A solid or semi-solid composition for administration to ruminants, comprising a virus capable of suppressing in vivo methane production in ruminants

Abstract

The present invention relates to the reduction of methane production in ruminants. In particular, the present invention relates to solid and semi-solid compositions for administration to ruminants, which contain substances capable of suppressing in vivo methane production in ruminants. The present invention also relates to controlled release boluses, animal licks and animal feeds, which contain solid and semi-solid compositions containing substances capable of suppressing in vivo methane production in ruminants. The present invention further relates to a method of orally administering to ruminants a bolus capable of suppressing in vivo methane production in ruminants, and a method of orally introducing into ruminants a substance capable of suppressing in vivo methane production in ruminants. The present invention also relates to a virus capable of suppressing archaea that produce methane in vivo in ruminants.

Description

Technical Field

[0001] Field of the Invention The present invention relates to the reduction of methane production in ruminants. In particular, the present invention relates to solid and semi-solid compositions for administration to ruminants, comprising a substance capable of suppressing in vivo methane production in ruminants. The present invention also relates to controlled release boluses, animal licks and animal feeds comprising solid and semi-solid compositions comprising a substance capable of suppressing in vivo methane production in ruminants. The present invention further relates to a method of orally administering to a ruminant a bolus capable of suppressing in vivo methane production in the ruminant, and a method of orally introducing into a ruminant a substance capable of suppressing in vivo methane production in the ruminant. The present invention also relates to a virus capable of suppressing archaea that produce methane in vivo in ruminants.

Background Art

[0002] Background Methane is produced as a byproduct of the digestive process in ruminants. The digestive process involves the intestinal fermentation of cellulose in the rumen, as a result of which ruminants can obtain nutritional benefits from foods such as forage. Methane production by ruminants accounts for approximately 81% of greenhouse gas emissions from the livestock sector, 90% of which results from rumen microbial methanogenesis. Intestinal fermentation caused by microorganisms produces a variety of metabolites utilized by the host animal and waste products containing hydrogen. Hydrogen is then utilized by other microorganisms, including methanogenic archaea, which are commonly referred to as methanogens. Representative methanogens found in ruminants include Methanobrevibacter millerae, Methanobrevibacter thaueri, Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanobrevibacter gottschalkii, Methanosarcina barkeri, Methanomicrobium mobile, Methanobacterium formicicum, and Methanobacterium bryantii. When methanogens metabolize hydrogen during the intestinal fermentation process in the rumen, the main byproduct is methane.

[0003] It is well known that the livestock industry is highly involved in global greenhouse gas emissions. Methane emissions from ruminants are a particularly significant contributing factor to these greenhouse gas emissions. World meat consumption is increasing, and the livestock industry is expanding to meet demand. Therefore, methane emissions from ruminants and the environmental impact of livestock production are expected to continue to increase in the future.

[0004] Numerous strategies are being considered to promote the reduction of methane production. These include providing extracts of plants, such as garlic and red algae, which are reported to reduce methane production. Also, certain chemicals, including antiprotozoal agents, aliphatic bromine compounds present in red algae extracts, oils from garlic extracts, and other plant-derived chemicals, are being considered as possible interventions to reduce methane production.

[0005] There is a need for new strategies to minimize the environmental impact of the livestock industry, particularly to minimize methane emissions associated with ruminant animals.

Brief Description of the Drawings

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[0007] Generalization of the Invention In a first aspect of the present invention, there is provided a solid or semi-solid composition for administration to a ruminant, wherein the composition comprises a virus capable of suppressing in vivo methane production in a ruminant. Also provided are a controlled-release bolus, a solid or semi-solid animal lick block, and animal feed comprising a composition comprising a virus capable of suppressing in vivo methane production in a ruminant. The semi-solid composition or animal lick block can be in the form of a colloid or a gel.

[0008] In a second aspect of the present invention, there is provided a method for reducing methane production in ruminants, comprising orally administering a bolus to a ruminant, wherein the bolus contains a virus capable of suppressing in vivo methane production in the ruminant, resulting in a controlled release of the virus to the ruminant.

[0009] In a third aspect of the present invention, there is provided a method for reducing methane production in ruminants, comprising providing an animal lick block or animal feed containing a virus capable of suppressing in vivo methane production in ruminants, wherein the virus is introduced orally into the ruminant when the ruminant ingests and digests components of the lick block or animal feed.

[0010] In a fourth aspect of the present invention, there is provided a virus capable of suppressing in vivo methane production in ruminants, wherein the virus targets archaea involved in methane production in ruminants.

[0011] The compositions and methods of the present invention provide effective and easily controllable means for reducing methane production from ruminants and thereby reducing greenhouse gas emissions from the livestock industry.

[0012] Definitions As used herein, the terms "comprising", "including" and "containing" mean that at least all of the recited elements must be present, but other elements not recited may also be present.

[0013] As used herein, the term "consisting of" means that only the recited elements must be present and no other elements not recited may be present.

[0014] As used herein, the term "semi-solid" for indicating the form of a composition relates to a composition having a viscosity and rigidity intermediate between the solid form and the liquid form of the composition. Exemplary semi-solid forms include, but are not limited to, gels, colloidal forms and creams.

[0015] The terms "licking block", "lick", "animal lick agent" and "animal licking block" are used interchangeably herein and mean a feed supplement source for animals adapted such that when the animal licks the feed supplement, the feed supplement is introduced into the animal. The feed supplement can contain minerals and nutrients.

[0016] The term "virus" according to the present invention includes double-stranded or single-stranded RNA or DNA viruses that infect bacteria, archaea, plant and / or animal cells. As used herein, the singular term "virus" can mean one or more viruses. Thus, the singular term "virus" encompasses both the singular and plural forms, i.e., it can mean one virus or multiple viruses.

[0017] The term "engineered virus" as used herein means a virus modified from its natural state so as to have enhanced activity or to be stabilized by covalent attachment to a substrate.

[0018] The term "archaea" as used herein means any unicellular prokaryote.

[0019] The term "substrate" in the context of the present invention is understood to mean any solid phase material to which a virus can be immobilized.

[0020] In the context of the present invention, "release period" means the length of time during which the active substance is released to the ruminant.

[0021] The terms "substance capable of suppressing methanogenesis" and "active substance" are used interchangeably herein and mean a substance capable of suppressing in vivo methanogenesis in ruminants. Thus, the term "virus capable of suppressing methanogenesis" means a virus capable of suppressing in vivo methanogenesis in ruminants.

[0022] As used herein, "inert substance" means a substance that cannot suppress methane production.

[0023] As used herein, "virus-free substance" means a substance that does not contain viruses. The virus-free substance may be, if desired, a substance generally administered to ruminants, such as minerals and / or anthelmintics.

[0024] As used herein, "controlled release" means that the active substance is released at a predetermined rate or at predetermined intervals. Controlled release includes sustained release and intermittent release.

[0025] As used herein, "sustained release" means that the release of the active substance is maintained throughout the release period. Sustained release includes embodiments in which the active substance is released at a constant dose throughout the release period, and also embodiments in which the active substance is released at varying doses throughout the release period.

[0026] As used herein, "intermittent release" means that the release of the active substance is discontinuous and there are intervals during the release period when the active substance is not released.

[0027] As used herein, "majority" means more than 50% (i.e., exceeding 50%). In some embodiments, this term means 60% or more. In some embodiments, this term means 70% or more, 80% or more, 90% or more, or 95% or more.

[0028] As used herein, the term "about" when applied to one or more values of interest refers to a value similar to the indicated reference value. In some embodiments, the term "about" means a range of values that are within 25% of the reference value in either direction (greater than or less than the reference value). In some embodiments, the term "about" means a value that is within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the indicated reference value (except where such a numerical value exceeds 100% of the possible values).

[0029] Detailed Description The present invention relates to a solid or semi-solid composition for administration to ruminants, comprising a virus capable of suppressing in vivo methane production in ruminants.

[0030] The present invention also provides a method for suppressing methane production in ruminants, comprising orally administering a bolus to a ruminant, wherein the bolus comprises an effective amount of a virus capable of suppressing in vivo methane production in ruminants and provides controlled release of the virus to the ruminant. The present invention further relates to a method for reducing methane production in ruminants, comprising providing an animal lick block or animal feed comprising an effective amount of a virus capable of suppressing in vivo methane production in ruminants, wherein the virus is introduced orally into the ruminant when the ruminant ingests and digests components of the animal lick block or animal feed.

[0031] The present invention also relates to controlled-release solid or semi-solid animal lick blocks and animal feeds comprising the compositions of the present invention. In some embodiments providing an animal lick block, the virus capable of suppressing methane production is covalently bound to at least a portion of the components of the animal lick block.

[0032] The present invention also relates to a virus capable of suppressing in vivo methane production in ruminants, where the virus targets archaea involved in methane production in ruminants. In some embodiments, the virus is an engineered virus produced for the disclosed methods, compositions, kits, and uses. In some embodiments, the engineered virus is modified from its naturally occurring state to have enhanced activity. In some embodiments, the engineered virus is modified to be stabilized by covalent attachment to a substrate. In some embodiments, the engineered virus is modified from its naturally occurring state to have enhanced activity and to be stabilized by covalent attachment to a substrate.

[0033] The virus of the present invention can be engineered to provide enhanced activity and performance. Conventional modification of a native virus using CRISPR and Cas9 can be performed to eliminate undesirable properties such as lysogeny. Specific examples include the elimination of the integrase gene within the virus characterized in the examples by SEQ ID NO: 1.

[0034] The present invention provides a means for suppressing methane emissions from ruminants and thereby reducing greenhouse gas emissions from the livestock industry. The ruminant can be a cow, goat, elaphine, or sheep (including calves or lambs). In particularly preferred embodiments, the ruminant is a cow.

[0035] Viruses that can suppress in vivo methane production in ruminants can block the metabolic pathway of methane production in ruminants and / or can have the ability to destroy the enzymes of enteric microorganisms in the rumen. For example, the virus can be involved in the metabolic pathway of methane in the rumen or can have the ability to destroy the enzymes of enteric microorganisms living in the rumen. In some embodiments, the virus is an inhibitor of methanogens. The virus can directly suppress the growth of methanogens or can indirectly suppress the growth of methanogens.

[0036] In some embodiments, the virus is an inhibitor of hydrogen production that promotes methane production. The virus can be a virus that directly suppresses the growth of hydrogen-producing archaea or bacteria. Alternatively, the virus can be a virus that indirectly suppresses the growth of hydrogen-producing archaea or bacteria.

[0037] In some embodiments, the composition comprises another active substance capable of suppressing in vivo methane production in ruminants, selected from brominated aliphatic compounds, carbohydrates, esters, lipids, peptides, enzymes, another virus, phlorotannins or combinations thereof. In a preferred embodiment, the composition comprises another active substance capable of suppressing in vivo methane production in ruminants, selected from bromoform, 3-nitrooxypropanol or hydroxypropionic acid or combinations thereof.

[0038] In a preferred embodiment, the virus capable of suppressing in vivo methane production in ruminants is a stabilized virus. For example, the virus is stabilized by immobilization on a substrate as described in EP 1496919 B1. This is advantageous as it ensures that the virus maintains its methane production inhibitory ability while improving its stability compared to free virus.

[0039] Viral stabilization can be achieved by binding the virus to a substrate through a covalent bond formed between the substrate and the virus. In some embodiments, the virus is immobilized through its head while keeping its tail in a free state. This ensures that the virus retains its activity because the tail of the virus is involved in the ability to recognize and infect specific bacteria and / or archaea. In some embodiments, the virus can be a bacteriophage. Suitable substrates include silica, chitosan, or animal or plant proteins. Such stabilization is carried out to enhance the stability of the virus. An example of a virus being stabilized by immobilization on a substrate through a covalent bond formed between the substrate and the virus is described in EP 1496919 B1.

[0040] Advantageously, in ruminants where methane production in the rumen is caused by specific methanogenic bacteria, the use of viruses provides a particularly effective means for targeted disruption of the methane production process. Unlike other methane inhibitors that result in a non-targeted approach and can cause toxicity to the rumen, viruses are specific to the target bacteria and / or archaea and thus have no problematic side effects for ruminants.

[0041] In a preferred embodiment, the virus is a bacteriophage that targets bacteria in the rumen, such as bacteria that produce hydrogen during fermentation, which acts as a fuel for methanogenic archaea. This is advantageous because the suppression of these bacteria has an impact on the ability of methanogenic bacteria that produce methane in the rumen, through extension.

[0042] In a preferred embodiment, the virus is a virus that targets archaea involved in methane production in ruminants. In a particularly preferred embodiment, the virus is a virus that targets metabolic processes leading to methane production. It has already been observed that the mixture of archaeal populations is different in naturally low-methane-emitting cows and high-emitting animals. Therefore, the use of viruses targeting specific archaea is advantageous, because it enables the targeting of archaea found in high-emitting ruminants without disturbing other members of the ruminant microbiota. Furthermore, the use of such viruses in the controlled-release compositions of the present invention enables the specific targeting of methane-producing microorganisms throughout the life of the ruminant. Thus, this provides a means of delivering the active substance without further human intervention.

[0043] It will be understood that viruses capable of suppressing in vivo methane production in ruminants can be isolated by those skilled in the art. For example, to isolate viruses targeting archaea, it is possible to prepare a culture of archaea in a suitable growth medium, such as 119 broth. Then, it is possible to collect a liquid from an environmental sample from a suitable virus source, such as bovine rumen fluid, rumen feces, municipal sewage, or an archaeal source that serves as an archaeal habitat (e.g., anaerobic soil containing peat and peatlands, hot springs, deep-sea hydrothermal vents). The virus can then be separated from the liquid, concentrated, and purified, for example, by PEG precipitation and ultracentrifugation. The resulting virus suspension can then be inoculated into the archaeal culture and the culture grown to allow viruses with specificity for archaea to initiate the cycle of infection and amplification. These viruses can be filtered from the culture and passaged repeatedly (i.e., repeating the previous steps with fresh archaea) to obtain pure and active virus. Following a similar procedure, it is possible to isolate other viruses, such as viruses active against other bacteria in the rumen.

[0044] Following the above procedure, a novel virus having activity against Methanobrevibacter smithii having the sequence set forth in SEQ ID NO: 1 was isolated. The isolation method is described in more detail in the Examples. In some embodiments, the nucleic acid of the virus capable of suppressing in vivo methane production in ruminants comprises at least 50% identity to SEQ ID NO: 1. In some embodiments, the nucleic acid of the virus capable of suppressing in vivo methane production in ruminants comprises at least 60% identity to SEQ ID NO: 1. In some embodiments, the nucleic acid of the virus capable of suppressing in vivo methane production in ruminants comprises at least 70% identity to SEQ ID NO: 1. In some embodiments, the nucleic acid of the virus capable of suppressing in vivo methane production in ruminants comprises at least 80% identity to SEQ ID NO: 1. In some embodiments, the virus capable of suppressing in vivo methane production in ruminants has a nucleic acid comprising at least 90% identity to SEQ ID NO: 1. In some embodiments, the virus capable of suppressing in vivo methane production in ruminants has a nucleic acid comprising at least 95% identity to SEQ ID NO: 1. In a particularly preferred embodiment, the virus has the sequence set forth in SEQ ID NO: 1.

[0045] TIFF2025523691000001.tif234152TIFF2025523691000002.tif241153TIFF2025523691000003.tif240151TIFF2025523691000004.tif240151TIFF2025523691000005.tif240150TIFF2025523691000006.tif240151TIFF2025523691000007.tif240151TIFF2025523691000008.tif240152TIFF2025523691000009.tif239152TIFF2025523691000010.tif239152TIFF2025523691000011.tif238151TIFF2025523691000012.tif239152TIFF2025523691000013.tif240154TIFF2025523691000014.tif240151TIFF2025523691000015.tif239151TIFF2025523691000016.tif239151TIFF2025523691000017.tif238152TIFF2025523691000018.tif239150TIFF2025523691000019.tif239151TIFF2025523691000020.tif239151TIFF2025523691000021.tif239152TIFF2025523691000022.tif240150TIFF2025523691000023.tif90151

[0046] The optimal concentration of a substance capable of suppressing methane production in ruminants can be determined using challenge studies that experiment with various concentrations. In some embodiments, the concentration of the virus capable of suppressing methane production in ruminants is greater than 10 2 pfu / ml, preferably greater than 10 3 pfu / ml, 10 4 pfu / ml, or most preferably greater than 10 6 pfu / ml.

[0047] In preferred embodiments, the composition, bolus, animal lick block or animal feed results in sustained release of a virus that can suppress in vivo methane production in ruminants. Thus, the composition, bolus or animal lick block provides controlled release of a predetermined dose of virus over a predetermined release period to the ruminant. Sustained release compositions, boluses or animal lick blocks are advantageous because virus delivery is provided over the entire release period, which means that methane suppression persists over the entire release period.

[0048] In some embodiments, the dose of virus is maintained constant over the entire release period. This is advantageous as it ensures that a constant level of virus is present in the ruminant. Thus, a certain amount of virus is present in the rumen, resulting in a constant level of methane production suppression.

[0049] In other embodiments, the composition, bolus, animal lick block or animal feed results in intermittent release of the virus. Thus, there are intervals during the release period when no virus is released to the ruminant. This embodiment of the invention allows the administration of the virus that can suppress methane production by the ruminant to be dispersed and separated by recovery intervals, and optionally, time is available for the ruminant to recover between administrations of the virus. In other embodiments, the composition of the present invention comprises two or more viruses that can suppress in vivo methane production in ruminants and is configured to release the viruses alternately, where each infects target archaea by attaching to the host by binding to a different receptor to prevent or overcome the development of resistance.

[0050] In some embodiments, the present invention relates to an animal lick block comprising the composition of the present invention, and also to a method for reducing methane production in ruminants, comprising providing an animal lick block comprising a virus capable of suppressing in vivo methane production in ruminants. Since animal lick blocks are commonly used to supplement ruminant feed with additional nutrients and vitamins, this is advantageous. Thus, by providing an animal lick block comprising the composition of the present invention, it is possible to administer, as appropriate and freely, an active substance for reducing methane, including a virus, to ruminants. Continuous access to the composition is made possible. The composition is then digested by the ruminant, resulting in controlled release of the active substance in the rumen over the release period.

[0051] In some embodiments, the present invention relates to an animal feed comprising a virus capable of suppressing in vivo methane production in ruminants, and also to a method for reducing methane production in ruminants, comprising providing an animal feed comprising a virus capable of suppressing in vivo methane production in ruminants. Since animal feed is used to feed ruminants, this is advantageous. Thus, by providing an animal feed comprising a virus capable of suppressing in vivo methane production in ruminants, it is possible to orally administer an active substance for reducing methane, including a virus, to ruminants during their normal feeding. The composition is then digested by the ruminant, resulting in controlled release of the active substance in the rumen over the release period. In some embodiments, the animal feed is solid or semi-solid. In a preferred embodiment, the animal feed is in the form of a suspension, where the suspension comprises a virus suspended in a liquid.

[0052] Animal feed and animal lick blocks can be stabilized by formulating (formulating) them in a composition having a high sugar content, for example, more than 40%, suitably more than 60%, most suitably more than 65%, preferably more than 68%. The high sugar concentration maintains the stability and activity of the virus. Molasses formulations are convenient sugar formulations for use in the compositions of the present disclosure.

[0053] In some embodiments, the release period is about one week. In some embodiments, the release period is about one month. In some embodiments, the release period is about one year. In a preferred embodiment, the release period is about two years.

[0054] Suitably, the composition has a release period selected according to the expected lifespan of the ruminant. Thus, in the case of calves, the release period can be 24 to 36 months, preferably 24 to 30 months. In the case of lambs, the release period can be 6 to 8 months.

[0055] In some embodiments, the present invention relates to a bolus comprising a solid or semi-solid composition, the composition comprising a virus capable of suppressing in vivo methane production in ruminants, the bolus being configured to effect controlled release of the virus to the ruminant. The bolus can be orally administered to the ruminant. Applicators for administering the bolus to ruminants such as cows are well known to those skilled in the veterinary art. The bolus can be administered to the ruminant after it has reached an appropriate size. In the case of calves, this can be when it exceeds 100 kg in weight and / or at about 2 to 3 months of age. In the case of lambs, this can be when it exceeds 15 kg in weight and / or at about 5 to 8 weeks of age.

[0056] In some embodiments, a single bolus can be administered to deliver a controlled dose over the lifetime of a calf. In other embodiments, it is possible to administer a bolus to provide a certain dose over an initial period and then administer subsequent boluses. This depends on the release period of each bolus and the desired total period over which the virus is to be delivered to the ruminant.

[0057] In some embodiments, the composition, bolus, animal lick block or animal feed is adjusted to provide a predetermined amount of virus to a ruminant. Thus, the combination of controlled release and a predetermined amount of virus allows for complete control of virus delivery to the ruminant. This ensures that an effective dose of the virus can be provided to the ruminant over the entire release period. The predetermined amount can be adjusted according to the release characteristics of the composition, bolus or animal lick.

[0058] In some embodiments, the present invention relates to a bolus comprising a solid or semi-solid composition, the composition comprising a virus capable of suppressing in vivo methane production in a ruminant, the bolus being configured to effect controlled release of the virus to a ruminant, and also to a method of orally administering these boluses to a ruminant.

[0059] In some embodiments, both the core and the shaft are soluble in the liquid found in the rumen, which is advantageous as it allows the bolus to completely dissolve within the rumen of the ruminant.

[0060] In some embodiments, the core comprises metal. In some embodiments, the core consists of metal. In some embodiments, the shaft comprises metal. In some embodiments, the shaft consists of metal. In some embodiments, both the core and the shaft comprise metal. In some embodiments, both the core and the shaft consist of metal.

[0061] In a preferred embodiment, most of the bolus is coated with a coating that prevents liquid, such as the liquid in the rumen of a ruminant, from entering the bolus. In this arrangement, the entry of liquid from the ruminant into the bolus can be carefully controlled. This is because the liquid can only enter the bolus through the uncoated portions.

[0062] When the bolus comprises a core and a shaft, in a preferred embodiment, the bolus is coated on all surfaces except the upper part of the shaft, so that fluid can penetrate the bolus only at the upper part of the shaft. This results in the release of virus from the bolus at a constant predetermined rate when liquid from the lumen penetrates into the bolus.

[0063] In some embodiments, the bolus further comprises a virus-free substance. In a preferred embodiment, a virus capable of suppressing methane production by ruminants and the virus-free substance are arranged to be released at different times. In one such arrangement, the virus and the virus-free substance are arranged in alternating layers, so that as the bolus dissolves, a certain dose of the virus is released, then a certain dose of the virus-free substance is released, and so on, resulting in intermittent release. In another embodiment, the bolus contains two or more viruses capable of suppressing in vivo methane production in ruminants and is configured to release the viruses alternately, where each infects target archaea by attaching to the host by binding to different receptors in order to prevent or overcome the development of resistance to infection.

[0064] In a particularly preferred embodiment of this arrangement, the bolus comprises alternating layers of virus and virus-free substance, so that intermittent release is provided as liquid from the lumen penetrates into the bolus. This arrangement is advantageous because the bolus provides controlled release of a predetermined dose of virus capable of suppressing in vivo methane production in ruminants, allowing its administration to be carefully controlled. In a preferred embodiment, the virus-free substance comprises one or more feed supplements such as vitamins and minerals, and / or therapeutic substances such as anthelmintics.

[0065] The bolus may include a core and a shaft extending therefrom. This arrangement is shown in Figure 1. The bolus in Figure 1 includes an exit port screen 101, a density element 102, and a semipermeable membrane 103. The bolus contains a drug 104, a placebo portion 105, and an osmotic tablet 106.

[0066] In another embodiment, the bolus can be tubular, as shown in Figures 2a and 2b. This is advantageous when a shorter release duration is required because the surface area available for product release is substantially constant in the absence of a coating. In this embodiment, the bolus can also include a member that allows for sustained release of the virus over the required release period, such as those described herein. The tubular bolus can have a uniform distribution of the virus throughout its structure or, as shown in Figure 2c, it can have layers of different substances.

[0067] In a preferred embodiment, the composition further includes one or more additional substances selected from brominated aliphatic compounds, carbohydrates, lipids, peptides, other viruses, and phlorotannins that can suppress in vivo methane production in ruminants. In a particularly preferred embodiment, the composition includes a combination of at least two viruses. The virus can be stabilized as described above. This is advantageous because archaea and bacteria have developed numerous defense mechanisms against viruses and are known to generate resistance. Therefore, in order to minimize the effects of such resistance, it is desirable for the formulation to include a cocktail of two or more active substances.

[0068] In a preferred embodiment, the composition comprises a first active substance and a second active substance, and the first and second active substances are arranged such that they are released at different times. This can be achieved by varying the viral composition in different production batches or by manufacturing a delivery composition having alternating layers of active substances. In a preferred embodiment, each active substance comprises a combination of two or more viruses. This is advantageous for further minimizing resistance by exposing ruminants to alternating cycles of different viruses.

[0069] Examples A proof of concept was achieved by isolating viruses having activity against methanogenic archaeal microorganisms and comparing in vitro methane production by archaeal microorganisms in the presence or absence of these viruses. The methods and experimental results are described below.

[0070] Methanobacterium bryantii (DSMZ 862) and Methanobrevibacter smithii (DSMZ 861) were purchased from the Leibniz Institute DSMZ - German Collection of Microorganisms and anaerobically cultured at 37 °C for 48 - 72 hours in 119 broth medium supplemented with rumen fluid in the presence of H2 / CO2 (pressure of 0.5 - 1 bar), as recommended.

[0071] Viruses having activity against Methanobacterium bryantii and Methanobrevibacter smithii were isolated from fresh bovine rumen fluid slaughterhouse specimens concentrated to final concentrations of 10% (w / v) and 0.5 M, respectively, by filtration and addition of polyethylene glycol (PEG) and sodium chloride, as described below.

[0072] First, virus particles were separated from the rumen fluid, concentrated, and purified using PEG precipitation and centrifugation. The purified virus suspension was inoculated into an archaeal culture in 119 broth, and the culture was incubated for 48 hours to allow the archaeal-specific virus to initiate the cycle of infection and amplification.

[0073] After incubation, the methane production was measured using gas chromatography - mass spectrometry (GC-MS). Cultures inoculated with the virus preparation were compared to cultures that were identical except not treated with the virus. The measurable decrease in methane production in the cultures exposed to the virus preparation confirmed the presence of active virus. Viruses released during the infection cycle were recovered from the cultures by filtration through a 0.22 μm filter that allowed separation of virus particles from archaea in the culture medium.

[0074] Subsequently, the recovered virus suspension was used to inoculate fresh cultures of the host archaeal cells as described above. This process of infection, replication, extraction, and reinfection (termed passage) was repeated 4 times and 6 times to obtain viruses herein designated as virus isolates 1 and 2, which have activity against Methanobacterium bryantii and Methanobrevibacter smithii, respectively. Cultures of Methanobacterium bryantii and virus isolate 1 that were passaged 4 times to obtain a clonal population of the virus were subjected to transmission electron microscopy. The resulting images are shown in Figure 3. Cultures of Methanobrevibacter smithii and virus isolate 2 were passaged 6 times to obtain a clonal population of the virus and transmission electron microscope images were obtained. The resulting images are shown in Figure 4.

[0075] The virus having activity against Methanobrevibacter smithii (virus isolate 2) was a novel virus having the sequence set forth in SEQ ID NO: 1.

[0076] Example 1 Virus isolate 1 containing 500 μL of rumen-derived fluid was added to a culture of Methanobacterium bryantii that was growing exponentially under the aforementioned recommended growth conditions. Heat-inactivated virus isolate 1 was added to a second culture. Also, a control culture without added virus was prepared with equivalent growth of Methanobacterium bryantii. The cultures were incubated for 48 hours, and then the methane production was measured by gas chromatography-mass spectrometry (GC-MS). The results of this experiment are shown in Table 1 and Figure 5.

[0077] [Table 1]

[0078] The data of this example show that a virus isolate having activity against methane-producing archaea can reduce its methane production in an ex vivo environment. The lack of inhibition in the heat-inactivated sample further demonstrates that an active virus is required for the inhibition of methane production.

[0079] Example 2 The rumen functions as a chamber, providing a hydrogen-rich anaerobic environment for the microbial fermentation of ingesta. This environment was reproduced with an apparatus including Hungate tubes that provide an airtight environment containing bovine rumen fluid. The rumen fluid for these experiments was obtained from animals within 30 minutes after euthanasia and used immediately. A constant H2 / CO2 gas mixture was supplied to the chamber at a ratio of 80 / 20% (v / v) each to establish the same anaerobic conditions as in bovine rumen. A fixed volume of any of the following was supplied to each flask.

[0080] (i) Heat-treated rumen fluid containing 119 broth medium, (ii) Heat-treated rumen fluid, and a live culture of Methanobrevibacter smithii in 119 broth medium, (iii) Heat-treated rumen fluid, and a live culture of Methanobrevibacter smithii in 119 broth medium, and virus isolate 1.

[0081] The heat treatment of the rumen fluid was carried out at 80 °C for 5 minutes. In each case, the majority (60%) of the fluid in the device contained the heat-treated rumen fluid. In each treatment, anaerobic incubation was carried out at 37 °C for 48 hours in the presence of an H2 / CO2 gas mixture (pressure of 0.5 - 1 bar). Subsequently, gas samples from each artificial rumen were analyzed by GC-MS to compare the methane concentrations in each artificial rumen. The results of this experiment are shown in Table 2 and Figure 6.

[0082]

Table 2

[0083] The results indicate that in experiments using the in vitro rumen model, the heat-treated rumen fluid did not produce any methane. In contrast, the rumen fluid containing a live population of archaea microorganisms resulted in methane emissions. However, this emission was measurably reduced by the presence of a viral isolate 2 that specifically infects Methanobrevibacter smithii present in the rumen fluid. Therefore, it is possible to reduce methane emissions in the rumen of cows and other ruminants by introducing the selected anti-archaeal virus.

[0084] Numbered embodiments of the present disclosure 1. A solid or semi-solid composition for administration to a ruminant, comprising a substance capable of suppressing in vivo methane production in the ruminant.

[0085] 2. A bolus comprising a solid or semi-solid composition, wherein the composition comprises a substance capable of suppressing in vivo methane production in the ruminant, and wherein the bolus is configured to effect controlled release of the substance to the ruminant.

[0086] 3. A solid or semi-solid animal lick block comprising a composition comprising a substance capable of suppressing in vivo methane production in the ruminant.

[0087] 4. An animal feed comprising a composition comprising a substance capable of suppressing in vivo methane production in ruminants.

[0088] 5. A method for reducing methane production in ruminants, comprising orally administering a bolus to a ruminant, wherein the bolus comprises a substance capable of suppressing in vivo methane production in ruminants and effects a controlled release of the substance to the ruminant.

[0089] 6. A method for reducing methane production in ruminants, comprising providing an animal lick block or animal feed comprising a substance capable of suppressing in vivo methane production in ruminants, wherein the substance is orally introduced into the ruminant when the ruminant ingests and digests a component of the lick block or animal feed.

[0090] 7. The composition or method according to embodiment 3, wherein the substance is covalently bonded to one or more components of the animal lick block.

[0091] 8. The composition, bolus, animal lick block, animal feed or method according to any one of the preceding embodiments, wherein the ruminant is a bovine, sheep, goat or elaphine animal, preferably a bovine animal.

[0092] 9. The composition, bolus, animal lick block, animal feed or method according to any one of the preceding embodiments, wherein the composition is a solid composition.

[0093] 10. The composition, bolus, animal lick block, animal feed or method according to any one of the preceding embodiments, wherein the composition is a semi-solid composition, preferably a gel or colloid.

[0094] 11. The bolus or method according to any one of the preceding embodiments, wherein the controlled release is a sustained release.

[0095] 12. The bolus or method according to any one of the preceding embodiments, wherein the controlled release is intermittent release.

[0096] 13. The composition, bolus, animal lick block, animal feed or method according to any one of the preceding embodiments, wherein the composition is adapted to release a predetermined dose of the substance to a ruminant animal.

[0097] 14. The composition, bolus, animal lick block, animal feed or method according to any one of the preceding embodiments, wherein the substance comprises an inhibitor involved in the metabolic pathway of methane production in the rumen.

[0098] 15. The composition, bolus, animal lick block, animal feed or method according to any one of the preceding embodiments, wherein the substance comprises an inhibitor of methanogenic bacteria.

[0099] 16. The composition, bolus, animal lick block, animal feed or method according to any one of the preceding embodiments, wherein the substance is capable of destroying the enzymes of the gut microbiota in the rumen.

[0100] 17. The composition, bolus, animal lick block, animal feed or method according to any one of the preceding embodiments, wherein the substance comprises a brominated aliphatic compound, a carbohydrate, a lipid, a peptide, a virus, a phlorotannin or a combination thereof.

[0101] 18. The composition, bolus, animal lick block, animal feed or method according to any one of the preceding embodiments, wherein the substance comprises bromoform, 3-nitroxypropanol, hydroxypropionic acid, a virus or a combination thereof.

[0102] 19. The composition, bolus, animal lick block, animal feed or method according to any one of the preceding embodiments, wherein the substance comprises bromoform.

[0103] 20. The composition, bolus, animal lick block, animal feed or method according to any one of the preceding embodiments, wherein the substance comprises a virus.

[0104] 21. The composition, bolus, animal lick block, animal feed or method according to embodiment 19, wherein the virus is a virus that targets a metabolic process involved in methane production.

[0105] 22. The composition, bolus, animal lick block, animal feed or method according to embodiment 20, wherein the virus is stabilized.

[0106] 23. The composition, bolus, animal lick block, animal feed or method according to embodiment 22, wherein the virus is stabilized by immobilization on a substrate.

[0107] 24. The composition, bolus, animal lick block, animal feed or method according to embodiment 23, wherein the virus is immobilized via its head.

[0108] 25. The composition, bolus, animal lick block, animal feed or method according to any one of the preceding embodiments, wherein the substance comprises a combination of at least two brominated aliphatic compounds, carbohydrates, lipids, peptides, viruses and phlorotannins.

[0109] 26. The composition, bolus, animal lick block, animal feed or method according to embodiment 24, wherein the substance comprises a combination of at least two viruses, preferably, at least two of the viruses are stabilized.

[0110] 27. The composition, bolus, animal lick block, animal feed or method according to any one of the preceding embodiments, wherein the composition further comprises an inert substance.

[0111] 28. The composition, bolus, animal lick block, animal feed or method according to any one of the preceding embodiments, wherein the composition further comprises a second substance capable of suppressing in vivo methane production in ruminants.

[0112] 29. The composition, bolus, animal lick block, animal feed or method according to embodiment 27 or 28, wherein a substance capable of suppressing methane production by ruminants and an inert substance and / or a second substance capable of suppressing in vivo methane production in ruminants are arranged to be released at different times.

[0113] 30. The bolus according to any one of the preceding embodiments, wherein most of the bolus is coated with a coating that prevents the intrusion of liquid into the bolus.

[0114] 31. The bolus according to embodiment 30, wherein the bolus includes a core and a shaft extending therefrom, and a substance capable of suppressing methane production by ruminants is arranged around the shaft.

[0115] 32. The bolus according to embodiment 31, wherein the bolus further includes an inert substance, and wherein the substance capable of suppressing methane production by ruminants and the inert substance are arranged in layers around the shaft of the bolus.

[0116] 33. The bolus according to embodiment 31 or 32, wherein the core and shaft of the bolus are soluble in the liquid found in the rumen of ruminants.

[0117] The foregoing disclosure provides exemplary embodiments of the invention and is not intended to be limiting. It will be understood that various other modifications and changes are possible to the invention.

Claims

1. 1. A controlled release bolus, solid or semi-solid animal lick block, or animal feed comprising a composition, the composition comprising a virus capable of suppressing in vivo methane production in ruminants, wherein the virus targets archaea involved in methane production in ruminants, and wherein the composition in the controlled release bolus is solid or semi-solid.

2. 1. A method of reducing methane production in a ruminant comprising orally administering to the ruminant a controlled release bolus or providing to the ruminant a solid or semi-solid animal lick block or animal feed, wherein said bolus, animal lick block or animal feed comprises a composition, wherein said composition comprises a virus capable of suppressing in vivo methane production in a ruminant; Here, the virus targets archaea involved in methane production in ruminants, wherein the composition in the controlled release bolus is a solid or semi-solid; and wherein the virus is orally introduced into the ruminant when the ruminant ingests and digests the lick block or a component of the animal feed. method.

3. 10. The animal lick block of claim 1 or the method of claim 2, wherein the virus is covalently bound to one or more components of the animal lick block.

4. 3. The bolus, animal lick block or animal feed of claim 1 or the method of claim 2, wherein the ruminant is a cow, sheep, goat or elafin animal.

5. 5. The bolus, animal lick block or animal feed or method according to claim 4, wherein the ruminant animal is a bovine animal.

6. 3. The bolus, animal lick block or animal feed of claim 1 or the method of claim 2, wherein the composition is a gel or a colloid.

7. 3. The bolus, animal lick block or animal feed of claim 1 or the method of claim 2, wherein the controlled release is sustained release or intermittent release.

8. 3. The bolus, animal lick block or animal feed of claim 1 or the method of claim 2, wherein the composition is adapted to release a predetermined dose of virus into a ruminant.

9. 3. The bolus, animal lick block or animal feed of claim 1 or the method of claim 2, wherein the virus is stabilized.

10. 10. The bolus, animal lick block or animal feed or method of claim 9, wherein the virus is stabilized by immobilization to a substrate.

11. 11. The bolus, animal lick block or animal feed or method of claim 10, wherein the virus is immobilized via its head portion.

12. 3. The bolus, animal lick block or animal feed of claim 1 or the method of claim 2, wherein the composition further comprises a substance capable of inhibiting in vivo methane production in ruminants selected from one or more of brominated aliphatic compounds, carbohydrates, lipids, peptides, other viruses and phlorotannins.

13. The composition comprises a virus that targets archaea involved in methane production in ruminants; including in combination with a virus that can suppress methane production in ruminants, 13. A bolus, animal lick block or animal feed or method according to claim 12.

14. 14. The bolus, animal lick block or animal feed or method of claim 13, wherein the virus is stabilized.

15. 3. The bolus, animal lick block or animal feed of claim 1 or the method of claim 2, wherein the composition further comprises a non-virus-containing substance.

16. 16. The bolus, animal lick block or animal feed or method of claim 15, wherein the virus targeting archaea involved in methane production in ruminants and the non-virus-containing substance are arranged to be released at different times.

17. 17. The bolus, animal lick block or animal feed or method of claim 16, wherein the non-virus containing substance is a mineral and / or an antiparasitic agent.

18. 10. The bolus of claim 1, wherein a majority of the bolus is coated with a coating that prevents liquid from entering the bolus.

19. 20. The bolus of claim 18, wherein the bolus comprises a core and a shaft extending therefrom, and the virus capable of suppressing methane production by ruminants is disposed around the shaft.

20. 20. The bolus of claim 19, wherein the bolus further comprises a non-virus-containing substance, and wherein the virus and non-virus-containing substance targeting archaea involved in methane production in ruminants are layered around the shaft of the bolus.

21. 21. The bolus of claim 19 or 20, wherein the core and shaft of the bolus are soluble in the fluid found in the rumen of a ruminant.

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