2) of 4% or 6% formalin. Either of these procedures is effective.

Direct spraying of feces on the surface of the litter with 500 ml/.37 m2 (4 ft2) of either 4% or 6% formalin will destroy salmonellae. Salmonellae in the litter cannot be destroyed by spraying the litter surface. A break period between the formalin applications is apparently undesirable, because contamination seems to build up in the litter, and subsequent formalin applications do not seem to be effective.

Formalin tends to volatilize into the air and


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TABLE 3. Culture results of litter and sentinel chicks placed on litter containing well mixed contamination sprayed three times -with three levels of formalin with litter turnover before each spray (Experiment 3)

Formalin concentration for mixed treatment

2% Treated

Untreated controls

4% Treated

Untreated controls

6% Treated

Untreated controls

Negative controls

Littera

Chicksb

Litter Chicks

Litter Chicks

Litter Chicks

Litter Chicks

Litter Chicks

Litter Chicks

Litter Chicks

5

0/2 0/10

2/2 8/10

0/2 0/10

2/2 10/10

0/2 0/10

2/2 10/10

0/1 0 /5

Days after sentine

7

1/2 1/10

2/2 10/10

0/2 0/10

2/2 10/10

0/2 0/10

2/2 9/9

0/1 0/5

:1 chick placement

12

2/2 0/10

2/2 10/10

0/2 0/10

2/2 9/9

0/2 0/10

2/2 9/9

0/1 0/5

14

2/2 0 /9

2/2 10/10

0/2 0/10

2/2 9/9

0/2 0 /9

2/2 9/9

0/1 0/5



with time, very low levels remain in treated substances such as litter. This explains why we were successful in direct culture of the treated litter. In earlier stages of this work the treated litter was diluted before culture, but our later experience showed that this dilution was not necessary because the salmonella organisms could be cultured satisfactorily from treated as well as untreated litter.

The use of a nalidixic acid resistant S. typhimurium culture was of great advantage in these experiments. Similar cultures have been used by other workers in both liquid (Benazet and Carrier, 1980) and solid (Smyser and Snoeyenbos, 1979) media containing nalidixic acid. By use of enrichment broth (TBG) and solid selective agar (BG) containing 100 /ig/ml of sodium nalidixate, we were able to obtain pure colonies of salmonella at will without any contaminants. Use of this technique, which should see wide application, also eliminates all concern with any salmonella or other organism being introduced into the experiments from feed or other sources, because these, if introduced, would be totally inhibited by the sodium nalidixate and only the salmonella resistant to sodium nalidixate would be able to grow. The potential of salmonella isolation is considerably enhanced by the use of a salmon

ella strain resistant to nalidixic acid. The uneven numbers of sentinel birds in the

tables are due to the fact that some of the birds died during the experiments. Most death losses, which were due to S. typhimurium infection, were in the untreated control pens where the salmonella contamination was apparently at a much higher level than in the treated pens.

Evidently the new litter used in these experiments is unlike the builtup litter used in most poultry houses. Because new litter is less destructive of salmonella contamination than old litter, the use of new litter was felt to be an advantage. With builtup litter, excessive caking may be a problem that could complicate and interfere with formalin effectiveness. Final recommendations must await additional research that is planned with both old and new litter and with selected formalin applications.

The methods proposed here for evaluating the efficacy of various disinfectants and chemicals in killing salmonellae in litter may serve as a model for studies in this field. In the past standard procedures have not been available for this purpose. With the use of these methods additional data should be developed on the relative efficacy of various treatments in destroying disease agents in litter and other substances.


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TABLE 4. Culture results of litter and sentinel chicks placed on litter surface sprayed with one level of formalin after well-mixed contamination (Experiment 4)

for surface treatment

6% Treated

Untreated controls

Negative controls

Littera

Chicksb

Litter Chicks

Litter Chicks

Litter Chicks

5

1/3 0/15

3/3 11/15

0/1 0/5

Days after sentin

7

2/3 5/15

3/3 14/14

0/1 0/5

lei chick placement

12

3/3 14/14

3/3 14/14

0/1 0/5

14

3/3 15/15

3/3 14/14

0/1 0/5



ACKNOWLEDGMENTS

The author is indebted to S. T. Benson for his expert technical assistance in conducting these experiments. Appreciation is also expressed to M. C. Kumar and G. H. Snoeyenbos for their helpful advice and counsel during these studies.

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Formalin Destruction of Salmonellae in Poultry Litter