Strain megasphaera elsdenii and its application

FIELD: chemistry, biochemistry.

SUBSTANCE: invention relates to area of microbiology and biotechnology and can be applied in agriculture. Novel strain Megasphaera elsdenii is characterised by ability to utilise lactate with degree from 40% to 90% even in presence of sugars. Strain with said ability is obtained by method, which includes culturing of sample of paunch liquid. Novel strain Megasphaera elsdenii is applied in method of treatment and prevention of lactic acidosis of ruminants, in composition of veterinary preparation for treatment and prevention of said disease, in method of milk productivity increasing and in method of increasing ruminants fattening efficiency, in method of increasing growth rate and reduction of time of ruminants fattening, in method of reducing morbidity, induced by digestion disorders, and death rate among ruminants from lactic acidosis, as well as in method of increasing efficiency of conversion of ruminants diet based on concentrated forages when transferring ruminant to said diet.

EFFECT: obtaining ability to carry out wide-scale treatment and prevention of lactic acidosis.

17 cl, 3 dwg, 18 tbl

 

The technical field to which the invention relates.

The invention relates to a new strain of Megasphaera elsdenii and its application. The invention also relates to products and methods with the inclusion of this strain. The invention also concerns a feed for ruminant animals and the preparation and method of prevention and treatment of lactic acidosis in ruminants.

Prior art

Lactic acidosis

Lactic acidosis is a disorder of digestion in ruminants, which can occur with a sudden excessive consumption easy formatiruem carbohydrates, in particular, the transition from forage on high-calorie or high-calorie concentrates with a high content of starch. The disease is characterized by accumulation of organic acids, especially lactic acid in the rumen (Dawson & Allison, 1988). Studies have shown that the main cause of lactic acidosis is a strong imbalance between the number of bacteria that produce lactic acid and bacteria, utilizing lactic acid, which occurs when a sudden increase of the share of easy formatiruem carbohydrates in the diet (Slyter, 1976).

Ways to control the population of bacteria in the rumen to prevent lactic acidosis by introducing a material containing a large number of utilizing lactic acid bacteria, was proposed for de is atility, but never practiced on a large scale. Increase utilization of lactate in the rumen were carried out by the introduction of fluid from the rumen pre-adapted animals (Allison et al., 1964; Braun et al., 1992) and the introduction of pure or mixed strains of bacteria that utilize lactate (1251483, Wilker et al., 1971; 3857971, Abdo & Cahilly, 1974; 4138498, Das, 1979; 5380525, Leedle et al., 1991; Hession & Kung, 1992; Robinson et al, 1992; Wiryawan & Brooker, 1995).

Some of these feed additives containing strains of live bacteria were patented (1251483, Wilker et al., 1971; 3857971, Abdo & Cahilly, 1974; 4138498, Das, 1979; 5380525, Leedle et al., 1991), but little or not found commercial application. In three of these patents (1251483, Wilker et al., 1971; 3857971, Abdo & Cahilly, 1974; 4138498, Das, 1979) culture was obtained in the fermenters for continuous cultivation during the initial inoculation their scar fluid. However, animal donors were not necessarily adapted to the diet with a high content of concentrates. Moreover, there is no information about the pH tolerance of these crops. In another patent (5 380 525, Leedle et al., 1991) cultures were isolated at a pH of 5.3, directly or indirectly, after enrichment of ruminant animals that are adapted to a diet with a high content of concentrates.

The incidence of subacute and acute acidosis in dairy cattle

Subacute rumen acidosis is a common and serious health problem and production in the dairy industry is nasty, since dairy cows are usually fed with feed containing a large amount of grains. Subacute and acute rumen acidosis are just different degrees of the same problem. Acute rumen acidosis occurs in a more severe form, and can be seriously impaired physiological functions. Sick animal is depressed, usually ataxite, does not accept food, pupils dilated and heartbeat casino. May cause diarrhea, the animal may fall and die within 2-5 days after lesion (Nordlund, 1995). Acute acidosis is characterized by a sharp decrease in rumen pH (≤5,0), a strong increase in the concentration of lactic acid and a strong decrease in the number of protozoa (Nocek, 1997).

Symptoms of subacute rumen acidosis is very different from the symptoms of acute acidosis. Modern economic system group content and group feeding dairy cattle make it difficult to identify these symptoms as individual cow with such problems is usually not noticeable within the group. In herds with subacute rumen acidosis manifested some or all of the following symptoms: laminated, alternating diarrhea, poor appetite or cyclic food intake, high rates of rejection in the herd on a vaguely-defined health problems, poor body condition, despite adequate energy intake, AB the processes for no apparent reason and hemoptysis (coughing blood) or epistaxis (bleeding from nose). Most of these symptoms are secondary due to acidosis and most of them do not appear within weeks or months after the onset of acidosis. Unlike beef (fed) cattle, dairy cows kept in for several years, so the control of acidosis, it is important to increase profits.

Chronic laminitis, apparently, is the most sustainable clinical signs in the herd with subacute rumen acidosis. Despite the fact that the relationship between acidosis and laminitis is not entirely clear, it is recognized clinically and demonstrated in research experiments (Kelly & Leaver, 1990; Manson & Leaver, 1988; Nocek, 1997). In addition, the majority of farmers, veterinarians and nutrition specialists tend to underestimate and even to tolerate abnormal occurrence of a condition known as laminitis and lameness in herds of dairy cows. Examination in Minnesota showed that the average frequency of lameness is 15% with a range of 0-33% (Nordlund, Garret & Oetzel, 1995). Studies in Europe have shown that lameness is the third on the cost of treatment in dairy cows after mastitis and reproduction (McDaniel & Wilk, 1989). Thus, treatment of acidosis is of great importance.

The main symptom of subacute acidosis is reduced feed intake and reduced efficiency of milk production. Pedastry the acidosis, due to difficulties in diagnosis, usually ignored, because other problems, such as poor management, poor quality feed, etc. cause large losses for many farmers, therefore, he is omnipresent, especially in high producing dairy herds.

Due to the high frequency of nutritional and metabolic disorders among high producing dairy cows, nutritional approaches to improve performance by using a grain-based feeds are focused on the prevention of dysfunction scar by controlling the production of acid or encourage more efficient growth of microorganisms. Currently an important role in this respect play a feed additive (Hutjens, 1999). Applying the cultural strains of yeast, specifically stimulating the growth of bacteria, utilizing lactic acid is of great interest, and a recent survey showed that yeast culture used in 33% of highly productive herds of Wisconsin. The results of various studies indicate that the strain Yea Sacc 84170 particularly well suited to modify to fermentation in the rumen and animal productivity, when it is used in silage with a high level of lactate in the feed with a high content of nutrients (Dawson, 1995). The results on productivity, however, is not very stable. In the USA the value is ü add yeast is 4-6 cents per cow per day (Hutjens, 1999). The ionophores, due to their ability to prevent the growth of important producers of lactic acid may also play a role in the fight against subacute acidosis. Although their cost is relatively low (1-2 cents per cow per day, Hutjens, 1999), there is some resistance to the use of ionophores because of several recent cases of toxicity ionophores. In addition, the ionophores are not registered in the United States for use in feed for dairy cattle.

Experimentally established that some bacteria are promising as a direct fed microorganisms (DFM) for ruminants, but they did not find commercial applications for several reasons. For example, Megasphaera elsdenii (ME) is the primary disposal of lactate in the body adapted rumen of cattle consuming forages with high grain content. In the transition from forage to the diet with a high content of concentrates a number of ME is often not sufficient to prevent lactic acidosis. Kung and market Church (1995) showed that adding ME strain 159 prevents the accumulation of lactic acid when fed well fermentable carbohydrates. Robinson et al. (1992) showed that the addition of another strain ME (A) prevents lactic acidosis castrated bulls.

Despite the fact that the costs associated with subclinical rumen acidosis, it is difficult determine the shape, possible costs in the dairy industry is huge (Hall, 1999). By conservative estimate Donovan (1997), the cost of subclinical acidosis in dairy industry in the U.S. range from 500 million to 1 trillion. dollars a year.

Elsden and Lewis (1953) first described a large, strictly anaerobic, gram-negative, producing fatty acids stationary cocca isolated from the rumen of sheep. However, the initial isolate was lost and has not been described in detail phenotype. After a few years Elsden al. (Elsden et al., 1956) identified the body, reminiscent of the original strain of the contents of the rumen of sheep. Characteristics of this organism did not coincide with the description of any known species, but in view of the studied small numbers of isolates, the authors refrained from attributing it to a new species and genus, and called it LC. Gutierrez et al. (1959) observed similar body scars bloated cattle and concluded that it meets the definition of the genus Peptostreptococcus, proposing to establish a new kind P.elsdenii. Subsequently, Rogosa (1971) showed that the isolates of type LC gram-negative and therefore should not be included in the genus Peptostreptococcus. He proposed to move P.elsdenii in a new genus Megasphaera and a new combination M.elsdenii, isolate LC1 according Elsden et al. (1956) as a model strain. M.elsdenii is simply gone anaerobic, which occurs mainly in the rumen of young people who's Pets and animals, receiving feed with a high content of concentrates, in which the fermentation of lactate pronounced. This organism is also sometimes was isolated from the feces of a human (Sugihara et al., 1974), and he sprayway lactate mainly to butyrate, propionate, isobutyrate, valerate, CO2H2and sometimes trace amounts kaproata (Stewart and Bryant, 1988). Because the microorganism M.elsdenii not subject to catabolite repression by glucose or maltose as Selenomonas, which also utilizes lactate and occurs in the rumen, its contribution to the catabolism of lactate is particularly improved when fed soluble carbohydrates (Stewart and Bryant, 1988).

In U.S. patent 3956482 (Hahn et al., 1976) described a method of increasing the milk production in ruminant animals, including the introduction in the rumen of dairy cows microorganisms, producing acetate, consisting of a mixture of 0-4% M.elsdenii, 30-42% Streptococcus bovis, 3-10% Lactobacillus acidophilus, 12-20% Bifidobacterium adolescentis, 18-44% of Bacteroides ruminicola and 3-12% of Butyrivibrio fibrisolvens, cultivated and adapted to the nutrient medium.

The main disadvantage of the invention disclosed in the above patent, is relatively high content (30-42%) Streptococcus bovis, which together with Lactobacillus is the main cause of lactic acidosis in ruminants. In addition, this mixture contains relatively few M.elsdenii (0-4%), and the introduction of this mixture is more likely to increase or cause lactic acidosis rumen, and not to prevent the Li to cure it. Moreover, the mixture is exposed to the atmosphere, so that most of M.elsdenii dies. Furthermore, the mixture of microorganisms is much more difficult to control than pure culture.

In U.S. patent 4138498 (Das, 1979) described feed additive for the introduction of the ruminant animal to prevent or minimize lactic acidosis while translating them to forage on starch, which includes bacterial strain M.elsdenii mixed with palatable feed additive. M.elsdenii - strict gone anaerobic, so the lack of feed additives disclosed in this patent, in addition to the disadvantages set forth below, is that M.elsdenii is exposed to the atmosphere, which leads to a rapid decrease in the number of viable cells in addition.

In U.S. patent 5380525 (Leedle et al., 1991) described a biologically pure culture M.elsdenii NRRL-18624 and its use in facilitating the adaptation of ruminants from forage or pasture to high-calorie food, enriched with starch. This culture suffers from the disadvantages set forth below.

In U.S. patent 5529793 (Garner et al., 1996) described a mixture of bacteria that produce lactic acid and bacteria that utilize lactate, type M.elsdenii in the dry mixture or in the diet of fattening animals to improve feed utilization by ruminants animals. The disadvantage of this invention is that M.elsdenii very strict gone anaerobic, is therefore its use in dry food leads to the death of most cells.

The applicants have assessed the above strains M.elsdenii and came to the conclusion that they are, in General, are not suitable for commercial applications and large-scale prevention of lactic acidosis in ruminants due to the following disadvantages of these strains, namely that they:

- not highly active and has not adapted to the proliferation in the rumen of animals on a diet with a high content of concentrates;

- not capable of proliferation at relatively low pH values less than 5.0 and even 4.5, which is defined as acute acidosis;

- not resistant to antibiotics, the ionophores usually added to the diet of fattening;

- not able to preferred use of lactate as a carbon source even in the presence of soluble carbohydrates, such as glucose and maltose.

Other disadvantages of these strains are that they:

- have a relatively low growth rate, i.e. less than 0,938 h-1;

- do not have the ability to grow on reducing sugars, and lactate;

- have a relatively low rate of accumulation of biomass, i.e. less than 0.39 g(l×h)-1;

- not resistant to ionophores;

mainly produce propionate and butyrate, but not acetate.

The purpose of the invention

Thus, the aim of the present invention is to obtain new strains M.elsdenii and their application, and t is the train medicines and methods on the basis of the isolated strains, with the help of which the above disadvantages can be overcome or at least minimized.

The invention

In accordance with the first aspect of the invention provides a strain M.elsdenii (isolate CH4), deposited at NCIMB, Aberdeen, Scotland, UK under number NCIMB 41125.

In accordance with the second aspect of the invention provides a strain M.elsdenii that has almost the same sequence of 16S ribosomal RNA, as in strain M.elsdenii deposited at NCIMB, Aberdeen, Scotland, UK under number NCIMB 41125.

Strain M.elsdenii by the first and second aspects of the invention are additionally characterized by:

- capacity utilization of lactate effective even in the presence of sugars;

- resistance to ionophores;

the relatively high growth rate;

- ability to preferential production of acetate and

- ability to proliferation at relatively low pH values less than 5.0 and even 4,5.

In accordance with a third aspect of the invention provides a composition for facilitating the adaptation of ruminants from a diet based on roughage to high-calorie diet based on the concentrates, and the composition consists mainly of bacterial strain according to the first or second aspect of the invention.

In accordance with the fourth aspect of the invention provides a method of facilitating adaptati the ruminant animal diets based on forages for high-calorie diet based on concentrates, which includes a step of introducing into the rumen of these ruminant an effective amount of a bacterial strain according to the first or second aspect of the invention.

In accordance with the fifth aspect of the invention provides a feed additive for ruminants, comprising a carrier and an effective amount of a bacterial strain according to the first or second aspect of the invention.

Preferably the culture is placed in an anaerobic container.

In accordance with the sixth aspect of the invention provides a method of treatment of lactic acidosis rumen and prevention of one or more of the following, namely: lactic acidosis rumen, Romanita, a condition known as laminitis caused by lactic acidosis rumen, bloating and liver abscesses caused by lactic acidosis rumen, which includes a step of anaerobic introduction in the rumen some animal an effective amount of a bacterial strain according to the first or second aspect of the invention.

In accordance with the seventh aspect of the invention provides a veterinary medicine for the treatment of lactic acidosis rumen and prevention of one or more of the following, namely: lactic acidosis rumen, Romanita, a condition known as laminitis caused by lactic acidosis rumen, bloating and liver abscesses caused by lactic acidosis rumen, which includes an effective amount of a bacterial strain according to the first or second TSA is KTU invention.

In accordance with the eighth aspect of the invention provides a drug for the treatment of lactic acidosis rumen and prevention of one or more of the following, namely: lactic acidosis rumen, Romanita, a condition known as laminitis caused by lactic acidosis rumen, bloating and liver abscesses caused by lactic acidosis rumen in ruminants, which includes:

- inoculate the bacterial strain according to the first or second aspect of the invention, and

- separate environment for anaerobic cultivation;

moreover, the drug components are located in separate compartments of the anaerobic container, which anaerobic are connected to each other so that it was possible to inoculate the anaerobic environment of this culture.

In accordance with the following aspect of the invention provides a method of achieving one or more of the following improvements in ruminants, namely:

- increase the production of milk;

- improve the efficiency of fattening;

- increase in the rate of growth;

- reduce the time of fattening;

- reduce morbidity and mortality from digestive disorders;

- reduce the number of cases of lactic acidosis and related diseases;

- improve the efficiency of processing food and

- the ability to feed on a diet with a relatively high content of concentrates;

which includes article is Dios introduction in the rumen some animal an effective amount of a bacterial strain according to the first or second aspect of the invention. Preferably the culture is introduced anaerobic.

In accordance with the following aspect of the invention provides a method of isolating biologically pure culture more valuable rumen microorganism in a relatively shorter period of time than conventional methods, which involves the following stages:

- a sample of fluid and scar

- cultivation of the sample at a given nutrient medium;

moreover, the method is characterized by the fact that pre-select a few parameters selected from the group comprising the composition of the nutrient medium, the degree of dilution, pH, temperature, antimicrobial agents, gas environment, redox potential, nutrient deficiencies, and inciting organisms, so that they favored the more valuable the rumen microorganism to the detriment of less valuable to the rumen microorganisms.

Hereinafter the invention will be described in more detail by the following examples and the accompanying drawings.

Brief description of figures

Figure 1. A graph of the growth rate utilizing lactate organisms at different pH values.

Figure 2. A graph of the growth rate (h-1) utilizing lactate isolates on glucose medium at different pH values.

Figure 3. Phylogenetic tree M.elsdenii of the present invention.

Evidence for the efficiency of carrying out the invention

In accordance with the present invention organisms capable of utilizing lactic acid, was isolated directly from ruminant animals that are adapted to a diet with a high content of concentrates. The goal was the selection of cultures that has the best combination of characteristics for use as inoculum, amenable to mass cultivation and storage, for prophylactic and/or therapeutic treatment of lactic acidosis.

In order to utilize lactate bacteria were effective, they must be highly active and adapted to breeding in the rumen of animals on a diet with a high content of concentrates. Organisms must be able to reproduce at pH values lower than 5.0. Selected isolates should also be resistant to antibiotics-ionophores, which are usually added to the diet of fattening. Lactate should be used primarily as a source of carbon even in the presence of soluble carbohydrates, such as glucose and maltose, which are usually found in large quantities in the feed with a high content of concentrates.

Methods

1. Animals used in the selection

Samples were taken of the contents of the rumen of animals that have passed the preliminary selection for utilizing lactate bacteria, namely: dairy cows with fistula in the Department of nutrition of milking the x cows Council for agricultural research (Irene, South Africa), as well as beef cattle Chalmar Beef (Pretoria, South Africa), which was scored at the end of the fattening period. All animals were adapted to the diet with a high content of concentrates, which increases the amount of naturally occurring bacteria that utilize lactate.

2. Sampling and sample preparation

Samples of the rumen contents of cows were taken around 9:00, after the cows fed and milked. Samples of the rumen contents of meat animals received 15-30 min after slaughter of animals. Plastic bottles with screw caps were filled to capacity scar fluid filtered through two layers of cheesecloth. Scar fluid is transferred directly into the fermenter.

3. pH-auxostat

System for flow-through culture Bioflo 1 firm, New Brunswick Scientific has been modified in the pH auxostat by transformation supports the pH of the pump in the pump environment. The pH was measured using pH-electrode Schott S23158 connected to the pH meter and the titrator model 302 by Digital Data Systems. When the pH was increased above the specified value was added weakly buffered medium to the desired value. The working volume of the vessel for culturing was 270 ml Maximum degree of dilution for a given organism under cultivation in auxostat is a measure of the maximum growth rate this is the body under these conditions.

4. Selection utilizing lactate bacteria of the rumen using auxostat

4.1 the cultivation Conditions and the environment

Filtered scar liquid used for the initial filling of the fermenter (270 ml) and activated titrator for adding sterile environment (environment 1) in culture in proportion to the increase in pH of the culture. Wednesday 1 represented the environment is partly a certain composition without scar fluid containing: Na-lactate (70%), 10 g/l; peptone, 2 g/l; KH2PO41 g/l; (NH4)2SO4, 3 g/l; MgSO4·7H2O, 0.2 g/l; CaCl2·2H2O, 0.06 g/l; vitamins (paradoxethereal, 4 mg/l; pyridoxamine, 4 mg/l, Riboflavin, 4 mg/l, diaminohexane, 4 mg/l nicotinamide, 4 mg/l; D-calcium Pantothenate, 4 mg/l; 4-aminobenzoic acid, 0.2 mg/l; Biotin, 0.2 mg/l; folic acid 0.1 mg/l; cyanocobalamin, 0.02 mg/l); Na2S·9H2O, 0.25 g/l; cysteine, 0.25 g/l; antifoaming additive, of 0.07 ml/l and monensin, 10 mg/l Na-lactate and the solution of minerals introduced into the tank titrator and autoclaved for 60 minutes Peptone was dissolved in 300 ml of dist. H2O and autoclaved separately in the flask to 1.0 l of the firm Schott with drainage at the bottom, equipped with a glass pipe Quick-fit. A solution of vitamins sterilized by filtration before use, as both reductant. After autoclaving through the tank titrator missed anaerobic gas t is the significance of the night and added the other components separately after cooling. The pH value was brought to the desired value using 5N HCl.

Started flowing cultivation until then, until he observed a pure culture under the microscope. Selected sample from the fermenter sterile syringe, which is hermetically closed and transferred to an anaerobic Cabinet (Forma Scientific, model 1024). One drop of culture was stehovani on a Petri dish containing 2% agar in the medium 1. Then incubated at 39°C during the night and carried a single colony using a sterile needle and syringe in a fresh environment 1, located in the vial 30 ml After incubation at 39°C for 24 h, the culture was transferred into several Kosyachkov containing medium 1, and incubated over night. These Kosyachkov kept in liquid nitrogen for long term storage.

4.2 the Rate of growth of isolates in the closed volume of the fermenter

The rate of growth of the isolates was tested by the method of cultivation in closed volume and measuring the increase in optical density over time. Put on the schedule of values of the natural logarithm of optical density (OD) depending on time and used the linear portion of the graph to determine the slope, which represents the maximum growth rate of the organism. Determination of the rate of growth in a confined space spent in chemostate on the culture that bred sterile environment to obtain a highly diluted is Uspenie culture, and stop feeding environment, starting the cultivation in a confined space. The advantage of cultivation in chemostate for this work is the lack of lag-phase, since all cells are viable and adapted to this environment.

4.3 Analytical methods

Volatile fatty acids were determined by gas chromatography on a gas chromatograph Carlo Erba GC4200 with a flame detector and a column Tupelo 1-1825 (Supelco Inc., Bellefonte PA, USA). Working conditions: carrier gas - nitrogen, gases, flame of hydrogen and air, the column temperature is 175°C, injector temperature of 200°C. For integrating peaks used a data processing system Barspec (Barspec Systems Inc., Rehovot, Israel). As an internal standard was pavlikova acid. Utilization of D - and L-isomers of lactate was determined enzymatically (Test set combination 1112 821, Boehringer Mannheim Gmbh, Mannheim).

5. Isolation of bacteria by the method of sieving on cups

5.1 Cultural environment

Lactate medium incubated with scar fluid (IRFL) allocation method of sieving on cups consisted of 400 ml incubated clarified scar fluid (Olumeyan et al., 1986) fed alfalfa sheep, 371 ml of distilled water, 2 g of peptone (Merck), 15 g agar, 100 ml of 10% solution of D,L-sodium lactate, 100 ml of 0.04% solution of bromocresol Magenta, and 25 ml of minerals containing 40 g/l KH2PO4, 120 g/l (NH4)2 SO4, 8 g/l MgSO4·7H2O and 2.4 g/l CaCl2·2H2O. Lactic acid (90% wt./volume) used for setting the pH to 5.5 before autoclaving at 121°C for 25 minutes After sterilization, the medium was cooled in a water bath at 50°C with the transmission of an anaerobic gas mixture. Aseptically added to 2 ml of each of the reducing agents: Na2S·9H2O (12,5% wt./volume) and cysteine·HCl·H2O (12,5% wt./volume). Because the environment IRFL not completely selective for utilizing lactate bacteria included bromocresol purple for best detection. When disposing of lactate there is a change in the ionic balance in the immediate vicinity of the colony, which causes an increase in pH. An increase in pH over 6.3 manifests itself as a change in color from yellow to purple in the concentric area of culture.

The acid resistance was determined on the cups with IRFL-agar at initial pH values of 4.4, 5.0 and 5.5 to.

Resistance to ionophores tested on cups with IRFL-agar containing 10 ppm of ionophores, commonly used in feed with a high content of concentrates, namely: monensin (Sigma) and lasalocid (Sigma). The repression utilization of lactate soluble sugars tested on cups with IRFL-agar containing maltose or glucose at a final concentration of 10 g/L. of a Positive result, i.e. purple concentric zone around the colony indicates NATO the speed of separation of the grounds due to the utilization of lactate exceeds the rate of formation of acids from added sugar. The isolates were also skanirovali on agar medium IRFL without lactate, but with the addition of maltose or glucose at a concentration of 10 g/l to determine the utilization of these two sugars.

The rate of growth on maltose and glucose were determined in environments similar to the environment of the SDL, but in which lactate was replaced by glucose or maltose at a concentration of 10 g/L.

5.2 Selection and screening method of sieving on cups

Samples for selection by the method of sieving on cups bred (Mackie & Heath, 1979) in an anaerobic Cabinet. Prepared cups for sieving environment IRFL with dilutions from 10-4up to 10-6and incubated anaerobically at 39°C. After 24 hours, the individual colonies with purple area was transferred into a liquid medium IRFL in microtubes 1.5 ml Inoculated microtube, showing the colour change to purple within 16 hours, was skanirovali on acid resistance, resistance to ionophores, repression by catabolites and utilization of glucose and/or maltose. Screening was performed by the method of printing (Lederberger & Lederberger, 1952) using multi-point needles for sowing, inocula 20 isolates on a set of 9 agar cups of different composition described above.

5.3 Determination of the rate of growth

The growth measure is whether, in the triple repetition, in the environment of the SDL, SDG or SDM to increase turbidity at 578 nm. Between measurements the vials were incubated in a water bath at 39°C. the Measurements were continued up until the turbidity was not reached its limit when a satisfactory correlation with the biomass. Inflicted on the graph of the natural logarithm of the optical density (OD) depending on the incubation time. The slope in the exponential growth phase, represents the specific growth rate was calculated by the method of linear regression using the software package for tabular calculations.

After this culture, cultivated on the environment of the SDL at pH 5,7, and incubated another 24 hours, and then took 9 ml storage by adding 1 ml of 10% (wt./volume) of NaOH for the analysis of the resulting end-products and disposal of isomers of lactate.

5.4 Physiological study of the growth of isolates of cups

Description of the fermenter. Started running system of cultivation with three fermenters with a capacity of about 250 ml each. Used the same peristaltic pump for feeding the medium with different speeds in three of the fermenter. The temperature of the cultures was maintained at 39°C. Through a medium and fermenters to pass 100% CO2to maintain anaerobic conditions. The pH of the cultures was maintained at a pH of 5.5 by the addition of 20% phosphoric acid as needed. The degree of dilution is to be ivali 70%, 80% and 90% of maximum growth rate. From each fermenter in a stationary state was collected aseptically sample in 80 ml of this sample was determined by dry weight of cells and residual lactic acid in the environment. The rate of accumulation of biomass as the product of dilution, and biomass at steady state was calculated by the actual values of the degree of dilution and dry mass. The yield coefficient of biomass, which is a function of the biomass concentration in the stationary state on the number of utilized substrate, was calculated as the residual values of lactic acid and dry matter.

6. Experiments on sheep to assess the ability of the isolate CH4 to prevent the accumulation of lactic acid in the rumen

6.1 Methods

In the first experiment 12 castrated rams catheter in the rumen (average live weight of about 40 kg) were divided randomly to experimental and control groups, each of which consisted of 6 animals. All animals were fed unlimited coarse fodder 21 days. On the 21st day they were starving 11 hours, after which they were given 1000 g corn feed and at the same time, was injected directly into the scar in 300 g of maltose syrup. After 1 hour, all animals consumed the corn was injected directly into the scar. Immediately after this, the animals of the experimental group was introduced into the rumen of 1×1011CoE CH4, while alive who tym control group was administered the same amount of cell-free filtrate from the preparation of CH4, i.e. without CH4. To determine the concentration of lactic acid in the rumen every 2 hours, taking samples of fluid from the rumen for 12 hours after a dose.

In the second experiment examined another group of 12 castrated lambs with the catheter in the rumen (average live weight 29 kg) without pre-feeding concentrate. They had free access to chopped hay from Eragrostis teff and protein-mineral mix. Lambs were divided randomly into two groups of 6 animals, i.e. experimental and control groups. On the first day of the experiment (day 1) all animals received in an unlimited number of the following ration: corn, 888; molasses, 69; urea, 17; limestone, 11; dicalcium phosphate, 6; salt, 4; ammonium sulfate, 4; mineral and vitamin premix with monensin, 1 (g/kg SV). On the first day of the transition concentrate on each animal experimental group was introduced into the rumen dose CH4 at 12:00, i.e. after 3 hours after feeding. Animals of the control group was administered the same amount of water. To determine the concentration of lactic acid samples from the rumen were taken at different times prior to the shift in concentrate (day -1) and on the 1st, 2nd, 3rd and 7th day after upgrading to concentrate.

7. Assessment isolate CH4 in high producing dairy cows

7.1 Cultivation utilizing lactate organisms experience with animals

The Braun fermenter patch BIOSTAT is with a working volume of 10 l was converted into a chemostat using a metering pump Watson-Marlow 505S, equipped with drive at 55 rpm, for supplying sterile environment of the 50-liter barrels made of stainless steel. The working volume was maintained constant by continuously feeding an excess of culture from the fermenter when exceeding the level of 10 l through a submerged tube using a peristaltic pump (Watson-Marlow 505S) in polypropylene 50-liter bottle, standing in the refrigerating chamber. The performance of this gathering of the pump was approximately 120% relative to the feeding environment of the pump. Excess volume that is output from the fermenter, consisted of anaerobic gas from the head space of the environment.

For concentration of the culture was used tangential filtration system (Millipore Pellicon)equipped with a Millipore filter HVMP on 0.45 µm (15 square feet) and peristaltic pump Millipore Masterflex Easy-Load.

For cultivation used the environment CSL4. The solutions of vitamins, reducing agents, minerals and trace elements were sterilized by filtration before adding to the capacity of the environment. After autoclaving the container was filled with anaerobic gas.

The production process was organized on a rolling basis. Were carried out two series of products, each of which gave enough cells for introduction of the group of animals on day 1. The number of stages of concentration was limited to one, is as a daily product obtained was collected in a 50-liter capacity. The dilution ratio was 0.4 h-1and "simple" between the parties was 50 minutes Before the first day of production was launched back cycle to 45 litres, which also contributed to the shift of culture in chemostat in the stationary state.

7.2 Experimental animals

Sixty high-yield dairy cows were divided into groups according to milk production during early lactation and body weight, and then within each group are randomly conducted one of the following experiments: 1) control diet containing 60% concentrates; 2) control diet containing 60% concentrate +CH4; 3) control diet containing 70% concentrates; 4) control diet containing 70% concentrate +CH4. Cows were injected dose of the organism CH4 at the hotel, after 10 days and 20 days after calving.

Registered the following options:

1) the daily intake of dry matter;

2) daily milk production;

3) the content of fat, protein and lactose in milk weekly, and

4) the body weight and General condition of the points on a monthly basis.

8. Statistical analysis

Data were analyzed using analysis of variance for completely random split into groups using the 5 subregions and districts. Data on milk production during the lactation was used as a covariant control is, and milk production expressed compared to the control. To determine significant differences when different impacts compared the results of the following pairs of experiments:

- +CH4 or CH4 (dose or no dose)

- The control diet consisting of 70% concentrates, against a control diet consisting of 70% concentrate +CH4.

- The control diet consisting of 60% concentrates, against a control diet consisting of 60% concentrate +CH4.

The differences were considered significant when p<0.10 value p<0,15 took up with the trends, unless otherwise specified.

9. The impact of adding Megasphaera elsdenii on health and performance of fattening animals

A homogeneous group of 448 excommunicated castrated bull breed Bonsmara (initial average live weight 215 kg) were divided randomly into 8 experimental groups according to plan factorial experiment 2×2×2 with the following factors: (1) adding CH4 (Yes or no); (2) adding ionophore (Yes or no) and (3) content of forage (high or low). Used the following rations and modes of feeding.

The ingredients of the experimental diets at the end of the adaptation period (from the 14th day to the end)
Ingredients Inclusion (% in total aft)
The high content of roughageLow content of roughage
Hay Eragrostis8,02,0
Corn flour27,030,0
Corn chopped32,035,0
Feeding molassesto 12.0to 12.0
Brewer's grain6,06,0
Wheat bran10,010,0
Cotton cake2,02,0
Urea1,0 1,0
Limestone1,51,5
Sol0,50,5
Adaptive power mode after the start of the experiment, when animals were given unlimited roughage before the transition to the final ration of fattening
DaysAdding hayThe amount of roughage (%) in the diet
High contentLow content
1-2Unlimited1812
3-4no1812
5-7no/td> 148
8-10no126
11-13no104
14 to the endno82
The nutrient composition of the final fattening ration (% in total aft)
NutrientsThe high content of roughageLow content of roughage
Dry matter89,689,8
Crude protein14.4Vthe 13.4
Starch 33,535,9
NDF fiber (neutral cleansing fiber)26,923,9
ADF-fiber (acid-cleansing fiber)9,58,7
Fats4,64,8

Animals were kept in small experimental pens for fattening. Each experience was 7 pens of 8 animals each. The animals were fed once daily in the morning in unlimited quantity. All bulls have passed a standard treatment upon admission to the shelter and gave them only long staple food for several days before they were injected CH4 (experience), or the same amount of water (control). Each experimental animal was poured into a disposable oral cavity 200 ml suspension of CH4 in the environment.

Culture CH4 received, inocula in the amount of 17.5 l sterile environment CSL6 (initial pH 5,20) 1000 ml fresh inoculum of CH4, which was pumped directly from the fermenter into the bottle and incubated overnight at 39°C. After the cultivation, the pH value of the culture was 6.63 and remained at the same level after 48 hours. Counting cells in culture CH4 was performed after incubation. Used a peristaltic pump to supply a dose of 200 ml in the oral cavity of the animal for 10 seconds out of the bottle 20 L.

Wednesday CSL6 for cultivation CH4 in the bottle 20 l
Wednesday CSL6 (sterilization 55 min)17.5 l
Sodium lactate971,25 g
The Indigo Carmine17,5 ml
Trace element solutionis 8.75 ml
The solution of minerals 5of 87.5 ml
Peptone17,5 g
Yeast extract17,5 g
Corn extract CSL598,5 g
Distilled water10688,2 g
10N KOH (dissolved in 5 l of water)58,3 ml
Components, sterilized by filtration and added before inoculation
A solution of vitamins35 ml
L-cysteine35 ml

Each pen was determined feed intake (daily/weekly), and for each animal, the weight (weekly or biweekly). The data were used to calculate feed conversion ratio (pen). Animals were examined daily, and if an animal showed signs of acidosis (diarrhea, bloating, depression), he was taken out of the corral and were subjected to the treatment, and then immediately returned to the appropriate corral.

Statistical analysis

Data were analyzed using the program 5 subregions and districts. Animals were divided into groups by weight. Examined the influence of CH4, ionophore and level of roughage in the format of a factorial experiment in a 2×2×2 method of analysis of variance (ANOVA). Data were acceptable normal distribution with homogeneous variance across groups. Mean values in the groups were compared using a protected Fisher's exact test, while taking the least significant difference (LSD) at the 5% level under the condition that the F-test in accordance with the ANOVA was significant at the 5%level.

10. Identification of isolates phylogenetic methods based on sequences of 16S genes pPHK

10.1 Isolates of bacteria and culturing conditions

Isolates M.elsdenii CH4 and SN initially allocated ismalone cows (Wiederhold, 1994), were provided by the authors. Typical strain M.elsdenii ADS 25040 was obtained from the American type culture Collection. Strains were grown on medium SDL, as described previously, and presumably identified as Megasphaera elsdenii (Wiederhold, 1994).

10.2 Amplification and sequencing of the genes 16S ribosomal RNA

Genomic DNA was isolated from bacterial cells using standard methods (Ausubel et al., 1988). Primers for the amplification of genes 16S pPHK were selected from a universal conservative for all eubacteria plots (table 1). PCR was performed using primers FD1 (covers provisions 8-26) and R11 (position 1384-1400). All provisions of the targets of the primers used for amplification and sequencing are on the numbering system for E. coli (Brosius et al., 1978). The reaction mixture for PCR contained 100 ál of approximately 200 ng DNA, 1 μm each primer, 200 μm of each nucleotide (dATP, dCTP, DSTF and dTTP), 50 mm KCl, 10 mm Tris-HCl (pH 8,4), 2.5 mm MgCl2 and 2.5 units of Taq polymerase (Boehringer Mannheim, Germany). The mixture was covered with mineral oil to prevent evaporation. The temperature profile consisted of 30 cycles: denaturation 1 min at 94°C, annealing for 2 min at 45°C and subsequent elongation for 3 min at 72°C amplifier (Hybaid, UK). Final elongation was performed for 6 min at 72°C. the homogeneity of the amplicons were analyzed by electrophoresis in agarose gel is (Sambrook et al., 1989). The PCR product was cut from the gel and purified using the set Wizard PCR Preps (Promega, USA) according to the manufacturer's instructions. Direct sequencing of double-stranded PCR amplicons and subsequent separation of the reaction products of the sequencing polyacrylamide gel mainly carried out by the method Dorsch and Stackebrandt (1992). Primers for sequencing are listed in table 1.

Table 1
The primers used for amplification and sequencing of the gene 16S pPHK. Sequences of primers have been published previously (Dorsch and Stackebrandt, 1992; Lane et al, 1985; Stackebrandt and Charfreitag, 1990; Hutson et al., 1993). The combination of these primers in General covers 1419 nucleotides of the gene 16S pPHK.
PrimerThe position of the target primerandThe sequence of primer (5'-3')
Reverse (antisense)
R11 (PCR)1384-1400CGGTGTGTACAAGGCCC
R11931174-1192CGTCATCCCCGCCTTCCTC
R13531336-1352CGATTACTGCGATTCC
R961/R7949-963TCGAATTAAACCACA
R5786-802CTACCAGGGTATCTAAT
R361/R1340-355CTGCTGCCTCCCGTAGG
The forward direction (sense)
FD1/F1 (PCR)8-26AGAGTTTGATCCTGGCTCA
F13531336-1352GGAATCGCTAGTAATCG
F361340-355CCTACGGGAGGCAGCAG
F961949-963TGTGGTTTAATTCGA
andAll provisions of the targets of the primers correspond to the numbering of E. coli (Brosius et al., 1978)

10.3 data Analysis

Obtained sequences of 16S rDNA were subjected to automatic comparison with the sequences obtained from the Project ribosomal database (RDP; Maidak et al., 1996)using the program CLUSTALW alignment (Genetics Computer Group, 1991). Sequence in the profile cut in order to standardize the size of the sequence of each organism on the Chennai profile mapping. The total number of positions of the nucleotides in the sequences included in the profile, as well 1388. In the profile mapping were included published sequences of a number of organisms found in the rumen (table 2). Dubious sequences in the profile mapping combined manually using the editor mapping Genetics Data Enviroment (GDE) (Smith et al., 1992). To test the phylogenetic kinship used the program fastDNAml (Olsen et al., 1994), which is based on the algorithm of maximum likelihood (Felsenstein, 1981). Using Treetool (GDE) was constructed phylogenetic tree. As external groups when building the tree served as Escherichia coli and Acinetobacter calcoaceticus.

Table 2
The organisms included in the profile matching using the program CLUSTALW. All sequences were obtained from the databases of the RDP and Genbank
Lactobacillus ruminis ADS 27780Streptococcus bovis ATCC 33317
Fibrobacter succinogenes S85 was ATSS 1916Methanobrevibacter ruminantium ATCC 35063
Megasphaera elsdenii ADS 17752Methanobacterium formicicum DSM 1312
M.elsdenii ADS 25940Methanosarcina barkeri DSM 1538
M.elsdenii With the 4 Methanomicrobium mobile ATCC 35094
M.elsdenii SNPrevotella ruminicola ATCC 19189
M.cerevisiaeWolinella succinogenes ATCC 33913
Synergistes jonesiiEscherichia coli
Clostridium acetobutylicum ATCC 824Acinetobacter calcoaceticus ATCC 33604
Eubacterium cellulosolvens ATCC 43171Quinella ovalis
Eubacterium uniformis ATCC 35992Selenomonas ruminantium GA192
Clostridium polysaccharolyticum ATCC 33142Eubacterium limosum ATCC 8486

Results

The selection in the conditions auxostat

Selection utilizing lactate bacteria in conditions auxostat

Allocation 1. The contents of the rumen of the cow 8710 filling the vessel for culturing the fermenter was immediately exposed to fresh sterile selective environment when you run mode auxostat with increasing pH. First, the degree of dilution was about 0,53 h-1during the first two hours at pH 5,30. In the next two hours, the degree of dilution increased to 0.65 h-1. In order to increase the specificity of the selection, the pH was lowered to 5.0, which led to the decrease in the degree of dilution do,37 h-1. The cultivation was continued for another 24 hours, after which the enrichment culture were found only two morphological types. The degree of dilution decreased with increasing duration of cultivation after the initial 24-hour period and at the end of the selection, it was only 0.33 h-1.

A sample of the contents of the fermenter were dealt a stroke on agar medium under aerobic Boxing, and individual colonies containing pure culture, were transferred to the Kosyachkov with agar and kept in liquid nitrogen; this culture was designated as isolate CH1.

Selection 2. This selection from the contents of the rumen of the cow 8812 was observed degree of dilution of 0.25 h-1in the first 24 hours, and in the next 24 hours, the degree of dilution ranged from 0.34 to 0.41 h-1. After 48 hours of cultivation was not clear if a pure culture, and cultivation was extended for another 24 hours. The degree of dilution during this period amounted to 0.41 h-1and was isolated pure culture from the fermenter in the form of colonies from Petri dishes. This isolate was identified as an isolate CH2.

Allocation 3. The degree of dilution during this allocation was reduced from 0.28 to 0.21 h-1within 48 hours. The isolate obtained from the rumen of the cow 8708, identified as isolate CH3.

Selection 4. At first, the degree of dilution was about 0.38 h -1but within 4 hours it went down to 0.276 h-1and at the end of the 48-hour period, the degree of dilution was 0,197 h-1. The isolate obtained in this selection from the contents of the rumen of the cow 8826, identified as CH4.

The $ 5. At the end of the selection was dominated by spore-forming microorganisms and the experiment was terminated.

Selection 6. When this selection is the degree of dilution was decreased in the same way as with other secretions, and the final degree of dilution amounted to only 0,116 h-1. The isolate was obtained from the contents of the rumen fed cattle and received designation as CH6.

Selection 7. The contents of the rumen used for this selection was obtained from fed cattle. The degree of dilution was decreased from 0,142 to 0,106 h-1for the first 7 hours of the selection. The resulting isolate was identified as CH7.

Selection environments for research and development in terms of hemostat

When selecting utilizing lactate cultures was observed a continuous decrease of the degree of dilution, indicating that the composition of the medium was not optimal. For the first 24 hours at selection 7 the degree of dilution decreased from 0,142 to 0,106 h-1. Directly in the fermenter added a single dose in 5 ml of sterile fluid from the rumen and after 4 hours the degree of dilution peaked in 0,408 h-1. Then the degree of p is zbawienia slowly decreased to 0.15 h -1. This "pulse" method showed that in an environment lacking nutrients.

Another "pulse" experiment with the addition of 1 ml of a solution of vitamins has led to an increase in dilution only to 0.28 h-1. However, the introduction of a higher dose of vitamins led to the achievement of the maximum degree of dilution in 0,497 h-1that was higher than the adding of the rumen contents. Utilization of lactate reflected similar results, namely: without added vitamins utilization of D - and L-isomers of lactate was 22% and 86%, respectively, and with added vitamins - 68 and 91%, respectively. Wednesday 1 presented in the Methods reflects a modified version with a high concentration of vitamins. During another "pulse" experiment, it was found that yeast extract increases the output cells of the isolates.

The growth rate of Megasphaera elsdenii ADS 25940 and isolates from auxostat depending on pH

The growth rate of bacteria was determined using the pH-auxostat at different pH values ranging from 4.5 to 6.5, using a modified lactate medium. These growth rates were compared with values obtained by cultivation in a closed volume at certain pH values, and used the average values.

Typical strain of Megasphaera elsdenii ADS 25940 showed an increase in the rate of growth at a pH of from 4.5 to 6.0, after che is about followed a sharp decline in the rate of growth at a pH of 6.5 (figure 1). The maximum growth rate achieved in ATSC 25940, amounted to 0.66 h-1that corresponds to a growth rate of 0.6 h-1in the work Therion et al. (1981).

All isolates exceeded ADS 25940 on the maximal growth rate, especially at pH values of 5.5 and below (figure 1). The maximum rate of growth of all isolates was observed at pH 5.5, while the growth rate amounted to 0.66, 0,93, 0,938 and 0,864 h-1the isolates SN, SN, CH4 and CH3, respectively. From all isolates of CH4 were the most acid-resistant, reaching a growth rate in 0,389 h-1at pH 4.5, whereas the second kislotostojkuju body SN showed a growth rate of only 0,19 h-1at pH 4.5. There was a sharp decrease in the rate of growth at a pH between 5,5 and 6,0 three isolates, namely SN, CH4 and CH3. Isolate SN showed only small deviations in the growth rate at pH between 5,5 and 6,0 that resembles the behavior of ATSS 25940 at a pH between 5,5 and 6,5.

The growth rate of isolates from auxostat on glucose

The rate of growth of the three isolates was determined at pH 5.0, 5.5 and 6.0 V conditions fueled culture (figure 2). The growth rate of all three isolates were significantly lower in glucose than lactate. The most promising isolate on lactate, namely CH4, reached maximum growth rate only 0,24 h-1on glucose at pH 5.5 compared with 0,938 h-1on the lactate. Isolate SN had the AMA highest among isolates growth rate (0.33 h -1) on glucose at pH of 6.0.

The conversion of lactate isolate CH4

Isolate CH4 were cultured in chemostats with three degrees of dilution of 0.94, 0.83 and 0.75 h-1on lactate medium. In the stationary phase was sampled and analyzed for volatile fatty acids (AGV) and determined the utilization of lactate. We also carried out the cultivation in a confined space, selecting the sample in the stationary phase. Samples sterile environment is also analyzed for the content of the AGV and lactate. With increasing dilution, the relative formation of fatty acids were changed, namely, at low degrees of dilution was formed more butyrate and valerate and less propionate and acetate (table 3). At the highest dilution were formed very small amounts of butyrate in the complete absence valerate and only slightly more than the acetate and propionate. Utilization of lactate, as expected, decreased with increasing dilution. Under cultivation in the conditions of D=0.75 in more than 40% of lactate turned into AGV and, although the utilization of lactate was intense, most of the available energy was gone. When CH4 was cultured in a confined space, he formed mainly acetate and propionate. The concentration of formed AGV under cultivation in closed volume was much lower than expected, and only about what the explanation for this can be that, that CH4 disposes of the AGV at the lack of lactate.

Table 3
Volatile fatty acids formed by isolate CH4 from lactate under cultivation in chemostat at various degrees of dilution and under cultivation in closed volume
The degree of dilution of the
(h-1)
Volatile fatty acids (mm)Utilization of lactate (%)
aceticpropionicn-oiln-valeric
0,757,2215,77911,3476,38392,66
0,8310,04812,2930,4230,01253,54
0,948,52910,5170,271039,65
Vicious is the volume 10,6597,7370,266097,62

Selection and screening method of sieving on cups

More than 800 colonies of the nine samples from the scars of four animals dairy cattle and two individuals fed cattle were inoculable in microtube liquid IRFL. Of these 610 has changed the coloring of the environment on purple for 16 hours incubation. For further research were selected nineteen of prokreditovannym isolates because they met the requirements.

Four of the selected isolates - AW09, AW10, AW11 and AW12 had the ability to grow at an initial pH of 4.5. All the remaining fifteen isolates grew at an initial pH of 5.0, and was further breeding for selection of crops with appropriate properties, and selected the most rapidly growing at pH 5.0.

All nineteen isolates were resistant to the ionophores monensin and lasalocid in concentrations of 10 ppm, utilized lactate in the presence of maltose and glucose and were able to grow on glucose and maltose. All these nineteen isolates represented gram-negative cocci (±1,8 µm), occurring in pairs or chains.

Physiological characteristics of the isolates

All isolates AW utilized as L-or D-isomer of lacto is a, since both isomers are almost completely recycled after incubation in the environment of the SDL within 24 hours. The results show that the isolates represent a fairly homogeneous group, so further studies were selected only certain isolates. Five isolates AW the growth rate on glucose at pH 5.8 ranged from 0.38 to 1.05 h-1with an average value of 0.66 h-1(±0,298). When testing isolates of AW education AGV from DL-lactate was found that they form acetic, propionic, n-butyric and n-valeric acids in the following proportions: 2:1,5:1:1,3. Some isolates AW formed trace amounts methylmalonic acid. The maximum rate of accumulation of biomass nine isolates ranged from 0.31 to 0.43 g (l×h)-1. The highest rate of accumulation of biomass was AW15, followed CH4 and AW01 at the level of 0.39 g (l×h)-1. The output of the dry weight of cells per 1 g disposed of lactic acid ranged from 0.1 to 0.17 in these nine isolates.

Presumptive identification of isolates

The obtained isolates were subjected to presumptive identification and those that corresponded to the morphological typing as strains of Megasphaera elsdenii, was used for further characterization.

Tests on sheep to assess the ability of the isolate CH4 to prevent accumulation of elochnoy acid in the rumen

The results of the first tests on sheep is presented in table 4.

Table 4
The concentration of lactic acid in the scar fluid of sheep fed roughage, with a sudden transition to the concentrates and the introduction of the rumen CH4 (experience) or placebo (control) at the same time
Time after injection of CH4 (hour)The concentration of lactic acid (g/l)
experience with CH4Control
0<0,1<0,1
20,31,4
60,83,6
80,55,2
100,46,1
120,25,9

The results of the second test on sheep is presented in table 5.

Table 5
The concentration of lactic acid in the scar fluid of sheep fed roughage, with a sudden transition to a diet of concentrates (unlimited) and the introduction of the rumen CH4 (experience) or placebo (control) 3 hours after the first production of concentrates
Day experienceTime of dayThe concentration of lactic acid in the rumen (mmol/l)
08:0012:0015:0019:00
-1CH41,40,70,60,7
control1,50,90,50,7
1CH40,70,52,12,4
control0,50,21,51,8
2 CH42,41,01,51,2
control5,013,615,217,4
3CH41,61,00,71,0
control7,84,53,52,5
7CH41,81,61,01,2
control2,01,52,01,5
14CH41,51,51,21,0
control1,82,01,2that 0

In both tests the introduction of CH4 caused a marked and significant (p<0.01 for both tests) the decrease in the concentration of lactic acid in the rumen compared with the control. In both tests the control animals after the sudden addition of easily fermentable substrate in the rumen was manifested the expected sharp increase in the concentration of lactic acid in the rumen. In contrast, the levels of lactic acid in the treated scars CH4 animals remained more or less on the same level as before the addition of substrate. This clearly suggests that a large part of the produced lactic acid is utilized CH4.

Assessment isolate CH4 in high producing dairy cows

The most important data on productivity are presented in table 6 and 7. Data were analyzed separately for all the cows (15/experience) and high producing cows (10/experience), respectively.

td align="center"> 0,43
Table 6
The effect of the isolate CH4 on the productivity of dairy cows within 80 days after calving (all cows)
OptionsExperience1The p value for pairwise comparison
12 34+CH4-CH41 and 23 and 4
The number of cows in the experience15151515---
Dry matter intake (kg/day)24,624,123,122,20,280,590,32
Milk (kg/day)36,434,0to 33.832,20,100,160,34
Fat (%)with 3.273,293,573,230,170,850,03
Protein(%)3,103,103,14of 3.070,930,23
Weight6626086186120,020,0040,73
Status points2,802,482,452,280,060,080,36

Experience 1: the control diet containing 70% concentrate +CH4

Experiment 2: control diet containing 70% concentrates-CH4

Experiment 3: control diet containing 60% concentrate +CH4

Experience 4: the control diet containing 60% concentrates-CH4

Table 7
The effect of the isolate CH4 on the productivity of dairy cows within 80 days after calving (highly productive animals)
OptionsExperience1The p value for pairwise comparison
123 4+CH4-CH41 and 23 and 4
The number of cows in the experience10101010---
Dry matter intake (kg/day)24,625,424,322,60,440,430,06
Milk (kg/day)39,335,935,234,80,130,060,82
Fat (%)3,233,24of 3.563,210,200,910,06
Protein(%)3,103,103,153,020,28 0,930,11
Weight6445976236250,110,020,90
Status points2,71of 2.262,342,440,200,020,61

The impact of adding Megasphaera elsdenii on animal health and efficiency of fattening

The number of live bacteria CH4, enter one animal was 2×1011colony-forming units per dose per animal in all experiments for fattening. The most important results are presented in table 8-11. The period from the third to the fifth week when the feeding is generally considered the most critical in terms of nutritional adaptation. During the first and second week of the diet still contains a high proportion of roughage, which gradually decreases. The consumption of concentrates begins at a relatively low level and gradually increases. Only starting from the third week diet contains the lowest level of roughage (and the highest level of concentrates) and pot is eblana food is increasing rapidly. By the beginning of the sixth week is usually considered that animals have adapted to the diet. Weeks three through fifth are indeed a critical period of adaptation of animals to high levels of concentrate.

Table 8
Average daily feed intake (kg intake per animal per day) with the introduction of CH4 versus control (without addition of CH4) in different periods of fattening
The fattening periodConditions of experienceA value of pSE
Experience (CH4)Control (without CH4)
1-2 weeks7,307,150,350,109
3-5 weeks10,1810,020,300,103
1-13 weeksfor 9.649,500,290,092

Total feed consumption was slightly (but not significantly) higher in on uchivshih CH4 animals compared to a control. Within 3-5 weeks castrated calves not receiving ionophor, but treated CH4 consumed significantly (p<0,05) more feed than the control (10,56 against 10,12), whereas animals treated with ionophor, there was no effect of CH4. Also during this period, the calves are fed a diet low in roughage and receiving CH4, tended (p<0,15) to greater feed intake than the control (10,14 against 9,80), whereas the animals receiving the diet with a high content of roughage, there was no effect of CH4.

Table 9
Average daily gain (kg per animal per day) with the introduction of CH4 versus control (without addition of CH4) in different periods of fattening
The fattening periodConditions of experienceA value of pSE
Experience (CH4)Control (without CH4)
1-2 weeks1,761,810,510,053
3-5 weeks2,091,97/td> 0,040,040
1-13 weeks2,192,200,670,019

Overall average daily gain (BSC) in a critical period of 3-5 weeks was significantly (p=0.04) higher in animals treated CH4 than in the control experiments. Within 1-2 weeks, all animals not treated CH4, a diet low in roughage gave significantly (p<0,05) lower MOP than a diet with a high content of roughage (1,61 against 2,02). However, animals treated CH4, BSC declined slightly on a diet low in roughage compared to a diet with a high content (1,70 against 1,83). Within 3-5 weeks in animals not treated with ionophor, but treated CH4, MOP was significantly (p<0,05) higher than in control animals (2,15 against 1,96).

Table 10
The feed conversion ratio (kg feed per kg weight gain) with the introduction of CH4 versus control (without addition of CH4) in different periods of fattening
The fattening periodConditions of experienceA value of pSE
Experience (CH4)Control (without CH4)
1-2 weeks4,254,120,430,111
3-5 weeks4,895,140,04of 0.081
1-13 weeksof 5.065,020,350,027

In General, animals treated CH4 showed a significant (p=0.04) improved feed conversion ratio (FCR) by about 5% compared with the control animals within 3-5 weeks. Within 1-2 weeks, all animals not treated CH4, when the diet is low in roughage FCR was significantly (p=0.06) higher (less desirable)than in the diet with a high content (4,42 against 3,83). However, in animals treated with CH4, the KKK was not significantly higher when the diet is low in roughage compared with a high content (4,24 against 4,25).

Table 11
The number of cases (including frequent removal of the same animal) and the number of the abdomen is s (multiple launch the same animal was mistaken for one), when animals are taken out of the corral and was treated for acidosis and swelling
Conditions of experienceONLY
Experience (CH4)Control (without CH4)
The total number of vyvedeni (cases)Low content. roughage62127
High content. roughage6410
TOTAL:122537
The total number of breeding animalsLow content. roughage41519
High content. roughage527
TOTAL:91726

Only p is half of the total number of animals receiving CH4, suffered from symptoms of acidosis (one or more times) in comparison with control. The same pattern was observed when considering the total number of cases of acidosis. It is also clear that the acidosis is more often caused by a diet low in roughage, and the introduction of CH4 filmed this problem with a diet low in roughage.

Identification of isolates phylogenetic methods in accordance with the gene sequences of 16S pHK

The results of the comparative sequencing showed that the isolates of the authors of the present invention, representing more phenotypically homogeneous group, identical to 97-99% (table). Table 13 shows the position of the characteristic nucleotides that are suitable to distinguish from each other the two new isolates Megasphaera elsdenii and strains ADS. 22% of the differences in the nucleotides at these four strains caused by insertions and deletions. The main differences in nucleotide sequences of these strains are observed in the positions of the nucleotides 529-536 and 1105-1120 (table 13). The great similarity between sequences of different strains M.elsdenii is consistent with their common phenotypic characteristics (Wiederhold, 1994), which is further reflected in their close phylogenetic relationship. Strains of the species M.elsdenii sequence similarity with M.cerevisiae is only 91-92%, and these two species is different form clusters on the phylogenetic tree. The cluster M.elsdenii branches into two monophyletic groups originating from the same ancestor taxonomic units, common ancestor. Common ancestor, from which evolved M.cerevisiae, however, appeared before the ancestor from which evolved the cluster M.elsdenii. A small length of the branches between strains M.elsdenii and the corresponding ancestor also indicates that they appeared later than the more highly branched cluster M.cerevisiae.

Table 12
The matrix of similarity to the sequences of 16S rDNA M.elsdenii and M.cerevisiae. The similarity score between sequences based on the comparison of all 1388 unambiguously aligned positions of the nucleotides. % G+C refers only to the relevant combined sequences of 16S rDNA
ADS 17752ADS 25940SNCH4%G+C
M.cerevisiae92,092,091,591,554,3
ADS 2594099,098,598,154,4
CH4of 98.298,199,054,9
SN97,7of 54.8
ADS 1775253,1
Table 13
Typical provisions that define the different isolates and strains M.elsdenii
Nucleotides
PositionaADS 25940b CH4SNADS 17752
87GGAndG
105TT
170TT
221TCT
241GAndAndG
283AndGG And
418And***
529-530CG**CG
533-536CG**CGACCGACGC**
539TT
550-552TASCGTCGTTAT
556GAndAndG
711G GG
718***G
850AndAndGAnd
1084AndGAndAnd
1105-1108TGGAAGGGAGGGTGGA
1117-1120TSSASSSTSSSTTSSA
1290And*And*
1297-1300AAGTCGGCAAGTCGGC
1396AndAndAnd
1425AndAndGAnd
1437GAndGG
1492TT
and the numbering System in E. coli (Brosius et al., 1978).
b typical strain.
The asterisk shows where the combination has been entered, the gap because of the deletion or insertion of nucleotides at any position posledovatelno and the corresponding isolates and strains.

The method of maximum likelihood, including finding the tree that gives the greatest probability of matching to the data obtained by sequencing (Felsenstein, 1981)was used to derive phylogenetic tree of the sequences included in the profile alignment (figure 3). This method has the advantage over traditional methods of sufficiency, which can lead to erroneous trees, if different descendants will occur with unequal velocities, in that it takes into account the possibility of evolution at different speeds in different lines (Felsenstein, 1981). Because the topology of the tree is also affected by the number of organisms used and the choice is obviously unrelated organisms for comparison (Stackebrandt and Ludwig, 1994; Stackebrandt and Rainey, 1995)profile of the combination was included a number of clearly related and apparently unrelated organisms occurring in the rumen. Later they were used to create the tree. Although phylogenetic tree was inferred only on the basis of incomplete (92%) of 16S gene sequences pPHK that could reduce the resolution between closely related organisms (Utaker et al., 1995; Li and Graur, 1991), it was shown that the overall topology of the trees obtained from both full of incomplete sequences, in General, coincide with each other (Van Camp et al., 1993; Vandamme et al., 1996).

Vandamme et al. (1996) reported the and, that different isolates can be considered as representatives of the same species if their sequence pPHK homologous more than 97%, they have phenotypic similarity and show a considerable degree of hybridization of DNA:DNA. Despite the fact that the relationship between similarity and DNA homology of 16S pPHK between different organisms not linearly, Fox et al. (1992) suggested that the actual identity of the sequences of 16S pPHK should mean that two organisms are members of the same species, because it is almost always confirmed by hybridization of the DNA:DNA. Although only the sequence data of 16S pPHK may not be sufficient in all cases, species identification, they are extremely useful to determine what kind presumably belongs strain, unless the species is represented in the database of sequences of the 16S pPHK. Strains with almost identical sequences of 16S pPHK should be attributed to one and the same "superwide on pPHK" or "complex species pPHK". Therefore, the described isolates of CH4 and SN, phenotypic presumably identified as strains of M. elsdenii, should be attributed to a single complex species pPHK, which will include reference strains of this species - typical strain ADS 25940 and strain ADS 17752. Because phylogenetic relationships the value of the corresponding isolates also coincide with their phenotypic characteristics, these isolates can be considered as strains of the species Megasphaera elsdenii. What sequence of 16S rDNA M.cerevisiae and M.elsdenii homologous only 92%, together with genetic and phenotypic data, confirms the division of the genus into two well-separated type.

Among rumen bacteria included in this study, most similar to the cluster Megasphaera, apparently, are Selenomonas ruminantium and Quinella ovalis. Obvious phylogenetic relationship between Megasphaera elsdenii and Selenomonas ruminantium is in accordance with the phenotypic and genetic properties common to these two organisms, such as close to the G+C content in DNA (53-54%), anaerobes, chemoorganotrophic metabolism and utilization of the same substrates (Stackebrandt et al., 1985; Stewart and Bryant, 1988; Haikara, 1992). Work Stackebrandt et al. (1985), who used the method of oligonucleotide cataloguing to identify phylogenetic relationships between species, confirms this phylogenetic relationship. On the other hand, Selenomonas ruminantium is also closely related to a relatively unknown body Quinella ovalis, which breeds in the rumen, when the animal gets food rich in sugars. These organisms, although they are not yet entered in the culture, have some physiological properties in common with those of the major selenomonas found in sheep (Stewart and Bryant, 1988). As expected, the most distant Rhodes the cursors of Megasphaera, occurring in the rumen, bacteria are included in the cluster archebacteria-methanogens, which are believed to have appeared about 600-800 million years ago (van Soest, 1994; Woese, 1987). The rate of evolution of these organisms also slower than Bacteria, and the primitiveness of this group clearly reflected in highly branched cluster of methanogens.

The recent divergence of different strains M.elsdenii, probably due to the improvement of their phenotype to adapt to a very selective conditions in the rumen. According to Woese (1987), the evolution of the phenotype of any organism is the process by which new or more effective characteristics for survival in a particular niche. Improvement should lead to the emergence of organisms with the universal metabolism, as in the case of Megasphaera. On the other hand, slower evolving methanogens compared them with the metabolic monotonous.

This study shows the suitability of the sequencing of the 16S rDNA to distinguish between closely related strains of the species M.elsdenii. In addition, it provides a phylogenetic framework for the identification of freshly isolated strains, already characterized phenotypically. These frames have special value when they serve as a basis for the development of species - and shtamposvarnyh probes designed for research in ecology rubs is.

Conclusions

Selection

Introduction bromcresol purple on Wednesday IRFL to help identify the bacteria that utilize lactate, was successful in the case of fast-growing bacteria that utilize lactate, which was the main interest in this study. In the early stages of incubation, they form a purple zone around the colonies, which clearly contrast with the yellowish background agarized medium. However, prolonged incubation the pH gradient around the colonies dissipeared due to diffusion of ions and the entire background is purple. Then it becomes difficult to distinguish slow-growing colonies, utilizing lactate from those organisms that grow on other sources of carbon that enters the rumen fluid.

M.elsdenii is not a dominant species that utilize lactate, animals on a diet with a high content of concentrates (Mackie et al., 1978; Mackie & Gilchrist, 1979; Mackie et al, 1984; van Gylswyk, 1990), but there are many reasons why he may prevail during the procedures of selection and screening.

Some were skanirovaniya colonies consisted of selenomonas and other morphological types. Most of these colonies was not selected because it does not have a positive sign that they can utilize lactate in the presence of soluble sugars. Russel & Baldwin (1978) showed the, in multisubstrate environment M.elsdenii V simultaneously uses glucose, maltose and lactate, but not sucrose. Marounek et al. (1989) showed that four strains M.elsdenii recycled lactate faster than glucose in media with two carbon sources.

Some other laboratories have studied the possibility of settling ruminants consuming forage with high levels of concentrate utilizing lactate organisms to prevent the accumulation of lactate, also worked with strains of M. elsdenii (Das, 1979; Leedle et al., 1991; Robinson et al., 1992; Kung & Hession, 1995; Wiryawan & Brooker, 1995). But it was impossible to compare the rate of growth of isolates and CH AW with strains from the literature to determine whether they have a higher growth rate and are more acid resistant, because no such data in the literature. However, the isolates AW and SN can be compared with the type strain M.elsdenii ADS 25940.

The isolates AW region of pH values, which were determined by the growth was not sufficient to determine the pH range for the selected strains or optimal pH for growth. However, it can be assumed that the optimum will be above a pH of 5.7 and the lowest value of pH is between pH 4.5 and pH of 4.9 for all but four isolates, as there was no growth on plates with medium IRFL at pH 4.5. This corresponds with the work carried out on the type strains of M. elsdenii ADS 25940. The pH range for a typical shtam is and M.elsdenii ADS 25940 is from 4.6 to 7.8 with optimum growth at pH 6,05 (Therion et al., 1982).

The isolates SN optimum pH for growth is between pH 5 and 6. In the area investigated pH values, the highest growth rate was at a pH of 5.5. Both groups of isolates on the SDL environment had a greater growth rate than the standard strain. The growth rate of strain M.elsdenii ADS 25940 in the environment of the SDL comparable with those obtained by previous researchers on lactate medium (Therion et al., 1982).

The rate of growth of the isolates on the lactate were higher than on glucose and maltose. This is consistent with previous research on M.elsdenii ADS 25940, in which it was found that the rate of growth on lactate medium at a pH between 5.0 and 6.5 was higher than in glucose medium (Therion et al., 1982). Outside this interval, the pH of the growth rate on glucose was higher. Study of substrate preferences of rumen bacteria showed that the growth M.elsdenii (159 on the lactate was slower than glucose and maltose, although the pH of the medium in this study was higher than 6.5 (between 6,75 and 6.9) (Russel & Baldwin, 1978).

The composition of the end products of digestion on lactate medium was set at four strains of M. elsdenii, including the model strain LC1 or ATSS 25940 (Marounek et al., 1989). These results showed the variability of the ratio of the formed fatty acids depending on the strain. Three strains formed a little valerianic acid no formed, whereas 22 mol.% the final products have M.elsdenii L8 was Valeriano the I acid (Marounek et al., 1989). Nine isolates AW, tested in this study did not show such a variation from strain to strain product valerianic acid, as it was the strains tested Marounek et al. (1989), but they were close to M.elsdenii L8, which was isolated from the rumen of calves fed a milk diet. However, the CH4 produced the same end-products of fermentation as a model strain.

From the point of view of the maximum speed of accumulation of biomass on the environment of the SDL strain AW15 would be best to get a large number of cells for experiments on animals and strains of CH4 and AW01 follow immediately afterwards. The time for producing 100 g dry weight of cells in the environment of the SDL in chemostat with a working volume of 5 l will be 1.9 days at AW15 and 2.1 days in CH4 and AW01.

The rate of growth of selected isolates on lactate compared with the type strain M.elsdenii. Selected strains of acid-resistant and can grow at pH values lower than 5.0. They are resistant to ionophores usually added during feeding, and can utilize both isomers of lactic acid even in the presence of glucose and maltose. The end product of the fermentation of lactate are volatile fatty acids (AGV), which is an important energy source in ruminant animals. Education propionate is particularly important in industrial fattening, since propionate is the main source of glucose for TC is her ruminants. Thus, the isolates possess the properties necessary for effective control of lactic acidosis in ruminants.

The growing utilizes lactate organisms was successful in the application environment, not containing liquid from the rumen. The only change to the original environment was to increase the content of vitamins and adding yeast extract to the medium. The excellent bacteria could survive on this medium at 4°C up to 20 days when used as the working culture.

Method using pH-auxostat for enrichment utilizing lactate bacteria of the rumen with a given combination of biochemical/physiological properties that make these bacteria are potentially more suitable for the prevention and fight against lactic acidosis in the fattening of animals, have been very successful. In most cases in the fermenter were established rapidly growing morphologically homogeneous population of two days after the start of the cycle. Subsequent inspections of cultures obtained from the contents of the fermenter by planting on cups, confirmed that these cultures have the required set of parameters.

Presumptive identification of isolates from enriched cultures showed that all of them, except one, belong to the species Megasphaera elsdenii.

Table 14
CPA is out the selection method with the use of pH-auxostat and standard method of sieving and screening on cups
The traditional method for cupsAuxostat
Duration (days)909
Time (hours)spent by man1807
Sample: the number of bacteria (CoE)12×101014×1012
The maximum specific growth rate (h-1)0,910,90
The biomass yield (g/l)0,600,59
The rate of accumulation of biomass g (l×h)-10,390,39

Because the selected strains were better than ATS 25940 at pH below 6.0, then they will be more suitable for pH values encountered in the rumen fed animals, which are usually less than a pH of 6.0. Moreover, the isolate CH4 has been the most suitable for experiments in fattening animals.

Cultivation utilizing lactate isolates on glucose was unacceptable due to low / min net and growth compared with the growth rate at the lactate.

Thirty cows, which were introduced isolate CH4, gave significantly more milk (p=0,10), had on average a higher body weight (p=0.02) and overall health (p=0.06). The fat content of milk from cows fed the diet with 60% concentrate +CH4, also significantly increased (3,57%) against 3,23%).

Tests on dairy animals

Dry matter intake was not changed, but milk production was significantly increased by 3.4 kg/day from 35.9 kg/day up to 39.3 kg/day (P=0.06)when cows were given food with 70% concentrates and introduced isolate of CH4. Milk production tended to increase (p=0.13), if you compare all of the cows fed with CH4, from cows not treated with it. The body weight and indices of body condition increased (p=0.02), when highly productive cows fed forage with a high content of concentrates was introduced isolate of CH4. Introduction CH4 cows fed forage with 60% concentrates, led to a significant increase in fat content of milk (p=0.06) tended to increase milk protein content (p=0,11). Components of milk play an important role in the existing schemes of payment for the milk.

Introduction to cows isolate CH4 significantly increased milk production and positive influence on milk composition, body weight and body condition. Dry matter intake did not change; thus, the results show that the introduction of the cows body SN creates a more favorable environment in the rumen, that leads to better utilization of nutrients.

Experiments in fattening animals

A significant improvement in average daily weight gain (BSC) and feed conversion ratio (FCR) during a critical period of fattening (3-5 weeks), and the General reduction in dyspepsia treated CH4 animals compared to control animals showed that the introduction of CH4 fed animals can produce the following effects:

- it contributes to adaptation during the transition from a roughage to concentrates, as was the case in these experiments. CH4 also softens the deterioration of the efficiency of a diet low in roughage compared to a diet with a high content of roughage in the early stages of adaptation;

- this method of application of CH4 effective because it allows its expression in accordance with the purpose;

- application of CH4 effectively prevents acidosis, what is directly seen by the decrease in the number of observed cases of acidosis and indirectly improvement in the critical phase of fattening, when the likely occurrence of acidosis. All this was observed in the diets and feeding regimes, which give a particularly high risk of acidosis (especially in the early stages of fattening), and was more pronounced when not used inatory;

- CH4 may be used as veterinary cf is DSTV, contributing to the prevention and/or treatment of disorders, as evidenced by the sharp drop in cases of acidosis when using CH4 as compared with control;

- CH4 can be used to improve the efficiency of fattening, including growth rate (thereby reducing the time required for fattening) and the efficiency of feed conversion;

- CH4 can be used to allow feeding on diets with high content of concentrates, i.e. use fewer forage, and to increase the speed of the transition from a diet with a high content of roughage in diets with a high content of concentrates, i.e. again to use less roughage. This is further confirmed by the observation in the early stages of feeding, when the negative effect of the low content of fodder compared with a high content significantly softened with the introduction of CH4.

The authors present invention also found that, compared with known strains M.elsdenii strain M.elsdenii CH4:

- high level and adapted to reproduce in the rumen of animals on a diet with a high content of concentrates;

- capable of reproduction at relatively low pH values below 5.0, and even at pH 4.5, which is defined as acute acidosis;

- resistant to antibiotics-ionophores usually added to the diet of fattening;

- able to pre-emptive use of lactate as a carbon source, even in the presence of soluble carbohydrates, such as glucose and maltose.

Next, the advantages of this strain are that:

he has a relatively high rate of growth, i.e. more 0,938 h-1;

- has the ability to grow the reducing sugars as well as lactate;

- has a relatively high rate of accumulation of biomass, i.e. more than 0.39 g(l×h)-1;

- resistant to ionophores;

- forms mostly the acetate, but not propionate and butyrate; and

- has a unique sequence of 16S rRNA and so is a new strain.

The authors of the present invention, moreover, found that the animals, which were injected maltose directly into the rumen, or suddenly translated from roughage to the high content of concentrates, show no measurable accumulation of lactate in the rumen with the introduction of CH4.

Moreover, high-yielding dairy cows with the introduction of CH4 yield of 2.4 to 3.2 litres more milk than control animals that were not injected CH4. Indicators of the condition of the body, as well as the body weight of cows that were injected CH4, were statistically significantly higher than in control animals.

It should be borne in mind that in respect of the microorganism according to the invention and its application for the changes in detail, not beyond the scope of the attached claims.

1. Strain M.elsdenii deposited at NCIMB, Aberdeen, Scotland, UK under number NCIMB 41125, characterized by the ability to utilization of lactate with a degree from 40 to 90% even in the presence of sugars, resistance to ionophores, high growth rate, ability to preferential production of acetate and ability to proliferation at a pH of less than 5.0.

2. Composition to facilitate translation of ruminant animals with diet-based forage on high-energy diet based on concentrated feed, consisting mainly of a strain according to claim 1.

3. Method of facilitating the transfer of ruminant animals with diet-based forage on high-energy diet based on concentrated feed, which includes the introduction in the rumen of these ruminant an effective amount of the strain according to claim 1.

4. Feed additive for ruminants, designed to facilitate the translation of ruminant animals with diet-based forage on high-energy diet based on concentrated feed containing the inoculum, consisting of an effective amount of the strain according to claim 1 and a carrier.

5. Feed additive according to claim 4, in which the carrier is sterile anaerobic nutrient medium, and in which the inoculum and nutrient medium are placed respectively in separate sterile is aerobnykh cameras of the same anaerobic container, the camera anaerobic connected to one another for inoculation of a nutrient medium with inoculum under anaerobic conditions.

6. Method for the treatment and prevention of lactic acidosis in ruminants and one or more associated conditions, including inflammation of the rumen caused by lactic acidosis inflammation of the hoof caused by lactic acidosis abscesses in the rumen as a consequence of excessive accumulation of gases in the rumen and liver, including anaerobic introduction to scar some animal an effective amount of the strain according to claim 1.

7. Veterinary treatment for and prevention of lactic acidosis in ruminants and one or more associated conditions, including inflammation of the rumen caused by lactic acidosis inflammation of the hoof caused by lactic acidosis abscesses in the rumen as a consequence of excessive accumulation of gases in the rumen and liver, containing an effective amount of the strain according to claim 1.

8. The method of increasing the milk productivity of ruminants, including the introduction in the rumen of these ruminant an effective amount of the strain according to claim 1.

9. The method according to claim 8, in which the strain according to claim 1 is introduced into anaerobic conditions.

10. The way to increase the efficiency of fattening in the case of feeding the ruminant animal diet based on concentrated feed, which includes the introduction in the rumen of these WW is cnyh animal an effective amount of the strain according to claim 1.

11. The method according to claim 10, in which the strain according to claim 1 is introduced into anaerobic conditions.

12. The method of increasing the growth rate and reduce the time of feeding of ruminants, including the introduction in the rumen of these ruminant an effective amount of the strain according to claim 1.

13. The method according to item 12, in which the strain according to claim 1 is introduced into anaerobic conditions.

14. The way to reduce morbidity caused by digestive disorders, and mortality among ruminants from lactic acidosis and related conditions and reduce cases of lactic acidosis and related conditions in ruminants, including the introduction in the rumen of these ruminant an effective amount of the strain according to claim 1.

15. The method according to 14, in which the strain according to claim 1 is introduced into anaerobic conditions.

16. Method for improving the efficiency of conversion of the diet of some animal on the basis of concentrated feed in some animal when translating it to the diet, including the introduction in the rumen specified some animal an effective amount of the strain according to claim 1.

17. The method of selection strain M.elsdenii capable of utilizing lactate with a degree from 40 to 90% even in the presence of sugars, which includes stages:
a sample of the fluid of the rumen some animal, adapted to a diet based on concentrated feed, and
culturing the sample in a nutrient medium is selected from one or more of these mediums, as a culture medium not containing rumen fluid, lactacidemia environment incubated with rumen fluid (IRFL), the SDL environment SDG, the SDM environment, environment CSL 4 and Wednesday CSL 6,
using the following cultivation parameters:
a nutrient medium containing dH2O, Na-lactate, agar, bromocresol purple, peptone, KH2PO4, (NH4)2SO4, MgSO4, CaCl2vitamins, including paradoxethereal, pyridoxamine, Ca-D-Pantothenate, aminobenzoic acid, Biotin, folic acid and cyanocobalamin, minerals, Na2S, antifoaming agent, maltose, glucose, Indigo Carmine, yeast extract, CSL, KOH and L-cysteine,
pH in the range from 4.5 to 6.5,
the temperature from 4 to 50°C,
antimicrobial agents selected from the group consisting of lasalocid and monensin,
oxygen-free gas atmosphere containing CO2.



 

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3 ex, 2 tbl

FIELD: medicine.

SUBSTANCE: 24 hours prior to the examination, children aged 3 to 8 year old takes in 100 ml lactulose (Duphalac) with water of room temperature in amount 1-1.5 liters within 4-6 hours. The second dose lactulose (Duphalac) 100 ml follows with water of room temperature in amount 1-1.5 liters. To prevent overaerogenesis and to reduce discomfort, Espumisan (Simethicone) is prescribed in dosage 15 ml 3 times a day. For children aged 8 to 18 years old, there is prescribed 200 ml lactulose (Duphalac) with water of room temperature in amount 1.5-2.0 litres within 4-6 hours followed with the second dose of lactulose (Duphalac) 200 ml with water of room temperature in amount 1-1.5 litres. Additionally Espumisan (Simethicone) is introduced in dosage 15 ml 3 times a day or 3-6 capsules 3 times a day.

EFFECT: effective cleansing of gastrointestinal tract in children of different age, thus ensuring good visualisation of large intestine mucosa and decreasing drug load on organism of younger children, preserving normal microflora of large intestine and preventing overaerogenesis.

2 ex

FIELD: medicine.

SUBSTANCE: there is prescribed gluten-free diet with eliminating cereals and additional order of protein: meat 100.0 g, cottage cheese 100.0 g, or sour cream 100.0 g daily. Creon is dosed 10000 ME at mealtime within 2 weeks. It is combined with physiotherapy exercises (PTE) within 20-30 minutes. During afternoon, every second day circular douche is applied at pressure 1.5-2 atmospheres, water temperature 36-35°C for 3-4 minutes within therapeutic course 10-12 procedures. On another days, radon baths are taken at water temperature 36°C, dosed 0.75 kBq/l for 8-10 minutes within therapeutic course 8-10 procedures. Electrophoresis is applied daily with 50% Dimexide solution (±) for small intestine projection by transverse technique, current density 0.05 mA/cm2 within 12-15 minutes within therapeutic course 10-12. The BAPs VC-20, VC-22, E-36, Gi-4 are exposed to red and infrared light daily within 1.5-2 minutes per each point within therapeutic course 10-12 procedures.

EFFECT: reduced specific immunologic markers of autoimmune intestine inflammations and cytokine generation level, normalised immune response of an organism, normalised hormonal and exchange processes.

2 ex, 2 tbl

FIELD: medicine.

SUBSTANCE: invention relates to experimental medicine and concerns pyelonephritis prevention in experiment. During 10 days preparation "Ultrasorb" is introduced to experimental animals with food in terms of 3 g/day. Then pyelonephritis is modelled by rectal infection with uropathogenic strain of lactose-negative E.Coli at the background of cold stress.

EFFECT: absence or reliable reduction of kidney tissue inflammation in animals which obtained "Ultrasorb".

4 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to pediatrics and gastroenterology and can be used for treating intestine disbacteriosis in children less than 1 year old. For this purpose bifidumbacterin forte is introduced. First 2-3 days preparation is introduced by 25-30 doses per day in 4 takings. During next 3-4 weeks bifidumbacterin forte is introduced by 5 doses 2 times a day.

EFFECT: method allows increasing efficiency of treatment of disbacteriosis in children less than 1 year old due to application of definite scheme of preparation introduction first in optimally high dose mode, then in supporting doses.

FIELD: medicine.

SUBSTANCE: invention relates to medicine, in particular to gastroenterology, and applies to treatment of chronic duodenal obstruction. For this purpose erithromicin is introduced on account of 3.0 mg/kg of body weight 3 times a day. Additionally Alfadol-Ca is introduced in case of initial normocalcemia in dose 1 capsule 1 time a day. In case of initial hypocalcemia Alfadol-Ca dose is 1 capsule 2 times a day. Course of treatment is 3 months.

EFFECT: method ensures elimination of symptoms of chronic duodenal obstruction in patients with functional form of chronic duodenal obstruction in combination with syndrome of disturbed absorption and normalisation of calcium balance in organism.

2 dwg, 1 tbl, 2 ex

FIELD: medicine; gastroenterology.

SUBSTANCE: antibacterial therapy is presented with selective decontamination using antiklebsiellic bacteriophage within 10 days. After that prebiotic therapy including Hylak Forte, enzymatic agent and Linex in therapeutic dosage is combined with probiotic introduction. For dysbacteriosis of I stage Bifiform is introduced in dose 1 capsule 2 times a day, for dysbacteriosis of II and III stages dose is 2 capsules 2 times a day, combined with Lactobacterinum 5 doses once a day. Thus medicinal plant tincture is introduced including silverweed rootstock, salvia leaves, milfoil herb, St. John's wort herb, elecampane rootstock with roots, tickseed herb, mint leaves, fennel fruits and buckthorn bark at component ratio respectively 2:2:2:2:2:2:2:2:1 dosed 1\3 of glass 3 times a day, 30 min before meal, within 4 weeks.

EFFECT: method has no contraindications and provides fast normalisation of gastrointestinal tract microflora.

1 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention concerns medicine. A soft drink on the basis of herbs consists of the concentrated vegetative extract obtained from an admixture of herbs, chosen of Sida sps., Boerhaavia diffusa, Vitis vinifera, Tinospora cordifolia and Withania somnifera, along with green palm sugar, the agent causing a fermentation, and carbonised water, in which proportion Sida sps.: Boerhaavia diffusa: Vitis vinifera: Tinospora cordifolia: Withania somnifera in a powdery admixture makes (15-20 : (5-10 : (15-20 : (5-10 : (5-10); Proportion of wt/wt: green palm sugar: the concentrated vegetative extract makes from 1 : 3 to 1 : 4; wt/wt, a proportion of carbonised water: the admixture of the concentrated vegetative extract, green palm sugar and the agent causing a fermentation, makes from 1 : 3 to 1 : 5. Perform: (a) obtaining of parts of Sida sps., Boerhaavia diffusa, Vitis vinifera, Tinospora cordifolia and Withania somnifera; (b) crushing of parts of plants and their mixing with obtaining of a powdery admixture; (c) water addition to a powdery admixture from a stage (b) for obtaining of a water extract; (d) concentration of a water extract from the stage (c); (e) filtration of the concentrated extract from the stage (d); (f) admixture of green palm sugar to the filtered extract from the stage (e); (g) addition of Sacromyces strain and the agent causing fermentation, to the admixture from the stage (f); (h) fermentation of the admixture from the stage (g) during the period of time from 3 till 6 days; (i) filtration of the fermented admixture from the stage (h); (j) concentration of the fermented filtrate from the stage (i) with obtaining of the basic solution and (k) mixing of the basic solution (j) with the carbonised water in wt/wt proportion from 1 : 3 to 1 : 5 with obtaining of a soft drink on the basis of herbs.

EFFECT: creation of a soft drink on the basis of herbs.

11 cl, 5 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: invention relates medicine area, in particular, gastroenterology, and concerns treatment of GI tract dysbacteriosis. A set containing agents, possessing antagonist action on a GI tract pathogenic microflora and agents for restoration of a normal GI tract microflora (eubiotics and bacteritic preparations), and also independent GI tract electrostimulators are used for this purpose. The method of GI tract dysbacteriosis treatment combines a diet, the specified agents, and also electrostimulator insertion before and-or in the course of treatment.

EFFECT: optimum conditions for effect of eubiotics and bacteritic preparations at the expense of preliminary normalisation of GE tract motor and evacuation functions.

2 cl

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