Method for controlling Salmonella enteritidis in poultry

In vitro results indicate that trimethoprim may be ineffective against S. enteritides. However, in vivo results show that trimethoprim alone and in combination with polymyxin B sulfate is effective on S. enteritidis infections in poultry.

The present invention relates to methods for controlling Salmonella 
enteritidis in the test tube and in live poultry and, more particularly, 
to methods utilizing antimicrobial agents such as trimethoprim and 
polymyxin. 
BACKGROUND OF THE INVENTION 
Salmonella infections have been prevalent in poultry flocks for several 
decades. Recently, Salmonella enteritidis (S. enteritidis) infections have 
caused epidemics in the United States and the United Kingdom. Outbreaks of 
food poisoning in humans due to the consumption of grade A eggs have 
increased dramatically. Epidemiological and microbiological investigations 
have shown that the outbreaks have involved foods containing hen's eggs, 
and approximately 80% have been caused by S. enteritidis. See St. Louis, 
M. E. et al. 1988. The Emergence of Grade A Eggs as a Major Source of S. 
enteritidis Infections. J. Amer. Med. Assoc. 259:2103-2107 and Coyle, E. 
F. et al. 1988. S. enteritidis Phase Type 4 Infection: Association with 
Hen's Eggs. Lancet, Dec. 3, p. 1295-1296. 
The poultry industry urgently needs to find means to control infections of 
poultry flocks and eggs. The gastric tract of poultry is a major reservoir 
of human pathogens. Feed is often the source of contamination, which leads 
to intestinal infection or colonization in the chicken. In turn, virulent 
pathogens may enter the blood and oviduct by passage through the cecal 
mucosa. Much effort has been concentrated on the elimination of pathogens 
from the environment, feeds and gastric tracts of poultry. Extensive 
research has been performed on providing competitive flora, referred to as 
the "Nurimi Concept." However, infections still persist. 
In order to understand the present invention, it may be helpful to briefly 
review some features of bacteria and antibiotics. A bacterium is a 
unicellular microorganism which may exist either as a free-living organism 
or as a parasite. Bacteria, or a single bacterium, have a wide range of 
biochemical and pathogenic properties. 
Bacteria may be classified according to cell wall structure into two basic 
groups: gram-negative bacteria and gram-positive bacteria. Gram-negative 
bacteria have more complex cell wall structures than gram-positive 
bacteria. In particular, the cell wall of gram-negative bacteria have an 
outer membrane, which is absent from gram-positive bacteria. 
The outer membrane of a gram-negative bacterium includes inner and outer 
protein layers with phospholipid (a type of fat) disposed in a bilayer 
between the protein layers. The proteins may include matrix protein (also 
referred to as porin) which is transmembranal and may create pores in the 
membrane to allow diffusion of molecules through the outer layer. 
Lipoproteins may anchor the outer membrane to a peptidoglycan layer. The 
outer membrane, lipoprotein, peptidoglycan, and periplasmic space, which 
are all layers external to the inner membrane, may be referred to as the 
cell wall. 
The outer membrane of the cell wall includes lipopolysaccharide molecules 
which extend from the cell wall. An outermost portion of a 
lipopolysaccharide molecule is referred to as an O antigen polysaccharide. 
O antigens are specific polysaccharide side chains and are often the major 
antigenic determinants in gram-negative bacteria. 
The outer membrane may repel certain compounds. For instance, the 
lipopolysaccharide molecules are hydrophilic (water-loving). Hence, the 
lipopolysaccharide molecules may deter the approach of hydrophobic 
compounds. However, even hydrophilic compounds of a size greater than the 
pores created by the matrix protein may be excluded by the outer layer. 
Salmonella is gram-negative group of bacteria with more than 2,000 
serotypes. In biological nomenclature, Salmonella is a genus which 
contains only one species. The one species comprises seven subspecies. 
Each subspecies is divided into serovars. Serovars are further divided 
into serotypes. S. enteritidis has been identified as a serovar. 
An antibiotic is a naturally produced substance which is effective in 
inhibiting the growth of or destroying microorganisms. Antibiotics may be 
produced by fungi, bacteria, or other organisms. 
An antibiotic such as polymyxin may damage or kill a bacterium such as by 
altering its membrane permeability, impeding respiration or impairing 
certain transport phenomena such as electron transport of the 
microorganism. For example, an antibiotic may bind to the outer membrane 
of a bacterium and open holes in the membrane through which components of 
the cell may leak out, thereby killing the bacterium. In other words, the 
permeability of the outer and inner membranes for small-molecular weight 
compounds may be increased in the presence of antibiotics and the efflux 
of cytoplasmic components may occur. 
SUMMARY OF THE INVENTION 
A feature of the present invention is the provision in controlling of S. 
enteritidis in poultry, of introducing trimethoprim into the poultry. 
Another feature of the present invention is the provision in controlling S. 
enteritidis in poultry, of introducing trimethoprim and polymyxin B into 
the poultry. 
Another feature of the present invention is the provision in controlling S. 
enteritidis in poultry, of an antimicrobial mixture of trimethoprim and 
water. 
Another feature of the present invention is the provision in controlling S. 
enteritidis in poultry, of an antimicrobial mixture of trimethoprim, 
polymyxin B, and water. 
Another feature of the present invention is the provision in controlling S. 
enteritidis, of introducing trimethoprim into poultry 24 hours old and 
continuing to introduce the trimethoprim daily via the poultry's drinking 
water for about 15 days or until the poultry's normal intestinal flora 
matures. 
An advantage of the present invention is the prevention of S. enteritidis 
in poultry. 
Another advantage of the present invention is the removal of S. enteritidis 
from poultry.

DETAILED DESCRIPTION 
1. Verification of S. Enteritidis 
The S. enteritidis strains used in the in vitro and in vivo examples were 
obtained from the N.Y. State Health Department and were labeled as E40 and 
13076 strains. The E40 strain was originally isolated from a chicken 
ovary. The strains were identified as being in fact S. enteritidis by 
using the Kauffmann method which uses biochemical testing to differentiate 
the separate species, serotypes, or serovars. 
The Kauffman method recognizes that different types of bacteria have 
different O antigens and different H antigens. For example, S. enteritidis 
has O antigens identified by the numbers 1, 9, and 12, and H antigens 
identified by the letters g and m. 
According to the Kauffmann method, homologous antiserum, which is 
commercially available, is reactive with its respective specific antigen 
and causes bacterial cells which have that specific antigen to agglutinate 
or clump together. The agglutination reaction is readily seen with the 
naked eye on a microscope slide. Identification of a Salmonella species is 
mainly based on a series of agglutination reactions. The reactions are 
also run alongside of known negative controls for the comparison of 
agglutinated cells with non-agglutinated cells. 
2. Preparation of S. Enteritidis Cultures 
The S. enteritidis cultures used in the in vitro and in vivo portions of 
the present invention were prepared by growing approximately 10.sup.10 
(ten billion) cells in 10-20 ml of soy broth in 24 hours immediately prior 
to inoculation of either the agar plates or the live chicks. The soy broth 
was prepared as identified below. 
3 Preparation of the Soy Broth solution for S. Enteritidis cultures 
A one liter solution of Trypticase.RTM. soy broth was prepared by 
dissolving 30 grams of the soy broth powder in one liter of purified 
water. The solution was mixed thoroughly and stirred until the soy broth 
powder was completely dissolved. The solution was then autoclaved at 
121.degree. C. for 15 minutes to sterilize the broth by killing any viable 
microorganisms. 
The Trypticase.RTM. soy broth is available from BBL Microbiology Systems, 
Becton Dickinson and Co., Cockeysville, Md. For every 30 grams, the 
Trypticase.RTM. soy broth has a composition of 17.0 grams of pancreatic 
digest of casein, 3.0 grams of papaic digest of soybean meal, 5.0 grams of 
sodium chloride, 2.5 grams of dipotassium phosphate, and 2.5 grams of 
dextrose. Such a broth provides nutrients for the rapid reproduction or 
growth of S. enteritidis. 
4. Preparation of the Reagent Antimicrobial Starting Materials 
The trimethoprim and polymyxin B sulfate starting materials used in both 
the in vitro and in vivo testing were filter sterilized through 
non-pyrogenic, sterile, low protein binding, Acrodisc.RTM. disposable 
filter assemblies available from Gelman Sciences, 600 S. Wagner Road, Ann 
Arbor, Mich. The purpose of filter sterilizing the starting materials is 
to remove any viable microorganisms. The starting materials were filter 
sterilized instead of autoclaved because trimethoprim and polymyxin B 
sulfate are heat sensitive. 
5 Preparation of Agar Medium 
Agar is a mucilaginous material used as a base for bacterial culture media 
and selectively allows the growth of certain types of bacteria. Agar media 
used in this experiment were dehydrated Bacto.RTM. bismuth sulfite agar, 
dehydrated Bacto.RTM. XLD agar, and Bacto.RTM. Brilliant Green agar all of 
which were obtained from Difco Laboratories of Detroit, Mich. The 
Bacto.RTM. bismuth sulfite agar is represented by Difco as being a highly 
selective medium for the isolation of Salmonella, especially Salmonella 
typhi. The Bacto.RTM. XLD agar is represented by Difco as being a 
selective and differential primary plating medium for isolating and 
differentiating gram-negative enteric bacilli, especially Shigella and 
Providencia. The Bacto.RTM. Brilliant Green agar is represented as being a 
highly selective, primary plating medium for isolating Salmonella other 
than Salmonella typhi. In less than 24 hours, an individual Salmonella 
cell divides a sufficient number of times and grows on the agar medium to 
form a mass which is perceivable to the naked eye. That mass, in 
biological nomenclature, is a colony. 
Fecal samples were collected in pre-weighed, sterile tubes by expressing 
the sample from the birds' cloaca. Except when enriched, the fecal samples 
were diluted with 1 ml of buffered saline and dilutions thereof were 
streaked onto the two types of agar medium. The agar plates were then 
incubated for 24 hours at 37.degree. C. By measuring the amount of fecal 
sample obtained, the amount of dilution with the buffer, the amount of 
buffer with fecal sample mix placed on the plate after the dilution, the 
number of colony forming units in the fecal sample may be determined from 
the number of colonies counted on the plate. 
6. Preparation of Enrichment Broths 
An enrichment broth is a liquid selective medium that directly receives an 
undiluted fecal sample and permits Salmonella species of the fecal sample 
to multiply perhaps 100-700 times over 24 hours while remaining mobile in 
solution. The enrichment broth simultaneously inhibits reproduction of 
other bacteria. In contrast, a mucilaginous material such as bismuth 
sulfite agar receives only liquid samples such as diluted fecal samples. 
Agar media does not permit movement of the bacteria, which reproduce in 
single locations and grow from a single S. enteritidis cell or "colony 
forming unit" to a colony that is visible to the naked eye. 
The plated agar method is inaccurate when S. enteritidis counts are low 
such as when fecal samples of 0.5g or less contain five S. enteritidis 
cells or less. When the fecal samples are dispersed into a liquid medium 
(and hence diluted), it is often difficult to locate the five cells. 
Accordingly, such fecal samples are "enriched" in an enrichment broth 
before being plated on agar medium. 
With an enrichment broth, a portion of the fecal sample is deposited into a 
test tube containing 10 milliliters of the broth. The solution is then 
stirred and incubated for 24 hours at 37.degree. C. Over 24 hours, S. 
enteritidis cells may multiply 100-700 times. After the incubation period, 
the solution is stirred and a loopfull of the solution is streaked onto 
the bismuth sulfite and XLD agar medium and incubated another 24 hours. 
The agar plates are then observed to determine the presence or absence of 
S. enteritidis colony forming units. The observation of even one S. 
enteritidis colony forming unit is scored as a (+) sign in Tables 3 and 4, 
while the absence of any S. enteritidis colony forming units is identified 
by the (-) sign. The number of S. enteritidis cells per gram of fecal 
sample is not determinable with enrichment broths because the growth rate 
of the cells in the enrichment broths cannot be quantified accurately. 
Bacto Selenite Cystine Broth (SC) is a selective enrichment broth that 
permits Salmonella species to grow while reducing growth of fecal coli and 
enterococci. A dehydrated Selenite Cystine enrichment broth contains the 
following ingredients: 
______________________________________ 
Bacto Tryptone 5 g 
Bacto Lactose 4 g 
Disodium Phosphate 10 g 
Sodium Acid Selenite 4 g 
L-Cystine 0.01 g 
______________________________________ 
The Selenite Cystine Broth was prepared by suspending 23 g of the broth in 
one liter of distilled water. The solution was then heated to boiling to 
dissolve the broth. The solution was then dispensed in sterile test tubes 
and allowed to cool to room temperature. When cool, the test tubes were 
inoculated with the remainder of the fecal sample and incubated at 
37.degree. C. for about 24 hours. A loopfull of the culture was then 
streaked for isolation on each of the bismuth sulfite and XLD agar 
mediums. 
Bacto Tetrathionate Broth Base (Tet) is a selective enrichment broth base 
that also enriches members of the Salmonella group in the isolation of 
these organisms from infectious material. A liter of dehydrated Bacto 
Testrathionate Broth Base has the following ingredients: 
______________________________________ 
Proteose peptone, Difco 5 g 
Bacto Bile Salts 1 g 
Sodium Thiosulfate 30 g 
Calcium Carbonate 10 g 
______________________________________ 
A medium of Bacto Tetrathionate Broth Base was prepared by suspending 4.6 g 
in 100 ml of distilled water. The medium was heated to boiling and then 
cooled to room temperature. A 2 ml iodine solution, which was prepared by 
dissolving 6 g iodine crystals and 5 g potassium iodide in 20 ml of 
distilled water was added to the medium. Approximately 10 ml quantity of 
the medium was dispensed into sterile test tubes and inoculated with 
entire fecal samples. The solutions were incubated at 37.degree. C. for 24 
hours. A loopfull of the culture was then streaked for isolation on the 
bismuth sulfite and XLD agar mediums. 
7. In vitro Testing of Trimethoprim Alone and in Combination with Polymyxin 
B Sulfate 
A solution of Trypticase.RTM. soy broth, containing 2% agar, was prepared. 
The agar solution was autoclaved and subsequently given amounts of the 
respective antimicrobial compounds or combinations were added to give the 
final concentrations of the antimicrobials as noted in Tables 1 and 2. 
Each of the antimicrobial, soy broth agar solutions was then poured into a 
Petri dish and allowed to solidify at room temperature. After the 
antimicrobial soy agar cooled and hardened, each of plates (except for 
control example nos. 1 and 7) was inoculated with 0.1 ml of 24 hour 
culture of S. enteritidis by streaking the culture across the soy agar 
with the blade of a biological "hockey stick" which is a glass stick 
formed like a hockey stick. The plates were incubated at 37.degree. C. for 
24 hours, after which the agar was analyzed for S. enteritidis colonies. 
The soy agar plates of Table 2 (Examples 7-11) were incubated for 48 hours 
instead of 24 hours. 
TABLE 1 
______________________________________ 
Incubation of S. enteritidis for 24 Hours 
Number of 
S. enteritidis 
Antibacterial Compound or 
Colony Forming Units 
Combination in Trypticase 
per ml of Agar After 
Example 
Soy Agar 24 hours of Incubation 
______________________________________ 
1 Control (no antibacterial 
1.48 .times. 10.sup.9 
compound) 
2 Polymyxin B sulfate 
4.7 .times. 10.sup.3 
(1 ug/ml) 
3 Trimothoprim (1 ug/ml) 
1.0 .times. 10.sup.9 
4 Trimethoprim (1 ug/ml) 
1.6 .times. 10.sup.3 
and polymyxin B sulfate 
(1 ug/ml) 
5 Trimethoprim (3.3 ug/ml) 
10.sup.9 
6 Trimethoprim (6.6 ug/ml) 
10.sup.9 
______________________________________ 
TABLE 2 
______________________________________ 
Incubation of S. enteritidis for 48 Hours 
Number of 
S. enteritidis 
Colony Forming Units 
Antibacterial Compound 
per ml of Agar after 
Example or Combination 48 hours of Incubation 
______________________________________ 
7 Control (no antibacterial 
6.45 .times. 10.sup.8 
compound) 
8 Polymyxin B sulfate 
6.5 .times. 10.sup.8 
(1 ug/ml) 
9 Trimethoprim 5.5 .times. 10.sup.8 
(6.6 ug/ml) 
10 Trimethoprim 10.sup.8 
(10 ug/ml) 
11 Trimethoprim 10.sup.8 
(25 ug/ml) 
______________________________________ 
Examples 1-11 indicate that trimethoprim may be ineffective in vitro. As 
shown by Examples 3, 5 and 6, S. enteritidis appears to be resistant at 
trimethoprim concentrations of 1 ug/ml, 3.3 ug/ml and 6.6 ug/ml when 
incubated on soy agar for 24 hours. As shown by examples 9, 10 and 11, S. 
enteritidis appears to be resistant at trimethoprim concentrations of 6.6 
ug/ml, 10 ug/ml and 25 ug/ml. However, the S. enteritidis colonies of 
Examples 9, 10 and 11 were translucent, in contrast to the opaque colonies 
of Example 7. Hence, the colony morphology may indicate that trimethoprim 
may be partially affecting growth of the S. enteritidis cells. 
8. In Vivo Testing of Trimethoprim alone and in combination with Polymyxin 
B Sulfate 
Trimethoprim alone and in combination with polymyxin B sulfate was 
introduced to chicks via their drinking water supply. A first group of 
chicks received the antimicrobial mixtures before being inoculated with S. 
enteritidis to determine whether the antimicrobial mixtures were effective 
in preventing infection of S. enteritidis. A second group of chicks 
received the antimicrobial mixtures after being inoculated with S. 
enteritidis to determine whether the antimicrobial mixtures were effective 
in removing S. enteritidis infections. 
24 hour old Dekalb chicks were obtained from Sunnsyside Poultry Farm of 
Oregon, Wis. and placed individually into separate solid bottom cages. 
The chicks were fed antibiotic-free feed (Ralston Purina's "Start and 
Grow") which was given ad libitum. Tap water and/or the antimicrobial 
mixtures were supplied in 8 ounce quantities in pint Mason jars and 
changed daily. The chicks resided in the BioTron facility at the 
University of Wis., Madison, which is a facility with proper containment 
features for working with class III pathogens. 
A. Prevention of S. enteritidis in poultry 
On day 1, chicks 12-19 were fed trimethoprim alone and in combination with 
polymixin B sulfate in concentrations as identified in Table 3. On day 
two, each of the chicks 12-19 was inoculated orally with a 0.5 ml solution 
of a 24-hour culture of E40 S. enteritidis which had an absorbence reading 
of O.D..sub.660 0.71 by a Spectronic.RTM. 20 spectrophotometer available 
from Milton Roy Company of Rochester, N.Y. Each of the examples 12-19 
represents one chick. 
As shown in Table 3, fecal samples were expressed from chicks 12-19 on days 
2, 6, 7, 9 and 15 after introduction into individual cages. On days 2 and 
6, bismuth sulfite and XLD agar medium was used to determine the number of 
S. enteritidis colony forming units per gram of fecal sample. On day 7, 9 
and 15 the fecal samples were placed in enrichment broths before being 
plated onto agar medium to determine the presence of S. enteritidis. 
On day 6, the fecal sample of the trimethoprim-treated chick 18 contained 
less than 70 S. enteritidis cells per gram. The fecal samples of chicks 
13-16, treated with trimethoprim and polymyxin B sulfate combinations, 
contained from about 68 to about 238 S. enteritidis cells per gram. These 
results are in contrast to the 70,000,000 (seventy million) S. enteritidis 
cells per gram of fecal sample for the untreated chick 19. Growth of S. 
enteritidis as even inhibited in chicks 12 and 17 where the fecal samples 
contained 10,000 to 1,000,000 S. enteritidis cells per gram. 
Trimethoprim-treated chick 18 was apparently completely free of S. 
enteritidis on day 7 and continued to show negative results on days 9 and 
15. Chick 13, which was treated with a minimum amount of polymyxin B 
sulfate (10 ug/ml) and a significant amount of trimethoprim (250 ug/ml), 
also was apparently completely free of S. enteritidis by day 7, and also 
continued to show negative results on days 9 and 15. 
Chick 12 appeared to be near death on day 9 and was sacrificed. Fecal 
samples could not be expressed from chicks 14 and 15 on day 9, but on day 
15 the expressed samples from these chicks indicated that the chicks were 
free of S. erteritidis. Chick 16 showed negative results on days 7 and 9 
but S. enteritidis was detected on day 15. thick 17, treated with 
polymyxin B sulfate only, showed positive results on days 7 and 9, but was 
near death on day 15 and was sacrificed. 
TABLE 3 
__________________________________________________________________________ 
GROUP I: Prevention of S. enteritidis in Poultry 
Number of colony forming units per gram of chick fecal 
sample as determined with Bismuth Sulfide and XLD agars, 
and presence or absence of S. enteritidis as determined 
Antimicrobial 
with Selenite Cystine and Tetrathionate Broth enrichments 
Concentration 
on days following placement into separate cages 
In Drinking 
Day 2 Day 6 Day 7 Day 9 Day 15 
Water Supply 
Agar Agar Enrich't 
Enrich't 
Enrich't 
__________________________________________________________________________ 
Example 12: 
Polymyxin B 
BS 2.4 .times. 10.sup.6 
BS 1.9 .times. 10.sup.6 
SC(+) Sacrificed 
-- 
Sulfate XLD 
1.1 .times. 10.sup.6 
XLD 
1.2 .times. 10.sup.4 
Tet(+) 
(10 ug/ml) 
and 
Trimethoprim 
(100 ug/ml) 
Example 13: 
Polymyxin B 
BS -3 .times. 10.sup.3 
BS -2.3 .times. 10.sup.2 
SC(-) SC(-) SC(-) 
Sulfate XLD 
-3 .times. 10.sup.3 
XLD 
-2.3 .times. 10.sup.2 
Tet(- ) 
Tet(-) 
Tet(-) 
(10 ug/ml) 
and 
Trimethoprim 
(250 ug/ml) 
Example 14: 
Polymyxin B 
BS -5 .times. 10.sup.2 
BS 1.1 .times. 10.sup.2 
SC(+) * SC(-) 
Sulfate XLD 
-5 .times. 10.sup.2 
XLD 
1.1 .times. 10.sup.2 
Tet(+) Tet(-) 
(50 ug/ml) 
and 
Trimethoprim 
(100 ug/ml) 
Example 15: 
Polymyxin B 
BS 1.2 .times. 10.sup.6 
BS -2.38 .times. 10.sup.2 
SC(+) * SC(-) 
Sulfate XLD 
4.6 .times. 10.sup.5 
XLD 
-2.38 .times. 10.sup.2 
Tet(+) Tet(-) 
(50 ug/ml) 
and 
Trimethoprim 
(250 ug/ml) 
Example 16: 
Polymyxin B 
BS 1 .times. 10.sup.2 
BS -6.8 .times. 10.sup.1 
SC(-) SC(-) SC(+) 
Sulfate XLD 
1 .times. 10.sup.2 
XLD 
-6.8 .times. 10.sup.1 
Tet(-) 
Tet(-) 
Tet(+) 
(100 ug/ml) 
and 
Trimethoprim 
(100 ug/ml) 
Example 17: 
Polymyxin B 
BS -8 .times. 10.sup.2 
BS 4.8 .times. 10.sup.5 
SC(+) SC(+) Sacrificed 
sulfate XLD 
-8 .times. 10.sup.2 
XLD 
8.4 .times. 10.sup.5 
Tet(+) 
Tet(+) 
(100 ug/ml) 
Example 18: 
Trimethoprim 
BS 6.6 .times. 10.sup.3 
BS 7.0 .times. 10.sup.1 
SC(-) SC(-) SC(-) 
(250 ug/ml) 
XLD 
1.5 .times. 10.sup.4 
XLD 
7.0 .times. 10.sup.1 
Tet(-) 
Tet(-) 
Tet(-) 
Example 19: 
Control BS 5 .times. 10.sup.2 
BS 7.3 .times. 10.sup.7 
SC(+) SC(+) SC(+) 
(250 ug/ml) 
XLD 
5 .times. 10.sup.2 
XLD 
7.8 .times. 10.sup.7 
Tet(+) 
Tet(+) 
Tet(+) 
__________________________________________________________________________ 
Fecal sample could not be expressed 
Each of the following examples 20-23 represents fecal sample obtained or 
pooled from three chicks, each of which was placed in a separate solid 
bottom cage. Each of the birds with the exception of the control group 
(example 20) was supplied with its respective antimicrobial on Day 1 and 
inoculated orally with 0.5 ml of an .sup.E 40 S. enteritidis solution 
containing about 10.sup.8 CFU/ml. 
Fecal samples were expressed from the twelve chicks on days 4, 6, 8, 1, 13, 
15 and 19. S. enteritidis was prevented in the three chicks of example 22 
which were fed the polymixin B sulfate and trimethoprim combination 
through day 11. On day 11, the chicks of example 22 were taken off the 
antimicrobials and transferred to clean cages, but maintained in close 
proximity with infected chicks. The chicks of example 22, as expected, 
tested positive for S. enteritidis after the antimicrobials were 
discontinued. 
S. enteritidis in the chicks of example 21 was initially suppressed through 
at least day 8. The infection in these polymyxin B sulfate treated chicks 
returned on day 13 and was present at the end of the experiment on day 19. 
The infection in the trimethoprim treated chicks of example 23 was present 
throughout the experiment. However, as indicated by the results on days 6 
and 8, the infection was somewhat suppressed as the average number of 
colony forming units per gram in the control group ranged from 1.5 million 
to more than 20 million while the colony forming units of the fecal 
samples of example 23 ranged from 440,000 to 660,000. 
Therefore, in view of examples 12-23, it is concluded that S. enteritidis 
infections are preventable in chicks where the chicks are treated with a 
trimethoprim and polymyxin B sulfate combination. Trimethoprim utilized 
alone appears to at least suppress the infection and, as indicated by 
example 18, may also prevent the infection. 
TABLE 4 
__________________________________________________________________________ 
GROUP I: Prevention of S. enteritidis in Poultry 
Number of colony forming units per gram of chick fecal 
Antimicrobial 
sample as determined with Bismuth Sulfide, XLD, and 
Concentration in 
Brilliant Green agars, and presence or absence of S. 
Drinking Water 
enteritidis as determined with Selenite Cystine and 
Supply Tetrathionate Broth enrichments. 
__________________________________________________________________________ 
Day 4 Day 6 Day 8 Day 11 Day 13 
__________________________________________________________________________ 
Example 20 
(Control) 
BS 2.3 .times. 10.sup.3 
BS &gt;10.sup.4 
BS 1.1 .times. 10.sup.6 
BS SC(+) Tet(+) 
BS SC(+) Tet(+) 
No antimicrobials 
XLD 
1.8 .times. 10.sup.3 
XLD 
&gt;8.00 .times. 10.sup.6 
XLD 
1.6 .times. 10.sup.5 
XLD 
SC(+) Tet(+) 
XLD 
SC(+) Tet(+) 
BG 2.5 .times. 10.sup.3 
BG &gt;10.sup.4 
BG 1.9 .times. 10.sup.5 
BG SC(+) Tet(+) 
BG SC(+) Tet(+) 
[&gt;2 .times. 10.sup.7 ]* 
[1.5 .times. 10.sup.6 ] 
Example 21 
Polymyxin B 
BS 0 .times. 10.sup.2 
BS 2.4 .times. 10.sup.4 
BS 0 BS 2.00 .times. 10.sup.6 
Sulfate XLD 
0 .times. 10.sup.2 
XLD 
6 .times. 10.sup.3 
XLD 
0 XLD 
4.5 .times. 10.sup.5 
(100 ug/ml) 
BG 0 .times. 10.sup.2 
BG 1.7 .times. 10.sup.4 
BG 0 BG -- 
[1.2 .times. 10.sup.5 ] [4.0 .times. 10.sup.6 ] 
Example 22 
Polymyxin B 
BS 0 .times. 10.sup.2 
BS 0 .times. 10.sup.1 
BS 0 BS SC(-) Tet(-) 
BS SC(-) Tet(-) 
Sulfate XLD 
0 .times. 10.sup.2 
XLD 
0 .times. 10.sup.1 
XLD 
0 XLD 
SC(-) Tet(-) 
XLD 
SC(-) Tet(-) 
(100 ug/ml) and 
BG 0 .times. 10.sup.2 
BG 0 .times. 10.sup.1 
BG 0 BG SC(-) Tet(-) 
BG SC(-) Tet(-) 
Trimethoprim [0] 
(250 ug/ml) 
Example 23 
Trimethoprim 
BS 7.8 .times. 10.sup.5 
BS 6.3 .times. 10.sup.4 
BS 6.6 .times. 10.sup.4 
BS SC(+) Tet(+) 
(250 ug/ml) 
XLD 
1.9 .times. 10.sup.5 
XLD 
2.2 .times. 10.sup.4 
XLD 
6.1 .times. 10.sup.3 
XLD 
SC(+) Tet(+) 
BG 5.5 .times. 10.sup.5 
BG 3.5 .times. 10.sup.4 
BG 4 .times. 10.sup.4 
BG SC(+) Tet(+) 
[4.4 .times. 10.sup.5 ] 
[6.6 .times. 10.sup.5 ] 
__________________________________________________________________________ 
Day 15 Day 19 
__________________________________________________________________________ 
Example 20 
(Control) 
BS 1.2 .times. 10.sup.4 
BS SC(+) Tet(+) 
No antimicrobials 
XLD 
1.8 .times. 10.sup.4 
XLD 
SC(+) Tet(+) 
BG -- BG SC(+) Tet(+) 
[4.4 .times. 10.sup.4 ] 
Example 21 
Polymyxin B BS 1.8 .times. 10.sup.4 
Sulfate XLD 
7 .times. 10.sup.3 
(100 ug/ml) BG 8 .times. 10.sup.3 
[2.7 .times. 10.sup.4 ] 
Example 22 
Polymyxin B 
BS SC(-) Tet(-) 
BS SC(+)Tet(+) 
Sulfate XLD 
SC(-) Tet(-) 
XLD 
SC(+)Tet(+) 
(100 ug/ml) and) 
BG SC(-) Tet(-) 
BG SC(+)Tet(+) 
Trimethoprim 
(250 ug/ml) 
Example 23 
Trimethoprim 
BS SC(+)Tet(+) 
BS SC(+)Tet(+) 
(250 ug/ml) 
XLD 
SC(+)Tet(+) 
XLD 
SC(+)Tet(+) 
BG SC(+)Tet(+) 
BG SC(+)Tet(+) 
__________________________________________________________________________ 
*Bracketed numbers represent average number of CFU/gm of fecal sample whe 
dilution of the fecal sample is taken into account. 
B. Removal of S. enteritidis from poultry 24-31 
Chicks 24-31 were inoculated on day 2 with the same culture of S. 
enteritidis as used with chicks 12-19, but were not supplied with their 
respective antibacterial mixtures until day 4. Each of the examples 24-31 
represents one chick. 
As shown in Table 5, fecal samples were expressed from chicks 24-31 on days 
3, 7, 9, 13 and 15 after introduction into individual cages. On days 3, 7, 
9 and 13, agar medium was used in determining the number of S. enteritidis 
colony forming units per gram of fecal sample. On day 15, the fecal 
samples were placed into an enrichment broth for being plated onto agar 
medium. 
Examples 24-31 indicate that it is more difficult to remove S. enteritidis 
infections from poultry than it is to inhibit such an infection. However, 
the results of days 13 and 15 show that S. enteritidis infections may be 
treated or controlled. For example, the S. enteritidis infection was 
apparently removed from chick 27 on day 15. Moreover, the infection in 
chick 26 was substantially controlled; its fecal sample on day 13 
contained less than 180 S. enteritidis cells per gram in contrast to the 
640,000 to 3,000,000 S. enteritidis per gram of the fecal sample of the 
untreated chick 31. Furthermore, it should be noted that chicks 25 and 26 
showed at least one negative reading on day 15. 
TABLE 5 
__________________________________________________________________________ 
GROUP II: Removal of S. enteritidis from Poultry 
Number of colony forming units per gram of chick fecal 
sample as determined with Bismuth Sulfide and XLD agars, 
and presence or absence of S. enteritidis as determined 
Antimicrobial 
with Selenite Cystine and Tetrathionate Broth enrichments 
Concentration 
on days following placement into separate cages 
In Drinking 
Day 3 Day 7 Day 9 Day 13 Day 15 
Water Supply 
Agar agar agar Agar Enrich't 
__________________________________________________________________________ 
Example 24: 
Polymyxin B 
BS 7.4 .times. 10.sup.3 
BS 2.4 .times. 10.sup.3 
BS 2.0 .times. 10.sup.5 
Sacrificed 
Sulfate XLD 
5.7 .times. 10.sup.3 
XLD 
1.9 .times. 10.sup.4 
XLD 
1.8 .times. 10.sup.5 
(10 ug/ml) 
and 
Trimethoprim 
(100 ug/ml) 
Example 25: 
Polymyxin B 
BS 1.2 .times. 10.sup.4 
BS 1.9 .times. 10.sup.4 
BS 1.3 .times. 10.sup.3 
BS * SC(+) 
Sulfate XLD 
1.1 .times. 10.sup.4 
XLD 
1.3 .times. 10.sup.4 
XLD 
* XLD 
4.3 .times. 10.sup.5 
Tet(-) 
(10 ug/ml) 
and 
Trimethoprim 
(250 ug/ml) 
Example 26: 
Polymyxin B 
BS 1.8 .times. 10.sup.4 
BS 5.3 .times. 10.sup.2 
BS 5.5 .times. 10.sup.2 
BS 1.8 .times. 10.sup.2 
SC(-) 
Sulfate XLD 
2.3 .times. 10.sup.4 
XLD 
5.3 .times. 10.sup.1 
XLD 
* XLD 
1.8 .times. 10.sup.2 
Tet(+) 
(50 ug/ml) 
and 
Trimethoprim 
(100 ug/ml) 
Example 27: 
Polymyxin B 
BS 3.4 .times. 10.sup.4 
BS 4.9 .times. 10.sup.2 
BS * BS 8.2 .times. 10.sup.2 
SC(-) 
Sulfate XLD 
* XLD 
* XLD 
* XLD 
0 Tet(-) 
(50 ug/ml) 
and 
Trimethoprim 
(250 ug/ml) 
Example 28: 
Polymyxin B: 
BS 1.8 .times. 10.sup.4 
Found dead 
Sulfate XLD 
9.0 .times. 10.sup.3 
on day 5 
(100 ug/ml) 
and 
Trimethoprim 
(100 ug/ml) 
Example 29: 
Polymyxin B 
BS 2.1 .times. 10.sup.8 
BS 6.3 .times. 10.sup.3 
BS 2.5 .times. 10.sup.6 
Found dead 
Sulfate XLD 
1.8 .times. 10.sup.8 
XLD 
5.3 .times. 10.sup.3 
XLD 
2.3 .times. 10.sup.6 
on day 9 
(100 ug/ml) 
Example 30: 
Trimethoprim 
BS * Found dead 
(250 ug/ml) 
XLD 
* on day 6 
Example 31: 
Control BS 3.3 .times. 10.sup.4 
BS 9.4 .times. 10.sup.6 
BS * BS 3.0 .times. 10.sup.6 
SC(+) 
XLD 
* XLD 
9.2 .times. 10.sup.4 
XLD 
* XLD 
6.4 .times. 10.sup.5 
Tet(+) 
__________________________________________________________________________ 
*Fecal sample could not be expressed 
Each of the following examples 32-34 represents a pooled fecal sample from 
three chicks, each of which was placed into a separate solid bottom cage. 
Examples 32-34 reflect experiments conducted at the same time and in the 
proximity of examples 20-23. Hence examples 21-23 and 32-34 share the same 
control group, example 20. Each of the birds of examples 32-34 was 
inoculated orally on Day 1 with 0.5-1 of an S. enteritidis solution 
containing about 10.sup.8 CFU/ul. The antimicrobial solutions were 
supplied on Day 4. The fecal samples of day 4 were expressed prior to 
antimicrobial introduction. 
As with examples 24-31, examples 32-34 of Table 6 show that it is more 
difficult to remove S. enteritidis infections from poultry than it is to 
inhibit such infections. However, example 33 indicates that trimethoprim 
may suppress S. enteritidis infections as the fecal samples from these 
chicks on Days 5 and 7 contained 10,000 to 60,000 colony forming units per 
gram in contrast to the 470,000 to 100,000,000 colony forming units per 
gram of fecal sample of the control group. 
TABLE 6 
__________________________________________________________________________ 
GROUP II: Removal of S. enteritidis in Poultry 
Number of colony forming units per gram of chick fecal 
Antimicrobial 
sample as determined with Bismuth Sulfide, XLD, and 
Concentration in 
Brilliant Green agars, and presence or absence of S. 
Drinking Water 
enteritidis as determined with selenite Cystine and 
Supply Tetrathionate Broth enrichments. 
__________________________________________________________________________ 
Day 4 Day 5 Day 6 Day 7 Day 11 
__________________________________________________________________________ 
Example 20 
(Control) BS 2.3 .times. 10.sup.3 
BS 4.4 .times. 10.sup.4 
BS &gt;10.sup.4 
BS 8.0 .times. 10.sup.7 
BS SC(+) Tet(+) 
(No antimicrobials) 
XLD 
1.8 .times. 10.sup.3 
XLD 
3.5 .times. 10.sup.4 
XLD 
&gt;8.00 .times. 10.sup.6 
XLD 
2.2 .times. 10.sup.7 
XLD 
SC(+) Tet(+) 
BG 2.5 .times. 10.sup.3 
BG 4.9 .times. 10.sup.4 
BG &gt;10.sup.4 
BG 4.0 .times. 10.sup.7 
BG SC(+) Tet(+) 
[4.7 .times. 10.sup.5 ]* 
[&gt;2 .times. 10.sup.7 ] 
[1.7 .times. 10.sup.8 ] 
Example 31 
Polymyxin B 
BS 1.6 .times. 10.sup.3 
BS 3.5 .times. 10.sup.4 
BS 2.2 .times. 10.sup.7 
Sulfate XLD 
6 .times. 10.sup.2 
XLD 
1.9 .times. 10.sup.4 
XLD 
1.6 .times. 10.sup.6 
(100 ug/ml) 
BG 2 .times. 10.sup.2 
BG 3.0 .times. 10.sup.4 
BG 1.35 .times. 10.sup.7 
[7.3 .times. 10.sup.5 ] 
[9 .times. 10.sup.7 ] 
Example 32 
Polymxin B 
BS 12 .times. 10.sup.3 
BS &gt;10.sup.4 
BS 2.10 .times. 10.sup.7 
BS 2.1 .times. 10.sup.4 
Sulfate XLD 
1 .times. 10.sup.3 
XLD 
&gt;8.50 .times. 10.sup.6 
XLD 
6.2 .times. 10.sup.6 
XLD 
3.6 .times. 10.sup.3 
(100 ug/ml) and 
BG 1.2 .times. 10.sup.3 
BG &gt;10.sup.4 
BG 2.00 .times. 10.sup.7 
BG 1.4 .times. 10.sup.4 
Trimethoprim [&gt;8 .times. 10.sup.7 ] 
[2.1 .times. 10.sup.8 ] 
[1.0 .times. 10.sup.5 ] 
(250 ug/ml) 
Example 33 
Trimethoprim 
BS 1.1 .times. 10.sup.3 
BS 7 .times. 10.sup.2 
BS 3.7 .times. 10.sup.3 
BS 5.0 .times. 10.sup.5 
(250 ug/ml) 
XLD 
0 XLD 
3.7 .times. 10.sup.2 
XLD 
2.6 .times. 10.sup.3 
XLD 
-- 
BG 3 .times. 10.sup.2 
BG 8 .times. 10.sup.2 
BG -- BG 1.1 .times. 10.sup.5 
[1.6 .times. 10.sup.4 ] 
[5.6 .times. 10.sup.4 ] 
__________________________________________________________________________ 
Day 13 Day 15 Day 19 
__________________________________________________________________________ 
Example 20 
(Control) BS SC(+) Tet(+) 
BS 1.2 .times. 10.sup.4 
BS SC(+) Tet(+) 
(No antimicrobials) 
XLD 
SC(+) Tet(+) 
XLD 
1.8 .times. 10.sup.4 
XLD 
SC(+) Tet(+) 
BG SC(+) Tet(+) 
BG -- BG SC(+) Tet(+) 
[4.4 .times. 10.sup.4 ] 
Example 31 
Polymyxin B 
BS 3.1 .times. 10.sup.3 
BS 1.0 .times. 10.sup.5 
BS 1 .times. 10.sup.2 
Sulfate XLD 
3.3 .times. 10.sup.3 
XLD 
9 .times. 10.sup.4 
XLD 
1 .times. 10.sup.2 
(100 ug/ml) 
BG 2.7 .times. 10.sup.3 
BG -- BG -- 
[4.6 .times. 10.sup.4 ] 
[3.9 .times. 10.sup.5 ] 
[2.9 .times. 10.sup.2 ] 
Example 32 
Polymyxin B 
BS 8.8 .times. 10.sup.3 
BS 2.00 .times. 10.sup.5 
BS 1.2 .times. 10.sup.3 
Sulfate XLD 
6.9 .times. 10.sup.3 
XLD 
1.48 .times. 10.sup.5 
XLD 
4 .times. 10.sup.2 
(100 ug/ml) and 
BG 7.0 .times. 10.sup.3 
BG 1.7 .times. 10.sup.5 
BG 8 .times. 10.sup.2 
Trimethoprim 
[7.7 .times. 10.sup.4 ] 
[1.1 .times. 10.sup.6 ] 
[6.3 .times. 10.sup.3 ] 
(250 ug/ml) 
Example 33 
Trimethoprim 
BS 8.5 .times. 10.sup.3 
BS 2.9 .times. 10.sup.3 
BS 1.0 .times. 10.sup.3 
(250 ug/ml) 
XLD 
7.2 .times. 10.sup.3 
XLD 
2.6 .times. 10.sup.3 
XLD 
7 .times. 10.sup.2 
BG 6 .times. 10.sup.3 
BG 4.3 .times. 10.sup.3 
BG -- 
[6.1 .times. 10.sup.4 ] 
[1.7 .times. 10.sup.4 ] 
[3.9 .times. 10.sup.3 
__________________________________________________________________________ 
] 
*Bracketed numbers represent average number of CFU/gm of fecal sample whe 
dilution of the fecal sample is taken into account. 
The present invention may be embodied in other specific forms without 
departing from the spirit or essential attributes thereof; therefore, the 
illustrated embodiment should be considered in all respects as 
illustrative and not restrictive, reference being made to the appended 
claims rather than to the foregoing description to indicate the scope of 
the invention.