A COLICINOGENIC STRAIN OF E COLI PRIOR APPLICATION INFORMATION

This application claims the benefit of US Provisional Patent Application 60/796, 145, filed May 1 , 2006 BACKGROUND OF THE INVENTION

Post-weaning diarrhoea in weaned piglets is a significant production problem Pigs that suffer from diarrhea typically have poor growth and/or become dehydrated, and in some instances will die The problem is not so much an issue of mortality as it is one of lingering morbidity in the animal which means that it takes longer for the pig to be at the proper weight or condition to be shipped for slaughter, and hence is less profitable One of the most frequent causes of this disease is Escherichia coll The most frequently occurring serotype is K88+ which attaches to the enterocyte via an F4-type fimbrea Once attached it can made a variety of toxins which, when injected into the enterocyte, result in diarrhoea SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided an isolated colicin-producing E coll strain selected from the group consisting of UM- 17 and UM-19

According to a second aspect of the invention, there is provided a probiotic for treatment of E coll K88+-assocιated diarrhea comprising a colicin- producing E coli strain selected from the group consisting of UM-17, UM-19 and combinations thereof

According to a third aspect of the invention, there is provided a feed additive comprising a colicin-producing E coli strain selected from the group consisting of UM-17, UM-19 and combinations thereof

According to a fourth aspect of the invention, there is provided a method of treating E coli K88+ diarrhea comprising administering an effective amount of a probiotic comprising a colicin-producing E coli strain selected from the group consisting of UM-3, UM-17, UM-19 and combinations thereof BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 Competition between the pathogen and probiotic Bacteria were allowed to compete over a 24h period, then 100 μl was transferred to a new chube Over time, only the most competitive strain will survive

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described All publications mentioned hereunder are incorporated herein by reference

Described herein is the isolation of colicin-producing strains of E coll for use as probiotic treatments for the prevention of E coll K88+ diarrhea These strains of E coli, designated as UM-17 and UM-19 express a filament and produce colicin but produce no compounds toxic to the host animal and as such inhibit the growth of E coli K88+ It is of note that the above identified strains may be used alone or in combination and may also be combined with other treatments known in the art It is noted that deposits of these strains were made on April 30, 2007 and they were assigned the following numbers UM-17 - and UM-19-

According to one aspect of the invention, there is provided an isolated colicin-producing E coli strain selected from the group consisting of UM- 17 and UM-19 As will be appreciated by one of skill in the art, 'isolated' refers to the fact that the E coli strains have been removed from their native environment, as discussed below It is noted that these strains are also considered to be 'purified' or 'substantially purified' in that either all or substantially all of the bacteria are the same

In other embodiments, the above-described strains are utilized in the manufacture of a probiotic for treatment of E coli K88+-assocιated diarrhea In these embodiments, the probiotic comprises an effective amount of at least one colicin-producing E coli strain selected from the group consisting of UM-17, UM-19 and combinations thereof As will be appreciated by one of skill in the art, 'an effective amount' is an amount that is sufficient to accomplish at least one of the following inhibit E coli K88+ growth, reduce E coli K88+ growth rate within the animal reduce severity of symptoms associated with E coli K88+ infection and the like

In yet other embodiments, there is provided a method of treating E

coli K88+ diarrhea comprising administering to an animal in need of such treatment an effective amount of a probiotic comprising an effective amount of at least one colicin-producing E coli strain selected from the group consisting of UM-17, UM-19 and combinations thereof

The probiotic may be administered with the feed ration of the animal or may be combined with feed as well as other modes of administration of powders known in the art

The colicinogenic strains identified are unique because they are also able to ferment amylase and/or inulin These are both unusual phenotypes for Escherichia coli which means that it puts a downward pressure on E coli growth Our strains can ferment these sugars so that makes them more competitive in the gut For example the probiotic will work in pigs of between 14 and 56 days of age because the serotype K88 is only pathogenic in pigs of this age Thus, in some embodiments of the invention, the probiotic as described above is administered in an effective amount to pigs between approximately 14 and 56 days old

Fecal samples from pigs, hog lagoon manure, cattle and soil were plated out on E coli selective chromogenic agar After 18 h E coli (purple colonies) and non-E coli coliforms (blue pink, and white colonies) were selected E coli (purple colonies) were picked from each positive tissue and sub-cultured in LB broth, and recultured on E coli /coliform medium, and tested for reactivity to indole, methyl red, Vogues Proskauer, and citrate utilization From this process 863 strains were positively identified as E coli To determine which of these strains were able to produce a colicin active against E coli K88+, screening was done using a zone inhibition assay In brief, the producer strain was grown in a single colony on LB agar for 24 h so that a colony of approximately 5 mm was visable on the agar The indicator strain, K88+, was inoculated into soft LB agar (1%), mixed well, and then pored over the agar with the producer straιn(s) on it The plate was then allowed to incubate at 37 ⁰ C for 16 h If a producer strain was positive, there was a clear zone around it indicating lack of growth This procedure was carried out on all strains, and strains were ranked in descending order based on the size of the zone of inhibition The top 80 strains were then screened for the presence of heat labile, heat stabile and enterotoxin genes typically found in E coli K88 Of these 80 strains 20 were selected for further investigation based on

lack of toxins and inhibitory ability These strains were screened with the API 50 system (bioMeπeux) for the ability to ferment a range of 50 carbohydrates

The probiotic can be administered as a freeze dried powder, prepared using means well-known in the art In some embodiments, the powder may be coated, for example, alginate coated In preferred embodiments, the probiotic powder is administered to animals in need of such treatment, for example, feed animals such as weaned pigs, at risk of or suspected of E coll K88+ infection It is of note that in these embodiments, the powder may be a food additive included in the feed ration of weaned pigs This way the bacterium can be administered continuously, thus increasing its effect Alternatively, in other embodiments, the bacterium could be administered as a stable paste, for example in a starch carrier, in high concentration, given on the day of weaning Given that these strains have a carbohydrate selective pressure, the product may be most effective when fed with diets containing starch and/or inulin

The invention will now be further described by way of example However, the invention is not necessarily limited by the invention Example I Animals, Housing, and Experimental Design

A total of 45 Cotswold piglets weaned at 17 ± 1 d were obtained from the University of Manitoba's Glenlea swine research farm The initial BW of the piglets was 4 82 ± 0 6 kg Five pigs were euthanized before the start of the experiment in order to generate the base line data for microbiology and other parameters Forty piglets were divided into groups of two pigs per pen and were blocked on the basis of BW Each pen had a plastic-covered expanded metal floor In each block, three replicate pens were assigned to each dietary treatment in a completely randomized design Pigs had unlimited access to feed and water throughout the 2 wk study BW and feed disappearance were monitored daily and the results used to calculate ADG, ADFI, and G F ratio Room temperature was maintained at 29 ± 1 °C throughout the study Experimental Diets

Each pen was assigned to one of the 4 wheat-soybean meal-based diets consisting of a control with an antibiotic (C) and three diets with no antibiotics but containing UM-17 and UM-19 as the probiotics (PRO), 14% potato starch (PS),

or a combination of 14% potato starch and probiotics (PRO-PS) All experimental diets were formulated to meet NRC (1998) nutrient requirements for piglets weighing 7 to 12 kg (Table 1) Diets were mixed one week before the start of experiment

Bacterial Culture, Oral Challenge and Health Status

Two strains (UM-17 and UM-19) having colicinogenic properties and a rapid growth on starch were selected Results of in vitro competition assays revealed that UM-17 and UM-19 suppressed the E coll K88 growth in the presence of starch Fresh overnight probiotic cultures (50 ml of 9 x 10 ¹⁰ cfu/ml per pen) were mixed with fresh feed each morning (Table 1 )

Two E coli K88+ strains (2-12 and I-36) were maintained aerobically in Luna Bertani (LB) broth at 39 C An overnight culture was scaled up to 2 L by inoculating 5 ml of pre-culture A sub-sample was taken and serially diluted (10- fold) and plated on LB agar to obtain colony forming units of the inoculant On day 7 of the experiment (24 d old pigs), each pig received 6 ml (2 x 10 ⁹ cfu mL ¹ ) of a bacterial suspension contained in a syringe attached to a polyethylene tube held in the back of the oral cavity Severity of diarrhea was characterized by using the fecal consistency (FC) score method Fecal consistency scoring (0 normal, 1 , soft feces, 2, mild diarrhea, 3, severe diarrhea) was performed in a blinded fashion by two trained personnel with no prior knowledge of dietary treatment allocation The presence of blood in feces was checked daily In vitro competition assays

Isolates E coli UM-17 and UM-19 were evaluated by in vitro competition assays with E coli K88+ strain 2-12 UM-17 and UM-19 were made resistant to levofloxacin by repeated transfer in LB broth containing 1 μg ml ¹ of levofloxacin The MIC for levofloxacin was measured to be 0 05 μg ml-1 E coli 2-12 was not resistant to levofloxacin at 0 05 μg ml ¹ Individual strains were maintained on minimal medium which contained (g L ^"1 ) glucose, 5, Na ₂ HPO ₄ , 6, KH ₂ PO ₄ , 3, NH ₄ CI, 1 , MgSO ₄ , 0 12, CaCI ₂ , 0 01 For competition assays strains were transferred at least three times on minimal medium with the growth substrate to be assayed For example, for the competition assay with starch, the glucose in the minimal medium was substituted with starch The E coli 2-12 strain plus UM- 17 or UM-19 was inoculated into medium to give a final cell concentration of

approximately 10 ⁶ cfu ml ¹ and incubated at 37 ⁰ C for 0, 12, 24, and 36 h At the end of each incubation period, one ml of a well mixed culture was taken, serially diluted in triplicate in buffered peptone water, and plated onto LB-agar with or without 0 05 μg ml ^"1 levofloxacin at 37 ⁰ C for 16 h All competition experiments were repeated on three separate occasions Blood Sampling and Blood Parameter Measurements

On day 0, five pigs were euthanized and blood was sampled via cardiac puncture using hepaπnized vacuum container tubes (Becton Dickinson, Rutherford, NJ, USA) to generate the base line data for blood parameters and packed cell volume (PCV) Blood collections from the forty experimental pigs was done three times, a day before inoculation, 24 h, and 48 h post inoculation via jugular vein puncture using hepaπnized vacuum container tubes The blood was processed by centπfugation at 2,000 * g for 10 mm at 4°C to recover plasma Plasma samples were stored at -70 ⁰ C until required for plasma urea nitrogen (PUN) determination using a Nova Stat profile M blood gas and electrolyte analyzer (Nova Biomedical Corporation, Waltham, MA, USA) An additional blood sample was collected at the time of slaughter (day 14) via cardiac puncture and centrifuged at 3,000 x g for 20 mm at 5 ⁰ C to recover serum Chemical Analysis

Crude protein (CP) was analyzed using a Leco NS 2000 Nitrogen Analyzer (LECO Corporation, St Joseph, Ml USA) Gross energy (GE) was measured using a Parr adiabatic oxygen bomb calorimeter (Parr Instrument Co , Moline, IL, USA)

While the preferred embodiments of the invention have been described above it will be recognized and understood that various modifications may be made therein, and the appended claims are intended to cover all such modifications which may fall within the spirit and scope of the invention

Table 1. Composition and nutrient analysis of experimental diets (as-fed basis)

Diets'

Ingredients C Pro 14% PS Pro + 14%

Corn 4465 4465 2596 2596

Soybean meal 328 328 390 390

Whey powder 120 120 120 120

Limestone 05 05 075 075

Dical P 075 076 10 10

Vege Oil 50 50 50 50

Lys-HCI 019 019 019 019

Fish meal 30 30 10 10

Vitamin

Premix ² 05 05 05 05

Mineral Premix ³ 05 05 05 05

L-Tryptophan 01 01 01 01

SP250 ⁵ 001 - - -

Potato starch - - 140 140

Nutrient Analysis

CP 2137 2196 2109 2195

GE Kcal/Kg 433617 437314 416569 416897

¹ Diets C = control with antibiotics, Pro = control without antibiotics + probiotics, 14% = control without antibiotics + 14% potato starch, Pro + 14% = 14% + probiotics

² Provιded per kg of diet 9000 IU of vitamin A, 1 ,500 IU of vitamin D3, 18 mg of vitamin E ₁ 15 mg of vitamin K, 250 mg of choline, 30 mg of niacin, 275 mg of calcium pantothenate, 94 mg of B2, 2 mg of B6, 25 μg of B12, 80 μg of biotin, 05 mg of folic acid

³ Provιded per kg of diet 18ιmg copper, 110mg zinc, 02mg iodine, 110mg iron, 50mg manganese, and 03mg selenium

⁴ Aureo sp250. Chlortetracycline, Penicillin (as penicillin G Procaine), Sulfamethazine (Alpharma Inc., Fort Lee, NJ, USA).

Table 2. Performance of earl) -weaned pigs fed different experimental diets

Diets ¹

Pro +

Item C Pro 14% PS 14% SEM ² P

Initial BW, kg 4 62 4 70 4 84 4 90 0 24 0 8556

Final BW, kg 5 78 5 85 5 90 6 40 0 31 0 5030

ADG, g/d

BI ¹ 86 25 94 50 54 97 122 50 23 13 0 2674

AI ¹ 104 41 1 14 39 129 83 152 81 16 85 0 2368

ADγI ■ g/d

351 23 ^ab 353 15 ^a 281 85 ^a 413 70 ^b 42 39 0 2249

BI b

Al 451 68 436 80 455 44 512 48 37 49 0 5190

Gain 1 feed

BI 0 22 0 23 0 20 0 29 0 04 0 4638

AI 0 24 0 26 0 28 0 30 0 03 0 5084

¹ Diets as in table 1 ² pooled standard error of the means ³ BI = before inoculation ⁴ AI = after inoculation _ ^ab Means within rows without common letters differ (P < 0 05)

Table 3. Effect of dietar> treatments on fecal score data after inoculation w ith b.coli in earl) -weaned pigs.

Diets'

Item Pro +

C Pro 14%PS 14% SEM ² P

Fecal Score ³

O to 48h 0 45 0 50 0 86 0 81 0 22 0 4602

48 to 96h 0 99 ^a 1 ₂₃ ab 1 39 ^b 0 99 ^a 0 12 0 0835 0 to 96h 0 73 ^a 0 88 ^ab 1 15 ^b 0 90 ^ab 0 15 0 2784

¹ Dιets as in table 1 ² pooled standard error of the means

³ Fecal score 0, normal, 1 , soft feces, 2, mild diarrhea, 3, severe diarrhea ^ab Means within rows without common letters differ (P < 0 05)