In recent years, much attention has been directed to diseases caused by Mycobacterium avium subspecies paratuberculosis (MAP) in animals (Choidini RJ, Van Kruinigen HJ, Merkal RS: Ruminant paratuberculosis (Johne's disease) : The current status and future prospects Cornell Veterinarian (1984) 74: 218-262) and its potential transfer to humans (Choidini RJ: Crohn's disease and the mycobacterioses : a review and comparison of two disease entities Clin Microbiol Rev 2: 90-117).

The disease in cattle, which is known as Johne's disease, is endemic in food animal herds and has been identified in almost every developed country in Europe and in the USA. The infection results from exposure to and ingestion of contaminated pastures, water or feed. There is a long incubation period (up to two years) during which an infected cow may spread the infection in her manure to other animals in the herd, including young animals which are the most susceptible to infection. Death rates in infected herds may approach 10%. These losses, together with long periods of poor health and reduced performance in infected animals, have serious economic consequences. In the United States, between 20% and 40% of dairy cattle herds are infected with MAP, resulting in losses of at least US$1. 5 billion every year.

Treatment of infected cattle is difficult since M. paratuberculosis is resistant to antibiotics and vaccination is not effective with the current vaccines. The lack of accurate tests and the long incubation period produce additional problems for controlling the disease.

Since MAP is not classified as a human pathogen, milk and other products from animals infected with MAP may be continually entering the human food chain. One specific area that has been intensively debated in recent years is a possible link between MAP and Crohn's disease in humans (Andus T, Gross V: Etiology and pathophysiology of inflammatory bowel disease-Environmental factors Hepato-Gastroenterology (2000) 47: 29-43).

It has been suggested in the course of this debate that M. avium subsp. paratuberculosis may play a part in the onset and pathogenesis of Crohn's disease (Brugere-Picoux J: Maladie de Crohn: incertitudes sur la role étilogique de Mvcobacterium paratuberculosis Bull Acad Natl Med (1998) 182: 830-832). Although the causal relationship between M. paratuberculosis infection and Crohn's disease in humans has not been conclusively demonstrated, a reduction in overall levels of human exposure to the bacterium would nevertheless undoubtedly be of medical benefit to humans.

M. paratuberculosis is, for example, known to be closely related to various recognised human and animal pathogens, including Mycobactesium avium complex strains, which are a leading cause of morbidity and mortality in AIDS patients. The toxic products of M. paratuberculosis could also have pathological consequences in humans infected by the bacterium.

Human exposure to M. paratuberculosis occurs primarily through the consumption of infected milk products such as pasteurised or unpasteurised milk from infected animals, or of drinking water which has been contaminated by faeces of infected animals. Studies made by the former Ministry of Agriculture and Fisheries and Food (MAFF) in the UK have confirmed that M. paratuberculosis exists in pasteurised milk sold in the retail trade. Consequent exposure levels have been calculated at 0-5 cfu/litre pasteurised milk from affected animals ; however it has been suggested that actual or potential exposure levels may be 100-fold higher (Nauta MJ, van der Giessen JW : Human exposure to Mvcobacterium aratuberculosis via pasteurised milk a modelling approach Vet Rec (1998) 143: 293-29610).

Conventional milk pasteurisation methods, involving thermal treatment of milk for 15 seconds at 72°C, are inadequate for killing or inactivating MAP. An increase in the stringency of milk pasteurisation has been proposed; however, this would involve a change in existing milk product technology, and might adversely affect the quality of the milk products.

There remains therefore an outstanding need for methods for the reduction or elimination of MAP and/or other pathogenic mycobacteria from consumables for humans or animals, and for methods which will serve to reduce and/or eventually eliminate levels of MAP infection in animal herds and human populations. There is also an outstanding need for methods for reducing or inhibiting the transmission of mycobacteria from one infected animal to other animals or to humans, so as to curb the spread of diseases which may be caused or linked to infection by said mycobacteria.

According to one aspect of the present invention therefore, there is provided a method for causing the death or inactivation of mycobacteria in a consumable for animals or humans, comprising the step of introducing a lytic bacteriophage preparation to said consumable under conditions suitable for producing the lysis or inactivation of said mycobacteria in the consumable by said bacteriophage. Said bacteriophage may be introduced to the consumable before, after or simultaneously with the ingestion of said consumable by a human or animal.

According to another aspect of the present invention, there is provided a method for causing the death or inactivation of mycobacteria in a human or animal, comprising the step of administering a lytic bacteriophage preparation to said human or animal under conditions suitable for producing the lysis or inactivation of said mycobacteria by said bacteriophage.

More particularly, there is provided in accordance with this aspect of the invention a method for causing the death or inactivation of mycobacteria within the digestive tract of a human or animal, comprising the step of

introducing a lytic bacteriophage preparation into said digestive tract under conditions suitable for producing the lysis or inactivation of said mycobacteria by said bacteriophage.

According to yet another aspect of the present invention, there is provided a method for reducing or preventing the transmission of mycobacteria from one animal infected with said bacteria to other animals or to humans, comprising the step of introducing into the rectum of said one animal a quantity of a lytic bacteriophage preparation, under conditions suitable for producing the lysis or inactivation of said mycobacteria within the colon of said one animal by said bacteriophage.

Although mycobacteria such as MAP are typically located intracellularly in the macrophages of infected humans or animals, a large number of mycobacteria in the colon and the faeces of an infected animal or human, or in contaminated consumables, are exposed extracellularly, and are hence susceptible to lysis when treated with suitable lytic bacteriophage.

Such mycobacteria are therefore also often present in an extracellular state in the upper regions of the digestive tracts of humans or animals which have ingested the mycobacteria, for example in a contaminated consumable.

According to another aspect of the present invention, there is provided a bacteriophage preparation for use in the methods of the present invention; and a method for preparing said bacteriophage preparation. Said bacteriophage preparation may comprise a quantity of phage propagating cells which are non-pathogenic to humans and/or animals, such as M. smegnialis cells or saprophytic mycobacterial cells, which cells may preferably be infected with said bacteriophage. Preferably, said cells may be susceptible to lysis by said bacteriophage. Suitably, said cells may be fast- growing. The presence of such cells in said bacteriophage preparation will serve to increase the quantity of phage in said preparation, and will also serve to protect phage absorbed by said cells from degradation, for example by acid environments or by enzymes. Said method for preparing the

bacteriophage preparation may comprise the steps of obtaining or preparing a sample of said bacteriophage, mixing said bacteriophage with a culture of said cells, and leaving said bacteriophage and cell mixture before use for a time sufficient to allow infection of some or all of said cells by said bacteriophage, but not sufficient to allow lysis of a significant number of said infected cells. Typically, said time may be of the order of one to five hours, suitably at least two hours.

According to yet another aspect of the present invention, there is provided a bacteriophage kit comprising a packaged bacteriophage preparation in accordance with the invention, a separately packaged quantity of phage propagating cells which are non-pathogenic to humans and/or animals, such as M. smegmatis cells or saprophytic mycobacterial cells, and instructions for administering said cells to a human or animal or for adding said cells to a consumable for humans or animals before, after or simultaneously with the administration or addition of said bacteriophage preparation to said human or animal or to said consumable respectively.

According to yet another aspect of the present invention, there is provided a kit comprising a packaged consumable for humans or animals and a separately packaged bacteriophage preparation in accordance with the invention, and written instructions for introducing said bacteriophage preparation to said consumable before, after or simultaneously with the ingestion of said consumable by a human or animal, so as to cause the death or inactivation of mycobacteria in said consumable. Said kit may further comprise a separately packaged quantity of phage propagating cells which are non-pathogenic to humans and/or animals such as M. smegmatis cells or saprophytic mycobacterial cells, and written instructions for the addition of said cells to said consumable before, after or simultaneously with the addition of said bacteriophage preparation thereto.

According to yet another aspect of the present invention, there is provided a kit comprising a bacteriophage preparation in accordance with the

invention, and written instructions for administering said bacteriophage preparation to a human or animal, for example by way of introducing said bacteriophage preparation into the digestive tract of a human or animal, so as to cause the death or inactivation of mycobacteria within said human or animal. Said kit may further comprise a separately packaged quantity of phage propagating cells which are non-pathogenic to humans and/or animals such as M. smegmatis cells or saprophytic mycobacterial cells, and written instructions for the administration of said cells to said human or animal before, after or simultaneously with the administration of said bacteriophage preparation thereto.

According to yet a further aspect of the present invention, there is provided a kit comprising a bacteriophage preparation in accordance with the invention, and written instructions for introducing said bacteriophage into the rectum of an animal so as to cause the death or inactivation of mycobacteria within the colon of said animal. Said kit may further comprise a separately packaged quantity of phage propagating cells which are non-pathogenic to animals, such as M. smegmatis cells or saprophytic mycobacterial cells, and written instructions for the introduction of said cells into the rectum of said animal before, after or simultaneously with the introduction of said bacteriophage preparation thereto.

According to yet another aspect of the present invention, there is provided a consumable for animals or humans which comprises a bacteriophage preparation in accordance with the invention.

Yet another aspect of the present invention encompasses the use of a bacteriophage preparation in accordance with the invention in the manufacture of a medicament for the prophylaxis of a disease mediated by mycobacteria which may be introduced into a human or animal body by the ingestion of a consumable.

Said mycobacteria may, for example, be M. avium subsp. paratuberculosis, M. avium subsp. Silvaticum, M. scrofulaceum, or M. bovis.

Said bacteria may, for example, be capable of directly or indirectly stimulating, causing, exacerbating or mediating one or more bacterial diseases or infections in humans and/or animals, including Crohn's disease in humans or Johne's disease in animals. The elimination or substantial elimination of said mycobacteria from consumables for humans or animals in accordance with the invention will therefore be of significant medical benefit to humans and/or animals.

Advantageously, said bacteriophage preparation may comprise one or more mycobacteriophage which are capable of causing the lysis of mycobacteria, particularly mycobacteria in dairy products; such as, for example, mycobacteriophage TM4, R51, BG-1, Minetti, or a genetically or chemically modified derivative of one of the aforementioned strains. A deposit of mycobacteriophage TM4 has been made at the American Type Culture Collection (ATCC) at 10801 University Boulevard, Manassas, VA 20110-2209, USA. The structure, properties and preparation of mycobacteriophage TM4 are also described by Ford, ME et aL Mycobacteriophage structure and gene expression (Tuber Lung Dis 1998; 79 (2) 63-73) and Timme TL et al, Induction of bacteriophage from members of the Mycobacterium avium. Mycobacterium intracellulare. Mycobacterium scrofulaceum serocomplex (J Gen Microbiol 1984 Aug; 130 (Pt 8): 2059-66).

Said bacteriophage preparation may for example be prepared by infecting a suitable culture cell with said bacteriophage, propagating said infected culture cell, and allowing or promoting the ultimate lysis of said infected culture cell by said bacteriophage. Preferably, said culture cell is susceptible to infection and lysis by said bacteriophage, and propagates rapidly. Advantageously, said culture cell is non-pathogenic. Said culture cell may, for example, be an M. smegmats cell. A deposit of M. smegmats cells suitable for the purposes of the present invention has been made at the American Type Culture Collection (ATCC) at 10801 University Boulevard,

Manassas, VA 20110-2209, USA; designation mc (2) 155 ; accession no.

700084; (Trevisan) Lehmann and Neumann ATCC 607. The infection of M. smegmatis cells by mycobacteriophage TM4 is described in Foley-Thomas EM et al. Phage infection, transfection and transformation of Mvcobacterium avium complex and Mvcobacterium paratuberculosis (Microbiology 1995 May; 141 (Pt 5): 1173-81).

Said bacteriophage may be administered to a human or animal prior to, simultaneously with or following ingestion of said mycobacteria, for example by way of consumption of said consumable, by the human or animal, such that said bacteriophage are introduced to said consumable within the digestive tract of said human or animal. Said bacteriophage preparation may be administered to said human or animal orally.

Where it is desired that the bacteriophage should reach the lower part of the digestive tract of said human or animal for introduction to mycobacteria present therein, said bacteriophage preparation may preferably comprise a plurality of non-pathogenic phage propagating cells, such as M snzegmatis cells, which are infected with said bacteriophage. Said cells will serve to protect the phage against low pH conditions and enzymatic activity within the upper part of the digestive tract during passage of the bacteriophage preparation therethrough. Said cells will furthermore serve to promote and increase the accumulation of said bacteriophage in the lower part of the digestive tract of said human or animal.

Alternatively, said bacteriophage preparation may be introduced to an animal via the rectum, for example by means of a suppository or a suspension delivered via a catheter. Thus the bacteriophage preparation may encounter and cause the death or inactivation of said bacteria in the colon of said animal. The administration of bacteriophage into the rectum of an animal will help to reduce the quantity of bacteria in the excreta of the animal, thereby serving to curb the spread of bacteria to other animals or to humans. Optionally, a quantity of phage propagating cells which are non-

pathogenic to animals, such as M. smegrnatis cells, may be introduced into the rectum of said animal prior to, simultaneously with, or following the introduction of said bacteriophage preparation thereto.

Advantageously, said bacteriophage preparation may be adapted for human or animal consumption. Said bacteriophage preparation may, for example, comprise a liquid suspension, or a powder or tablet or capsule or chewing gum or lozenge, and may contain formulation components intended to protect phage or bacteria from low pH conditions and/or enzymes within the gastric tract.

In some embodiments, said consumable is intended for human consumption. Accordingly, the consumable may be any food or drink product which may comprise mycobacteria. Said consumable may be drinking water. In particularly advantageous embodiments, however, said consumable comprises a dairy product such as milk in liquid or powdered form, cream, butter, cheese, or a fermented milk product such as yoghurt, soured cream, soured milk or buttermilk.

In other embodiments, said consumable is intended for animal consumption. Accordingly, the consumable may comprise animal feed or drinking water. Advantageously, said consumable may be provided in sufficient quantity for administration to several animals simultaneously, for example for administration to an entire herd of cows.

Following is a description, by way of example only, of embodiments of the present invention.

Example 1 Phage accumulation Mycobacterium phage TM4 was propagated on Msmegniatis. This strain was selected because it is susceptible to lysis by TM4, is non- pathogenic, and has a growth rate about 6 times greater than that of M. paratuberculosis.

The M. smegmatis (mc2 155) cultures were grown with shaking at 37°C in Middlebrook 7H9 broth supplemented with albumin/dextrose complex and 0.05% Tween 80.

M. paratuberculosis bovine strain B2 was grown at 37°C in Middlebrook 7H9 broth adjusted to pH 5.9 and supplemented with oleic acid/albumin/dextrose complex and 0.05% Tween 80.1. Ogg ml-'ferric mycobactin J (Allied Monitor) was added to the M. paratuberculosis cultures.

200gui samples of each of the Mparatuberculosis and Msmegmatis cultures, containing about 1x10'cfu, were each incubated for 30 min at room temperature with 1001 of TM4 phage lysats. Middlebrook 7H9 soft agar (0.7%) was added to the incubation mixes, and the cells were plated on 7H9 Middlebrook medium by the soft agar layer method and incubated at 37°C for 3 days (Msmegitiatis) or 16 days (M. paratuberculosis). Routinely, 10-50 ml of phage suspended in PBS buffer was prepared from plaques formed on M. smegmatis lawns. The lytic activity of the phage was tested on M. paratuberculosis lawns prepared as above.

High-titre phage lysates (108-109 pfu/ml) were prepared by phage purification and concentration by tangential filtration module such as Pall- Gelman Macrosep 300K Omega.

Example 2 Determination of the time required for enhanced phage production with phage propagation bacterial host lml of saturated Mycobacterium smegmatis strain mc2155 culture was used to inoculate a fresh 50ml batch of Lab Lemco medium (Oxoid). The culture was incubated with shaking overnight for approximately 15 hours until an OD650 of between 0.1 and 0.3 was achieved. This culture was diluted to an OD650 of 0. 1, which represents approximately 1 x 107 cfu per ml of culture. Phage (R35) from a filtered stock solution (titre 3.0 x 109) were added to Sml of this culture to achieve a multiplicity of infection (m. o. i.) of

10, i. e. I x 10'phage added per ml of culture.

These 5ml samples in 15ml Falcon tubes were incubated on their sides at 37°C with gentle agitation (50 rpm in a shaking incubator). Samples were taken every 30 minutes and analysed for their cell density (OD650) and phage content (titre determination).

Titres were determined using standard dilution methods. lOul of each dilution was added to 400ul of saturated M. smegmatis culture. This mix was added to 4ml of molten top agar (Lab Lemco media containing 0.7% agar) cooled to 50°C. After brief mixing, this mix was poured onto Lab Lemco Agar plates and left to set. Plates were inverted and incubated for >15 hours in order for plaques to develop. The number of plaques on the appropriate dilution plate was used to determine the pfu content (titre) of the original sample.

Figure 1 shows the amplification of phage within the bacterial culture over the first 4 hours of incubation. The initial titre of phage at time 0, before phage absorption or amplification, is 1x108. The figure graphically illustrates the relationship between culture optical density and phage accumulation. Phage accumulation takes off between 3 and 4 hours after infection, and after 4 hours results in a 2 log increase in phage numbers.

Example 3 Susceptibility of the test mycobacteria cultures in milk to lysis by phage M. smegmatis and M. paratubercarlosis cultures are grown to exponential phase (OD600 = 0. 2; approximately 6.0x107 cfu mol-') in Middlebrook 7H9 medium and washed three times in growth medium without Tween. The bacterial cultures are then diluted threefold in fresh medium, and grown at 37°C to an OD600 of 0.1. These cultures are used as stocks to spike samples of pasteurised milk, producing spiked samples containing 10,100 and 1000 cfu ml''respectively.

Sufficient phage TM4 is added to 1 ml of each spiked milk sample to

produce a pfu: cfu ratio of 100: 1 in each case; and the resulting phage-treated milk samples are incubated at 37°C for 30 min. lml samples of spiked milk containing no phage TM4 are also prepared as non-phage-treated controls, whilst lml samples of unspiked milk are prepared as blanks. The blanks and non-phage-treated controls are also incubated at 37°C for 30 min.

Following incubation, mycobacteria are isolated from each of the phage-treated, non-phage-treated and blank milk samples by immunomagnetic separation using Dynobeads loaded with fibronectin (20gel of F-MB added to 1 ml of milk). Recovered Dynobeads are washed twice with phosphate-buffered saline and incubated with fibronectin at 2 mg/ml to displace bound bacteria. The supernatants from these incubations are plated without dilution on solid agar plates made of 7H9 Middlebrook medium containing Bacto Agar at 15 gl-'. This enables the numbers of bacterial colonies on the plates, and hence the activity of the phage within the phage- treated samples, to be assessed.