The present application relates to a composition comprising acid and lignosulphonate for addition to litter where the composition reduces the amount of unfavorable bacteria and mould in the litter. In particular, the application relates to the use of a composition comprising acid and lingo¬ sulphonate for addition to litter, exemplified by the breeding of birds, especially chicken, turkey and ostrich. Addition of this composition to the litter alters the bacterial flora and the content of mould both in the litter and in the intestinal bacterial flora of the animals in a positive manner, and thereby affects the health of the animals in a favorable way. Additionally, the composition acts as a binder of dust in litter, and thus reduces free dust. Use of the composition also reduces the evaporation of acid from the litter.

Litter is used as support for animals, both pets and breeding animals, which are accommondated in cages, pens and larger enclosures. Litter exists in several grades and is fabricated from different materials such as straw, hay, wood chips, sawdust, resirculated paper, treated resirculated paper, sand, mineral particles and turf. Litter made from most of the materials mentioned above, is presently in use.

Litter is, among other things, used as support for chickens, and in Norway only, there were in 1995 produced 22.7 millions chickens. Chickens are fed in large rooms where they walk freely on litter. Modern broiler species obtain a very rapid daily weight gain, and the chickens reach optimal slaughtered weight (approximately 0.9 kg sloughed) in 32-34 days.

Norwegian chickens generally demonstrate good physical health. The mortality is about 4-6% during the breeding. The first days, feeble chickens will die because they are not able to take up feed and water. Gastrointestinal infection caused by bacteria of the Clostridium species (necrotizing enteritis) gave a marked increase in the mortality during the first period subsequent to the removal of antibiotics from the feed. "Sudden death syndrome" or the Martedal's disease, due to heart problems caused by high growth intensity, is seen in most livestocks. The frequency of abdominal dropsy has diminished relative to the early days. The fraction of discarding at the slaughterhouses is about 1%.

While in most other countries, there were problems with Salmonella bacteria in consumers' eggs and the slaughter of poultry, the situation for this zoonotic disease in Norway is largely satisfactory. However, the bacterium Campylobacter is also often present in Norwegian carcasses. These bacteria usually play a more important role in human health than in poultry.

A potential problem in breeding of birds, especially chickens, is that the birds normally live close together in the production plants. In these plants, they usually have litter on the floor, automatic tap water nipples and automatic feed stations. The high density of animals implies that bacteria and diseases are easily transferred from one individual to another.

Consequently, antibiotic growth promoters (AGMs) in production of livestock is in use. AGMs are antibiotics added to animal feed at subtherapeutic levels to increase growth, improve feed efficiency and decrease the incidence of diseases. The use of antibiotics over time, both within human and veterinary medicine, has caused a large pressure on the microflora with consequent appearance of resistance to these antibiotics among pathogenic bacteria. This has resulted in banning and/or regulation of the use of AGMs in several countries and also increased interest in ecological animal husbandry. One example is the feed antibiotic avoparcin, which was frequently used in the past, but the use of this agent is now prohibited in Norway. The reason is that it has been demonstrated that bacteria having developed resistance against avoparcin simultaneously have developed resistance towards vancomycin, which is an important antibiotic in human medicine. In feed intended for poultry, and especially in feed for chickens, it is customary to add coccidiostatics (agents active against coccidia, i.e., unicellular intestinal bacteria) . Coccidiostatics and feed antibiotics are added prophylactic, and the doses given are not therapeutic.

Development of alternative products and improved handling is therefore necessary to eliminate the use of AGMs, but yet obtain the same productivity. Another aspect, to which one has to pay attention, is the importance of the normal bacterial flora. A well-established normal intestinal flora competes with pathogens and hence decreases the risk of salmonellosis, C. perfringens-associated lesions, campylobacteriosis or colibacillosis. In the breeding of birds and especially chicken production, litter is a potential reservoir and transmission vehicle for pathogens and potential pathogens. The normal intestinal flora of chickens comprises Lactobacillus species, Enterococcus spp., E. coll and C. perfringens.

Litter is important, both as a factor for thriving and as a infection vector for animals kept together in breeding farms, and also during feeding of chickens. The litter quality is varying widely. Experiments have shown that the pH in dry litter is approximately 4.4-5.2, while the pH in raw litter is 5.5-6.2. Dry litter is defined as litter with a moisture content of less than 20%. Raw litter may exhibit a moisture content exceeding 100% when calculated on the basis of dry weight. Litter can absorb water equivalent to 2.5-5.3 times its own weight depending on humidity and structure. Dry litter is practically sterile and displays antibacterial effect towards several bacteria, while raw litter, on the contrary, has substantial growth of bacteria and often fungus, and stimulates the growth of bacteria. The litter in the chicken houses normally becomes moist due to the droppings of the animals, however, the degree of moisture will vary depending on environmental control and animal density. The chickens may, in worst case, suffer from abscesses and wounds on their feet ("hock burns") due to the wet litter.

It has now, surprisingly, been found that a composition comprising acid and lignosulphonate can be used as additive to litter to alter the bacterial flora, both in the litter and in the intestinal flora of the animals, in a positive manner. In particular, a composition comprising acid and lingosulphonate can be added to litter which is utilized for the breeding of animals, both pets and breeding ani¬ mals, which are kept in cages, pens and larger enclosures. The use of the above mentioned composition has proved to have a positive effect on the normal bacterial flora of the chickens and thereby also on their state of health.

Compositions comprising acid and lignosulphonate were first described in Norwegian Patent No. 308983, granted on November 27, 2000, to Borregaard Industries Ltd. The composition is there described as a growth enhancing additive to compound feed containing an organic or inor¬ ganic acid, or salts thereof, with a beneficial effect on the treatment of compound feed, added a spent sulphite liquor from an acidic or neutral cellulose sulphite cooking, wherein the spent sulphite liquor has a pH in the range of 1 to 10, and the base used to produce the spent sulphite liquor is calcium, sodium, ammonium or magnesium, and the additive contains the organic or inorganic acid, or salts thereof, in an amount ranging from 10-90 percentage by weight, and the spent sulphite liquor from an acidic or neutral cellulose sulphite cooking in an amount ranging from 10-90 percentage weight, wherein the acid and the acidic or neutral cellulose spent sulphite liquor is optionally adsorbed on a suitable carrier in order to obtain a dry substance. This additive has favourable effects on the growth and health of pigs, first and foremost in that the addition of acid affects the digestive system beneficially. Also, the quality of the compound feed is improved in that the acid kills undesirable bacteria and microorganisms in the feed.

The organic or inorganic acids with a beneficial effect on the treatment of compound feed described in the above mentioned patent are formic acid, acetic acid, propionic acid, citric acid, hydrochloric acid, phosphoric acid or mixtures thereof.

The salts of these acids are ammonium, alkali, and/or earth alkali metal salts, especially NH4-, Na-, Ca- or Mg-.

By spent sulphite liquor from an acidic or neutral cellulose sulphite cooking is meant the spent sulphite liquor produced by treating timber with an aqueous liquid to which has been added sulphur dioxide and the aforementioned cations ammonia, sodium, calcium or magnesium. The main ingredient in the resulting spent sulphite liquor, after the separating out of cellulose, is lignosulphate. In addition, there are amongst others, mono or polymer sugars and inorganic salts .

The composition comprising acid and lignosulphonate for addition to litter encompasses more specifically an organic or inorganic acid, or salts thereof, added a spent sulphite liquor from an acidic or neutral cellulose sulphite cooking, in which the spent sulphite liquor has a pH value in the range of 1 to 10 and the base used to produce the spent sulphite liquor is calcium, sodium, ammonium or magnesium, and the additive contains the organic or inorganic acid, or salts thereof, in an amount ranging from 1-99 percentage weight and the spent sulphite liquor from an acidic or neutral cellulose sulphite cooking in an amount ranging from 1-99 percentage weight, where the acid and the acidic or neutral cellulose sulphite cooking optionally is adsorbed onto a suitable carrier to obtain a dry composition.

The beneficially influencing acids are formic acid, acetic acid, propionic acid, lactic acid, sorbic acid, citric acid, hydrochloric acid, sulphuric acid, phosphoric acid, or mixtures thereof. The salts are as mentioned above.

The composition may for example comprise:

Lignosulphonate concentrate: 40-60% (w/w)

Formic acid: 35-50% (w/w)

Propionic acid: 5-15% (w/w)

The following examples are meant to illustrate the invention without, in any way, limiting the scope of it:

Examples

Example 1

Addition of a composition comprising acid and lignosulpho¬ nate to litter

a) Composition:

Lignosulphonate concentrate: 40-60% (w/w)

Formic acid: 35-50% (w/w)

Propionic acid: 5-15% (w/w) b) Addition of composition to litter

bl) The composition is sprayed onto the application area with for example a manually driven spray apparatus or a tractor spray apparatus .

b2) The composition is mixed with litter prior to delivery to the application area or optionally before or after packaging.

Independent of field of application, recommended amount added to the litter is in the range of 1-20% and will depend upon the acid binding capacity of the litter. The pH in the litter can be used as an indicator to ensure that a proper dosing is added.

Example 2

Effect of composition comprising acid and lignosulphonate on C. perfringens and E. coli in litter for breeding of chickens

The experiment was conducted at Samvirkekylling in a room with 12 pens each containing 120 animals with 4 pens per segment. Standard feed without coccidiostatics was employed. There was no floor heating, and the exchange of air was reduced by about 50%. The composition (as given in Example 1) was added to the litter in such a way that the pH was reduced to 3. The pen was sprayed with a manually driven spray. The litter compositions were as follows: Product Raw litter Litter x Dry Litter x Dry wood chips + wood chips + Acid product Acid product

Segment I Segment II Segment III*

Type of timber Pine Pine Pine

pH 5.8 3.0 3.0

Moisture (%) 103.2 7.3 7.3

*The litters in Segments II and III were identical, however, in Segment III, the litter was replaced on day 12.

Slaughtering took place on day 32. On days 3, 6, 10, 13, 17, 20, 25, 27 and 32, combined samples of litter were collected from all pens. On these samples, there were performed analyses of pH and moisture and quantitative analysis of Clostridium perfringens and coliform bacteria. Feed consumption, weight gain and mortality were registered successively.

Results

Production data

Segment Segment Segment I II III Average weight (g) , day 28 1242 1289 1272 Feed consumption per kg weight 1.53 1.51 1.52 gain, day 0-28 Average slaughter weight (g) 1025 1038 1038 Dead animals, day 0-32 10 12 8 Discarded 5 2 3

There is a marked difference in alive weight at day 28 and slaughter weight between segment I and segments II/III. With regards to the feed consumption per kg of weight gain, there is little difference.

Initially, the litter in segment I had a moisture content just above 100%, while the litter in segments II and III had a moisture content of about 7%. On day 3, the moisture content of the litter in segment I was reduced to 23%. The difference in moisture content between the litters was balanced on day 6 and was at that time about 13%. The moisture content increased continuously during the experiment in all the litters to approximately 40% on day 32.

Initially, the litter in segment I had a pH of 5.8, while pH in segments II/III was 3.0. The pH on day 10 was 5.9 and 5.5 in segments I and segment II/III, respectively. On day 12, the pH values for all segments were equal. Replacement of the litter in segment III yielded a transient alteration in these parameters. At the end of the experiment, the pH value in all segments was 8.2.

The number of coliform bacteria quickly increased until day 20 (60-80 million), thereafter decreasing to 1 million and less on day 30. Replacement of the litter on day 12 yielded a lower and a little delayed peak (40 million) for segment III.

In general, the amount of C. perfringens was far greater in segment I than in segments II and III. In segment I C. per¬ fringens was detectable in the litter already from day 3. This level was more or less maintained until day 27. Thereafter, tendency to increase. In segments II and III, there was only a sporadic detection of C. perfringens, and this level was maintained until sacrifice. Replacement of the litter therefore yielded no additional effect on this parameter. Conclusion

The experiment shows that addition of the composition comprising acid and lignosulphonate to litter reduces the amount of C. perfringens substantially. It means that this litter does not function as an infection vector. If infection via the litter is as important for the development of necrotizing enteritis (NE) as indicated by the literature, this finding might be an important factor in the attempt to avoid using coccidiostatica in the chicken feed.

Example 3

The effect of composition comprising acid and lingo- sulphonate on the natural microflora and Salmonella in litter

Broiler litter from a commercial poultry house was treated with a composition comprising acid and lignosulphonate [as given in Example 1] , while an untreated house from the same site served as control.

Pine wood chips litter was obtained from a breeder house (20 weeks) located at North Carolina State University's Lake Wheeler Road Poultry Research Farm. The litter was initially sterilized by autoclaving at 121°C for 90 min. to eliminate the background microflora before inoculation with the Salmonella cocktail and initiation of the dose response study. The dried litter matter content was determined by drying 2.5 g litter samples for 15 hours in a forced air drying oven. The average dried litter dry weight was 84.57%.

Composite litter samples were taken on days 0, 7, 21, and 48 and analyzed for total aerobes, lactic acid bacteria, Salmonella populations, litter pH and moisture content. For the laboratory study, litter obtained from a commercial broiler house was autoclaved and subsequently inoculated with a 4-strain Salmonella cocktail to an initial population of 106 CFU per gram. The above mentioned composition was thereafter applied to the litter [mecha- nical blending] in varying amounts of O, 0.2, 0.4, 0.6, 0.8 and 1.0 % (dry litter wt. basis) and thoroughly mixed. Replicate composite litter samples were then taken from each dose treatment on days 3 and 7 post treatment and analyzed for Salmonella populations and pH. For the field trial, calculated mean values for pH and moisture content were 6.95 and 17.4 % in the treated house, while values for the control house were 7.84 and 20.8 %, respectively. Ave¬ rage total aerobes for treated and control houses were 9.86 and 9.84, while average lactic acid bacteria for treated and control houses were 8.77 and 9.07 log CFU per gram, respectively. Mean Salmonella populations for the treated and untreated houses were 2.39 and 4.83 log CFU per gram, respectively. Compared to the control, slight reductions in litter pH values (rang of 0.6 to 0.96 pH units) were observed in the treated samples. Salmonella populations were significantly reduced (day 1 - 1.4 to 1.8; day 3 - 1.1 to 1.2; day 7 - 0.4 to 1.0 log reduction) as treatment concentrations increased.

The results are shown in Tables 1-2. Table 1. Effect of composition comprising acid and lignosulphonate on pH values and Salmonella populations in poultry litter *

Day 0 Day 1 Day 3 Day 7 Treatment pH CFU/ml pH CFU/ml pH CFU/ml PH CFU/ml (%) 0.0 77..5544++00..4411 100501639 7.46±0.25 190683+124002 7.64+0.22 24700±15274 7.60+0.36 5800±3111 0.2 7.32+0.18 7.38±0.21 6600+424 7.38±0.13 3100+3677 7.34+0.11 2250+919 0.4 7.11+0.14 7.15+0.22 3900+2970 7.26+0.25 1800±707 7.20+0.15 750±354 0.6 6.71+0.38 7.19+0.19 6800+990 7.07±0.27 2085±1718 7.20±0.17 850±495 0.8 6.64±0.26 7.08+0.33 9500+4524 6.90+0.32 3100+566 7.07+0.20 750+354 1.0 6.94+0.44 6.77+0.29 3000+849 6.68+0.19 1550+636 6.90±0.27 550+778 " H to Table 2. Conversion of Salmonella population data to a logio basis and calculation of Log reductions in Salmonella populations

(day 0) Salmonella population (logio Treatment CFU/ml) ) dose Day 1 Day 3 Day 7 4.00 0.0 5.28* 4.39 3.76 0.2 3.82 3.49 3.35 0.4 3.59 3.26 2.88 0.6 3.83 3.32 2.93 0.8 3.98 3.49 2.88 1.0 3.48 3.19 2.74

Log- reductions 0.2 1.46** 0.9 0.41 0.4 1.69 1.13 0.88 0.6 1.45 1.07 0.83 0.8 1.3 0.9 0.88 1.0 1.8 1.2 1.02 *Log10 values (CFU/ml)

**Log reduction (control value - treated value, CFU/ml) .

Conclusion

The findings indicate that significant reductions in Sa- lmonella populations present in poultry litter can be achieved by the use of a composition comprising acid and lignosulphonate.

Example 4

Effect of treating poultry litter with composition comprising acid and lignosulphonate on the bacterial microflora

The object of the experiment was to quantitatively estimate the effect of treatment with a composition comprising acid and lignosulphonate of broiler litter on enhancing the litter microflora by reducing bacterial pathogens {Salmonella) and increasing potential beneficial microorganisms (i.e., lactic acid bacteria) . The total number of mesophilic aerobes, lactic acid bacteria and Salmonella spp. populations were estimated for litter samples treated with acid and lignosulphonate in comparison to untreated control samples. The litter samples were collected on day 0 (post treatment application) and after 7, 21 and 48 days post treatment.

For both treated and untreated houses at a commercial poultry farm (New Hampshire, USA) , about 50 g surface litter per sampling site (total of around 500 g per house) were taken and the 10 samples from each house combined in sterile WhirlPak bags. The samples were placed on ice until analyzed.

Procedures for counting total mesophilic bacterial populations

Aerobic plate counts indicate the total mesophilic bacterial population in a sample. Ten (10) g of each litter sample per house were placed into a sterile filter bag containing 90 ml PW (0.1% peptone water) . The bags were mixed for one minute, serially diluted in PW, and appropriate dilutions were placed on the surface of BHI (brain heart infusion) agar plates using a spiral plate method (Association of Official Analytical Chemists, 1990. Official methods for Analysis, 15th edition, AOAC, Arling¬ ton, VA.) . After incubation for 24 hours at 37°C, colonies were enumerated using a Protocol automatic colony counting system (Software Version 2.06.07, Synbiosis, Cambridge, UK) . The minimum detection limit for this method was 1 log/g of litter (10 organisms) . Procedures for counting Salmonella

Twenty-five (25) g of each litter sample per house were placed into a sterile filter bag containing 50 ml of BPW (buffered peptone water) . The bags were mixed for one minute. A MPN (Most Probable Number) procedure was employed to enumerate Salmonella populations. After mixing, the sample was serially diluted in BPW as needed (ICT1 to 10~6) and then 1 ml of each sample was transferred in triplicates to 9 ml BPW tubes, incubated at 37°C for 18 to 24 hours and then 0.1 ml of the appropriate dilutions of each tube transferred to triplicate 10 ml tubes of Rappaport Vassiliadis (RV) broth (Difco) used for selective enrichment. All RV broth tubes were then incubated at 420C for 24 hours. Following incubation, one loopful from each tube was streaked onto MLIA (modified lysine iron agar, selective medium for Salmonella) and incubated at 37°C for 24 hours. Suspect black colonies were picked and confirmed by testing on TSI agar (triple sugar iron) slants and agglutination using Salmonella poly-0 antiserum (Difco) . Populations of Salmonella spp. for each sample were determined using standard MPN tables. The minimum detection limit for the method was 1 log or 10 organisms/g.

Procedures for counting lactic acid bacteria

Ten (10) g of each litter sample per house were placed into a sterile filter bag containing 90 ml of PW. The bags were mixed for one minute, serially diluted in PW, and approperiate dilutions spiral-plated onto MRS agar plates. MRS agar, developed by deMan and Rogosa, is a selective medium for the cultivation and enumeration of lactobacilli. The plates were placed in plastic bags, flushed for 1 minute with CO2, and then the bag sealed. After incubation for 48 hours at 37°C, the colonies were enumerated using the Protocol automatic colony counting system (Software Version 2.06.07, Synbiosis, Cambridge, UK) . The minimum detection limit for this method was 1 log or 10 organisms per gram of litter.

Determination of pH

One (1) g of litter samples were added to triplicate 10 ml volumes of distilled deionized water, and pH determined using a pH meter and combination electrode.

Determination of moisture

Triplicate 2.5 g litter samples were placed in a 1050C oven and dried overnight. Moisture content was calculated as the difference in litter weight before and after heating.

Results

pH and moisture content

The treated litter samples had pH values of 6.69, 6.25, 7.43 and 7.44 for the 0, 7, 21 and 48 day samples, respectively. For the control samples, the pH values were 7.16, 6.86, 8.58 and 8.76 over the same time periods, respectively. As the birds aged, litter pH increased. However, pH increased at nearly double the rate for the control samples (slope = 0.32) as compared to the treated samples (slope = 0.17) . The average pH over the four sampling times was 6.95 and 7.84 for treated and control samples, respectively. pH differences between untreated and treated litter were 0.47 pH units on day 0 and 1.32 pH units on day 48. Litter moisture values also varied between treated and control samples. For the treated samples, moisture levels were 24.8, 12.7, 15.6 and 16.3 % for the 0, 7, 21, and 48 day samples, respectively. For the control samples, the moisture levels were 25.5, 21.5, 18.4 and 17.9 % over the same time periods, respectively. The average moisture level over the four sampling times was 17.4 and 20.8 % for treated and control samples, respectively. It appears that the treatment with the composition may- contribute to the reduction of moisture content in the litter. This lower moisture content could influence microbial populations, especially if the environmental 5 conditions are more stressful on the organisms.

Total mesophilic bacteria, lactic acid bacteria, and Salmonella spp. populations

The total mesophilic, lactic acid bacteria, and Salmonella spp. litter populations are summarized in Table 3. The o total mesophilic bacteria populations in the treated litter samples were log 9.87, 8.55, 10.92 and 10.10 CFU/g (colony forming units) for the 0, 7, 21 and 48 day samples, respectively. In comparison, the mesophilic populations in the control samples were log 8.15, 10.25, 11.04 and 9.94 s CFU/g for the same time periods. The overall mean mesophilic populations across all sampling times were log 9.86 and log 9.84 CFU/g for treated and untreated (control) samples, respectively. Although there were some fairly large population variations detected between treatments o through the first week (approaching nearly 2 logs on days 0 and 7), there does not appear to be any overall treatment effects on the total mesophilic bacterial populations occurring over the final two sampling periods. Lactic acid bacterial populations in treated litter were 7.98, 8.81, 5 9.98 and 8.32 CFU/g over the four sampling times, respectively. In comparison, the lactic acid bacterial populations of the control samples were log 8.35, 9.87, 9.60 and 8.47 CFU/g, respectively. The mean lactic acid bacterial populations were log 8.77 and log 9.07 CFU/g for 0 the treated and control litters, respectively. The treatment with the composition did not appear to have any consistent effect on this group of organisms. However, this does not appear to be the case with Salmonella. Salmonella populations of the treated litter were log < 1, 2.93, 4.63 5 and 1 MPN/g for the four sampling times. In contrast, the Salmonella populations for the control litter were log 3.41, 5.80, 6.08 and 4.03 MPN/g, respectively. The mean Salmonella populations across all sampling times were log ≤ 2.39 and log 4.83 MPN/g for the treated and control samples, respectively. The significant population differences detected between these two treatments ranged from 1.45 logs at day 21 to 3.03 logs at day 48.

Table 3. Effect of treatment with composition comprising acid and lignosulphonate of broiler litter on total mesophiles, lactic acid bacteria and Salmonella spp. popu- lations.

Litter Total Lactic acid Salmonella sampling mesophiles bacteria time Spp.

Treated Control Treated Control Treated Control

Log CFU/g --Log MPN/g—

0 day 9.87 8.15 7.98 8.35 < 1 3.41

7 days 8.55 10.25 8.81 9.87 2.93 5.8

21 days 10.92 11.04 9.98 9.60 4.63 6.08

48 days 10.10 9.94 8.32 8.47 1 4.03

Average 9.86 9.84 8.77 9.07 < 2.39 4.83 (STD) (0.58) (1.22) (0.87) (0.77) (1.75) (1.31)

Conclusion

The treatment with the composition comprising acid and lignosulphonate appears to have a significantly inhibitory effect on Salmonella. Example 5

Effect of composition comprising acid and lingosulphonate on intestinal bacterial flora balance

In this study, chickens were exposed to litter treated with a 7% composition comprising formic acid, propionic acid and lignosulphonate, and the influence of the treated litter on the balance of their normal intestinal flora {Lactobacillus spp. ; Enterococcus spp.; E. coli and C. perfrlngens) , with special interest on C. perfringens because of its participation in the development of necrotic enteritis (NE) , was examined.

Materials and methods

Animals, treatment and diet. 1728 one day old, healthy male chickens (breed: White Italian, type: Roff 208) from the hatchery Samvirkekylling, Solør, Kongsvinger, Norway) were employed and arbitrarily divided in 12 pens (5.35 m2) , each pen containing 144 animals. The pens were assigned either to control litter or the acidified litter alternately. The house had a monitored temperature of 300C, which was reduced a half degree per day to 220C. A lightning regime of 18 hours was employed. The relative humidity started at 70% and was decreased to 50%.

The litter was wood chips from dry pinewood (control) or litter treated with a mixture of sodium lignosulphonate, formic acid and propionic acid (acidified litter) . The added mixture constituted 7% of the litter weight and was sprayed onto the litter in the pens.

The chickens had ad libitum access to water and feed, which did not contain antibiotics or coccidiostatics. The feed was designed based on a necrotic enteritis disease model developed at The National Veterinary Institute in Oslo, Norway (M. Kaldhusdal, unpublished) to induce C perfringens associated enteritis and hepatitis (CPEHj . C. perfringens (100-500 CFU/g) was detected in the starter feed.

Intestinal sampling and processing. At 18-, 21-, 25-, 29-, and 32 days of age, five animals from each pen were randomly selected for sampling.

Penwise, the chickens were made unconscious by a blow to the head and then killed by cervical dislocation. Immediately after, the alimentary tract was dissected and intestinal contents collected from an approximately 30 cm long segment of the lower ileum, measured from the vitelline diverticulum and the caeca (one of the horns) into plastic 50 ml Falcon tubes (Corning Incorporated, NY, USA) , which were kept on ice until inoculation and incubation. If the chosen section from the lower ileum was empty or contained little material, the contents of the next 30 cm of the lower ileum was collected.

The samples were processed within 10-60 minutes after collection. They were weighed and serially diluted in 0.9% saline, and 0.1 ml of each sample was plated on selective media.

Bacterial counts from intestinal samples: C. perfringens was grown on 5% blood agar (MERCK 10886) anaerobically at 370C during 48 hours (MERCK Anaerocult® A) . The colonies appeared as large irregular colonies with a double zone of haemolysis.

Lactobacillus spp. was grown on Rogosa agar (MERCK 5413) anaerobically at 370C during 48 hours. These were enumerated by counting as white colonies.

Enterococcus spp. was grown on 5% blood agar (MERCK 10886) aerobically at 37°C during 24 hours. The colonies were small (1 mm 0) and presented an α-haemolysis. E. coli was grown on MacConcey agar (MERCK 5465) aerobically at 37°C for 24 hours. These were typical lactose-fermenting colonies.

The macroscopic image of the colonies of the bacteria was confirmed by the microscopic image of the bacteria after Gram staining.

Recording of data from intestinal bacterial counts. The figures from the bacterial counts were recorded as CFU.

Litter sampling and processing: Litter samples were collected from all the pens on day 4, 7, 11, 14, 18, 21, 25, 28 and 32 to measure their humidity, bacterial counts and pH.

- pH: was measured in the first dilution of the samples for bacterial count using a combined pH electrode (Model Knick Portamess 751 Calimatis, Knick, West Germany) .

- Humidity: The samples were weighed and placed in a varm cabinet at 1050C. After 5 hours, the samples were weighed again and the weight loss calculated. Humidity (%) = { (weight of sample before warm cabinet - weight after warm cabinet) / (weight after warm cabinet) } x 100.

- Bacterial counts: The samples were serially diluted in 0.9% saltwater and 0.1 ml of each plated onto selective media. C. perfringens was isolated on TSC agar for C. perfringens (OXOID CM587 with the addition of SR88 and SR47) after an incubation time of 24 hours in an anaerobic atmosphere at 370C. The colonies were black with an opaque halo indicating lecithinase reaction. E. coli was isolated on MacConkey agar (Difco 0075-17) after an incubation time of 24 hours in an aerobic atmosphere at 37°C. Performance: Feed consumption and body weight was recorded penwise on day 32.

Clinical observations: The chickens were inspected each day and the number of dead chickens was registered. At slaughter the number of discarded chickens was registered.

Statistical analysis: Due to skewness in the distribution, statistical analyses of intestinal bacterial counts were performed on logarithmically transformed data. The results were back-transformed and expressed as mean values and 95% Confidence Intervals (CI) constructed by using the Student procedure (Altman, D.G., 1991w. Practical statistics for medical research. London 2) . Additionally, Standard Deviation (SD) is given in brackets. Comparison of groups on assumed continuously distributed variables were performed by using analysis of variance with pen and age as covariates. Comparison of groups on categorical variables were performed by using contingency table analysis (Kleinbaum, D.G. 2003x. Applied regression analysis and other multivariate methods. Duxbury Press, Pacific Grove, CA, USA) . Differences were considered significant if the p- values were found less or equal to a level of 5%.

Results

Intestinal bacterial counts. The size of the bacterial populations in the ileum and caeca of the chickens exposed to the acidified and control litter are presented in Figure 1.

Ileum

C. perfringens. Chickens on both litters had an increasing number of C. perfringens in the intestine with time. The acidified groups had much lower counts (5.52) on day 18 than the control group (6.47), but these counts increased noticeably on day 21. In total, the acidified group has lower counts (p<0.01) and specifically at 18 (p<0.01) and 32 (p=0.06) days.

Lactobacillus spp. The counts increased with time in both groups. The acidified group had overall significantly lower counts (p<0.01) . On day 18, a significant difference (p<0,01) was observed between the two groups with a mean of 6.11 for the acidified group and logio 7 for the control group. The counts increased rapidly in the acidified group to day 21, reaching a level of 6.95, and reached similar levels as the control group at 25 days.

Enterococcus spp. The counts increased rapidly in both groups from day 18 to day 21, and remained stable for the rest of the test period. The acidified group had overall lower (p≤0.05) number of CFU/g, with lower counts at 18 (p=0.06) and 32 (p=0.08) days.

E. coll. No differences between the two groups was observed. There was an increase in the number of CFU/g from day 18 to day 21, followed by a decrease at day 25 and an increase the last two sampling days. The differences between the counts made the first day and last day, was higher for the control group than for the acidified group.

Caeca

C. perfringens. The development of population C. perfringens of the acidified group and the control group showed different patterns. At day 18, the acidified group has counts of 6.97, which oscillate first up and then down, and increase in the last two sampling days getting close to the level of the control group (7.64) . The control group has much higher counts (7.69) at day 18, and oscillate first down and then up and down again to end up at 7.7, very close to the level from day 18. It is important to notice that the acidified group has in general lower counts (p<0,01) than the control group through the entire test period, which are significant specifically at day 18 (p<0.01), 25 (p<0.01) and 29 (p-0.06) days.

Lactobacillus spp. The counts increased with time in both treatment groups. The acidified group has overall significantly higher (p≤0.05) counts, but only on day 18 there is a significant difference (p≤0.05) between the two groups with a mean of 7.54 for the acidified group and 7.12 for the control group.

Ente∑ococcus spp. No differences between the groups was observed. A rapid and continuous increase in the number of CFU/g is observed in both groups.

E. coli. No difference between the groups was observed. The counts follow the same pattern as in the ileum, but the oscillations were smaller. The difference between the counts conducted on day 18 and day 32 is higher for the control group than for the acidified group.

Litter parameters

The influence of litter pH and humidity on the presence of E. coli and C. perfringens in the litter is shown in Table 4. Table 4. Mean values of pH, humidity, C. perfringens and E. coli counts with SD in brackets.

pH Humidity (%) C. perfringens E. coli (Logio CFU/g) (Logio CFU/g) Acidified[ Control Acidified Control Acidified Control Acidified Control Day 1 2.8* 4.9* 22.42* 14.64* ND ND ND ND 4 5.2(0.58) 5.9(0.24) 14.5 (2.58) 16.1(3.45) 4.65 (1.21) 5.49(0.46) 5.61(1.29) 6(0) 7 5.8(0.53) 5.9(0.2) 15.7 (11.26) 17.7 (10.19) 4.1(0.97) 5.38(0.66) 6(1.81) 6.85(0.23) 11 5.9(0.29) 5.9(0.09) 17.6(10.38) 22.1(6.6) 5.62 (0.86) 6.07(1.04) 6.9(1.35) 7.12(1.07) 14 6(0.19) 5.9(0.11) 26.8 (8.74) 31.9(13.52) 5.97 (0.47) 6.11(0.38) 7.04(1.57) 6.37 (1.38) 18 6.2(0.33) 6.5(1.37) 35.1 (9.95) 38.6(10.34) 5.89 (1.01) 6.06(0.47) 6.71(0.63) 6.58(0.79) 21 6.8 (0.71) 6.8(1.0) 39.2 (7.42) 36.9(9.51) 5.12 (0.69) 5.37 (1.0) 6.73(1.24) 6.44 (0.14) 25 7.9(0.74) 7.9(0.75) 35.4 (5.44) 36.7 (4.4) 5.98 (0.32) 6.16(0.81) 6.09(1.0) 5.75(2.16) 28 8.4 (0.41) 8.4(0.48) 29.6(38.19) 38.313.0) 5.92 (1.28) 6.16(0.81) 3.95(1.92) 4.4(2.51) P 0.002 0.78 0.016 0.98 *Measurements taken before the litter was divided among pens pH. In both litters, pH reaches the same level. The acidified litter is significantly lower (p<0.01) than the control litter, but there is no effect of the acidified litter when ignoring the data from day 1.

Humidity. The humidity increased in both litters and drops down at the end for the acidified litter. There is not a significant difference between the litters.

C. perfr±ngens. Both litters showed the same development in bacterial counts but there is a significantly lower number (p<0.05) for the acidified litter. The number of CFU/g increased with time, but there is a noticeable decrease at day 21.

E. coll. No difference between the groups was observed. In both litters the counts increased until day 11 and they are maintained at the same levels until day 21, when they decreased rapidly.

Performance. The chickens exposed to the acidified litter had a larger alive weight (p<0.05) at day 32 and had consumed a larger amount of the feed (p<0.01) throughout the test period than the chickens in the control group. These results give no significant difference in the Feed Conversion Rate (FCR) between the two groups (Table 5) . The carcasses of the acidified group after processing were significantly heavier (p<0.01), with a mean of 1097 g, than the ones from the control group, with a mean of 1070 g.

Table 5. Mean values with SD in brackets of weight gain, feed intake and Feed Conversion Rate (FCR) measured at day 32 penwise, and the p-value.

Acidified Control p-value Weight (kg) 186..5 (13.8) 183.8 (2• 9) 0.41 Feed intake (kg) 338..9 (14.0) 327.6 (4.1) 0.004 FCR (kg feed 1.82 (0.09) 1.78 (0.03) 0.15 intake/kg gain Mortality and discarding. The mortality during the entire growth period was 18 animals (2.1%) in the control group and 21 animals (2.4%) in the acidified group. A total of seven carcasses (0.8%) were discarded at slaughter in the control group and three (0.4%) in the acidified group.

In the ileum, C. perfringens, Enterococcus species and Lactobacillus spp. had fewer counts in the group exposed to the acidified litter compared to the control group. In the caeca, only C. perfringens had fewer counts, while Lactoba- clllus spp. had higher counts. The most noticeable changes were observed in the ileum between day 18 and day 21.This can be explained by the change in the properties of the litter, which reached neutral values (pH > 6) already at day 14.

The mortality rate and discarding at slaughter was low in both groups, indicating that the chickens in the study had good health despite the alteration in the intestinal flora and the necrotic enteritis (NE) disease model applied.

Conclusion

The study demonstrates that acidification of litter by applying the composition, reduces the horizontal infection of chickens by pathogenic bacteria. The counts of C. perfringens and Enterococcus spp. in the intestine of the chickens are reduced by use of the said composition in the litter. This reduces the risk of developing clinical or subclinical NE and growth depression. The alteration in the balance of the intestinal flora did not have negative consequences on chicken's health or performance. No external corrosion damages on the feet of the chickens were reported. Example 6

The dust binding capacity of the composition in straw

The composition was assessed as a binder of dust in straw. Reduction of the amount of dust and free particles will also reduce the amount of infection vectors inhaled by the animals.

The composition consisted of calcium lignosulphonate (33.5%), propionic acid (8.2%) and Span 20 -sorbitane fatty acid ester (0.01%) .

Straw was chopped, squeezed through a conveyer screw and sprinkled through a cyclone equipped with three x two nozzles (nozzles on two levels) where the composition was added (20%, w/w) . Six (6) straw samples were collected and forwarded to the Arbeidsmiljøinstituttet (AMI), Copenhagen, for analyses and calculations. The samples were stored in the same room as the test plant until the time of testing. The plastic bag containing the straw contents was weighed, the contents placed in the drum and then the bag was weighed again to determine the total weight of straw in the drum. The rotational speed of the drum was 7 rotations per min (rpm) . The duration of test period was 5 min and the collected dust on the filters represents the entire test period.

From each run, samples on two filters were taken for gravimetrical and microbiological analyses. The results of the gravimetrical analyses are given as average concentration for the entire test period.

Gravimetrical analysis of dust in the drum. There was applied 25 mm diameter Teflon filters, pore size 3 μm. The filters were weighed in an acclimatized weighing room. Volume flow 1.9 litres per min (lpm) . Analysis of endotoxin. Subsequent to weighing, the filters were extracted and the total extract from the two filters analyzed for endotoxin content by kinetic LAL test.

Microbiological analysis of dust from the drum. Nucleopore filters, 25 mm diameter, pore size 0.4 μm, volume flow 1.0 1pm, were used. Earlier 1.9 lpm was applied, but this is only relevant to the detection limit. The two filters were combined before the analysis of cultivable microorganisms. The analytical resolution (corresponding to detection of 1 CFU) is 2500 CFU/m3 for the average of 4 individual straw samples, or about 6.4xlO6 CFU/g dust.

Dust elution. The dust in the outlet of the drum was collected on 140 mm diameter cellulose acetate filters, pore size 5 μm, and weighed. The results are shown in Table 6. Table 6.

ID Straw Dust Endotoxin Microorg. Microorg. Endotoxin Liberated dust kg mg/m3 EU/m3 CFU/m3 CFU/g dust EU/g dust g CA+prop+spa 3.4 4.00E02 4.89E00 0 0 8.18E04 2.66E-02 n 1 CA+prop+spa 2.9 4.82E03 4.15E01 0 0 1.16EO5 1.58E-01 n 2 CA+prop+spa 3.55 4.42E03 9.24E01 0 0 4.78E04 3.16E-01 n 3 CA+prop+spa 3.15 1.71E03 1.69E01 0 0 1.01E05 9.04E-02 n 4 Average 3.89E01 2.84E03 0 0 8.67E04 1.48E-01 SD 3.88E01 2.13E03 0 0 2.95E04 1.24E-01 Reference 1 2.9 1.04E03 4.49E05 9.91E06 9.49E06 4.30E05 2.95E00 Reference 2 2.45 1.11E03 8.87E05 1.53E06 1.38E06 8.02E05 2.67E00 Average 1.08E03 6.68E05 5.72E06 5.44E06 6.16E05 2.81E00 U) O SD 4.44E01 3.10E05 5.93E06 5.73E06 2.63E05 1.99E-01 Conclusion

A substantial effect on dedusting as well as bacterial con¬ tents was demonstrated between treated samples and reference samples.

Example 7

Reduced evaporation

Experiments conducted at Felleskjøpet Stange, show that the evaporation of acid (ppm) is reduced from 0.7 ppm to 0.2 pp when comparing the composition with concentrated formic acid.