Patent Application
20220062403
The present invention relates to the use of a bacteria-based compound in conjunction with an appropriate vaccine in the prevention and treatment of disease. More particularly, the present invention relates to the use of a compound such as that derived from a substance produced by gram negative bacteria such as, but not limited to, a lipopolysaccharide (LPS) of gram negative bacteria in conjunction with an appropriate vaccine in the prevention and treatment of an intestinal tract disease such as coccidiosis by way of an animal feed regimen administered from the day of hatch onward.
Substantial economic losses in the poultry industry are most often the result of disease. Diseases in flocks often result in reduced production volume or compromised quality of meat. Prevention and treatment of poultry disease adds significantly to poultry production costs. Some estimates place total losses as a result of poultry disease at more than 10% of all production costs.
Of the diseases known to strike poultry flocks, the most common are enteric diseases, which include coccidiosis, a disease caused by a parasite, the coccidian protozoa. Annual economic losses due to coccidiosis alone are estimated to exceed $3 billion per year and these costs are expected to increase due to a variety of reasons.
First, coccidiosis prevention today is accomplished mainly through the use of vaccines. A one-time administration of the vaccine is given very early in broiler life and, specifically, on the day of hatch. While this approach has shown some benefit, vaccines are known to suffer from variable effectiveness in controlling the disease over time. Experimentation has shown that a vaccine used in conjunction with a supplement such as a probiotic may improve outcome, but this approach adds to production cost.
Second, coccidiosis treatment today is accomplished conventionally through the use of antibiotics and ionophores, both of which are added costs in an industry with constricting margins. The use of antibiotics and ionophores is under pressure globally for a number of reasons, including environmental. Relatively recently, the European Union banned sub-therapeutic doses of certain antibiotics for use as feed additives. Synthetic treatment compounds and other chemical agents are known but are not as effective as conventional antibiotics.
Third, the above-noted pressure against using antibiotics and ionophores notwithstanding, one or both antibiotics and ionophores are often used in a series with a staggered approach (bioshuttling) in which live coccidiosis vaccine is administered at the hatchery followed by treatment with an anti-coccidial between two and three weeks of age.
Fourth, drug resistance to antibiotics, ionophores, and synthetic treatment compounds is increasing largely due to overuse thereby severely compromising the effectiveness of these treatments. There has been no approval of new drugs in any of these categories for many years.
Fifth, even if known treatments were still economical and effective, known approaches would still be regarded as unsatisfactory because the medication must be included in the animal's feed for the full duration of its lifespan to be fully effective. This requirement adds significant cost to feed for the entire growout period.
Accordingly, it is desirable to develop a treatment method that boosts the effectiveness of vaccines for use in the treatment of pathogenic infections such as, but not limited to, coccidiosis in animals without neutralizing the activity of the vaccine itself.
The disclosed inventive concept provides an improved treatment method for a broad variety of diseases in both animals and humans. The method includes combining one or more vaccines with a treatment compound set forth as part of the disclosed inventive concept that enhances vaccine efficacy. Unlike the unworkable combination of an anti-coccidia agent with a vaccine, the treatment compound may be combined with a vaccine because the treatment compound does not act on the pathogens directly. An added advantage of the disclosed inventive treatment method over known treatment methods is concomitant use with a vaccine without the risk of treatment resistance.
The vaccines used to control coccidiosis in poultry have historically been of the live coccidian vaccine variety. Known vaccines contain sporulated oocysts of various species. It is known that the parasites E. maxima, E. tenella, and E. acervulina generally exist in vaccines. The total number of oocysts present in each of today's vaccines differs significantly. The total number may be as low as about 200 oocysts per dose to as high as about 3000 oocysts per dose.
A number of methods are known for delivering live oocyst vaccines. These methods include delivery through drinking water, administration through feed by either spraying the feed or applying gel droplets to the feed, or administration at the hatchery through gel bead delivery, ocular vaccination, or spray cabinets, the latter being the most common method of administration in the United States today. When using the spray cabinet for coccidian vaccination, the chicks are a day old at the time of treatment.
When a compound, such as, but not limited to, the disclosed compound is used in conjunction with one or more vaccines, a synergistic effect is realized. The suggested compound is derived from a substance produced by gram negative bacteria such as, but not limited to, a lipopolysaccharide (LPS) of gram negative bacteria. The treatment compound is combined with one or more appropriate vaccines and is administered early in the life of an animal, providing a synergistic effect compared to the use of the treatment compound or the vaccine alone.
For a more complete understanding of this invention, reference should now be made to the accompanying figures. As set forth in many of the figures, the designation “No Tx, No Challenge” refers to a test in which no treatment was administered to a subject animal not deliberately infected with coccidiosis. The designation “No Tx, Cocci” refers to a test in which no treatment was administered to a subject animal deliberately infected with coccidiosis. The designation “Anti-cocci, Cocci” refers to a test in which the subject animal was infected with coccidiosis and the animal was administered an anticoccidial.
The designation “ZIVO all rations, Cocci” refers to a test in which the subject animal was infected with coccidiosis and the animal was administered a treatment composition according to the disclosed inventive concept. The designation “ZIVO starter & grower, Cocci” refers to a test in which the subject animal was infected with coccidiosis and the animal was administered a starter diet at 0-21 day of age. The designation “ZIVO starter, Cocci” refers to a test in which the subject animal was infected with coccidiosis and the animal was administered a grower diet at 22-35 days of age. The designation “ZIVO, grower & finisher, Cocci” refers to a test in which the subject animal was infected with coccidiosis and the animal was administered a grower and finisher diet at 36-42 days of age.
The accompanying figures are described as follows:
In the following description, various operating parameters and components are described for different constructed embodiments. These specific parameters and components are included as examples and are not meant to be limiting. Unless otherwise noted, all technical and scientific terms used herein are to be accorded their common meanings as would be understood by one having ordinary skill in the art.
The Compound Used in Treatment
The disclosed method of treatment preferably, but not absolutely, utilizes a compound generally derived from a gram negative bacteria such as, but not limited to, a lipopolysaccharide (LPS) of gram negative bacteria. By administering the compound early in broiler life, disease prevention and treatment via immune modulation are achieved.
As used herein, the term “algal culture” is defined as an algal organism and bacteria (one or more types) that grow together in a liquid medium. Unless expressly stated otherwise, the term “algal biomass” refers to the algal cells and bacterial cells (with the liquid culture medium removed). The “algal biomass” can be wet material or dried material.
Unless expressly stated otherwise, the term “algal supernatant” is defined as the culture medium in which the algal biomass is grown that contains excreted compounds from the algal biomass. Algal supernatant is obtained by growing algal biomass in culture medium for an appropriate length of time and then removing the algal and bacterial cells by filtration and/or centrifugation.
It is known that bacteria of the Variovorax genus and the Rhodobacter genus are metabolically versatile. Variovorax is a gram negative aerobic bacterium that can grow under a variety of conditions. It is part of the subclass Proteobacteria and is capable of metabolically utilizing several natural compounds generated by plants or algae. Rhodobacter can grow under a broad variety of conditions, including both photosynthesis and chemosynthesis. Growth can also be achieved under both anaerobic and aerobic conditions. Rhodobacter sphaeroides represents a gram negative facultative bacterium and is a member of the α-3 subdivision of the Proteobacteria.
Embodiments of the compound used in the treatment of disease as set forth herein include one or more LPS/Lipid A compounds produced by gram negative bacterial strains for use as selective modulators of the TLR4 signaling pathway.
The disclosed inventive concept involves any combination of three fundamental steps: (1) the gram negative bacteria produces LPS/Lipid A compounds; (2) the LPS/Lipid compounds modulate TLR4 activity; and (3) a downstream effect results in enhancement of innate and adaptive immune processes, thereby aiding in the treatment of coccidiosis.
In an embodiment, the LPS/Lipid A compounds used as selective modulators of the TLR4 signaling pathway are produced from a Variovorax paradoxus strain. The Variovorax paradoxus strain may be a naturally occurring strain found in an algal biomass and/or algal supernatant products. For example, the algal biomass may comprise the algal species Klebsormidium flaccidum. More specifically, the algal biomass culture may comprise the algal strain Klebsormidium flaccidum, var. ZIVO.
In another embodiment, the LPS/Lipid A compounds used as selective modulators of the TLR4 signaling pathway are produced from a Rhodobacter sphaeroides strain. Extensive studies have been undertaken regarding the structure and function of Rhodobacter sphaeroides. More focused studies have examined the photosynthetic characteristics of Rhodobacter sphaeroides. While it is known that lipopolysaccharides from Rhodobacter sphaeroides are effective TLR4 antagonists in human cells that prevent TLR4-mediated inflammation by blocking LPS/TLR4 signaling, the inventors employed a testing methodology to address multiple immune response mechanisms in poultry to arrive at the conclusion that an LPS compound derived from Rhodobacter sphaeroides proved effective as a treatment of coccidiosis in poultry. Research demonstrated that combining a TLR4 modulator with an activator of TLR2 (such as LPS from gram negative bacteria) appears to provide an anti-coccidiosis effect.
Accordingly, embodiments of the compound used in the treatment of disease according to the present disclosure are directed to one or more LPS/Lipid A compounds produced by a gram negative bacterial strain of the group Variovorax or the group Rhodobacter for use as selective modulators of the TLR4 signaling pathway. A specific embodiment of the disclosed inventive concept is directed to the use of an LPS/Lipid A compound used as a selective modulator of the TLR4 signaling pathway produced from the Variovorax paradoxus strain and the Rhodobacter sphaeroides strain.
The LPS/Lipid A compound employed herein may be obtained from the Variovorax paradoxus strain and/or the Rhodobacter sphaeroides strain by any suitable method, but in specific embodiments they are extracted using standard multi-step LPS extraction protocols, such as: (1) extracting freeze-dried bacteria with a solution of phenol/guanidine thiocyanate and collecting the water layer for freeze-drying; (2) resolubilizing the freeze-dried fraction in water; (3) ultrafiltration of the solubilized fraction to remove low molecular weight substances and salts; (4) affinity purifying the high-molecular weight fraction using a polymyxin B resin column such as Affi-prep polymyxin matrix material (Bio-Rad), from which an active fraction is eluted with 1 deoxycholate and, optionally; (5) performing additional purification using size-exclusion chromatography.
In some examples, multiple types of LPS extraction protocols are employed to obtain an LPS compound from the bacteria, and extraction procedures may be performed more than once. Once the LPS compound is extracted and purified from the bacteria, the Lipid A fraction may be prepared by acid hydrolysis or other suitable technique.
The one or more LPS/Lipid A compounds derived from gram negative bacterial strains, such as Variovorax paradoxus or Rhodobacter sphaeroides, may selectively modulate the TLR4 signaling pathway to alter inflammatory responses and to improve immune health in a variety of uses and applications. In an embodiment, the LPS/Lipid A compound derived from Variovorax paradoxus or Rhodobacter sphaeroides may be incorporated within an algal-based feed ingredient to improve gut health of poultry.
The disclosed LPS/Lipid A compound derived from Variovorax paradoxus or Rhodobacter sphaeroides may be used to improve the health of poultry through a variety of mechanisms.
Specific Treatment Compounds
The disclosed treatment compounds are based on one or more fresh water algal biomasses including bacterial strains as discussed above. More particularly, the algal biomass may include the Gram negative such as Variovorax paradoxus strain or Gram negative Rhodobacter sphaeroides strain.
The compounds share the common characteristic of the algal biomass referenced above and are used in animal treatment. The algal biomass-based products are fed to animals in a formulated diet such as a corn or corn-soybean meal (SBM) diet or are delivered in drinking water.
In all of is variations the ZIVO treatment compound is fresh water biomass containing gram negative bacteria and any products derived therefrom provided as animal drink and/or feed in combination of a feed additive, such as soy oil, preferably though not exclusively at a ratio of two parts soil oil to one part algal biomass. Once the biomass and feed additive are combined to the preferred premix level, the combined batch is poured or administered evenly into a ribbon mixer containing finished feed. The biomass is preferably provided in an amount of between about 0.1 lbs. composition per ton of finished feed to about 10.0 lbs. composition per ton of finished feed, is more preferably provided in an amount of between about 0.2 lbs. composition per ton of finished feed to about 5.0 lbs. composition per ton of finished feed, and is most preferably provided in an amount of between about 0.5 lbs. composition per ton of finished feed to about 2.0 lbs. composition per ton of finished feed. The ideal suggested and non-limiting ratio is about 0.5, 1.0, and 2.0 composition per ton of finished feed when used in conjunction with a vaccine.
Treatment Method
A non-limiting example of a method for the enhancement of a vaccine in poultry is set forth. It is to be understood that the following method is not intended as being the sole treatment method but is only exemplary. The studies set forth herein compare the disclosed inventive compound to the ionophorous anticoccidial drug, Salinomycin, and with day old coccidia vaccination. Treatment was provided to separate groups. However, no single group was treated with both an anticoccidial drug and a vaccine. All birds except a negative control group were challenged with Eimeria acervulina, E. maxima, and E. tenella on day 21.
Testing in all three studies set forth below was undertaken using a high oocyst dose vaccine, Coccivac-B52 (trademark, Intervet Inc.), a product sold by Merck Animal Health. According to the manufacturer, Coccivac-B52 contains E. acervulina, E. maxima, E. mivati, and E. tenella. The vaccine was mixed with water at a dosage of 240 mL of water per 1,000 doses. The vaccine-water combination was placed in a sealed vaccine container and mixed by stirring and inversion of the container. The mixed vaccine-water combination was sprayed onto each chick when one day old in a conventional spray cabinet. It was estimated that each chick ingested an equal number of droplets so as to result in the ingestion of about 240 μL dose per chick.
The live coccidia vaccination (Coccivac-B52 [Merck]) was administered to day old chicks in the studies according to the manufacturer's recommendation and the test groupings described in the tables to follow below.
First Study
The objective of the first study was to determine the most effective dose regime for the disclosed inventive natural product.
The rations consisted of non-medicated commercial-type broiler starter, grower, and finisher diets compounded according to NRC guidelines and contained feedstuffs commonly used in the United States. As a common regime rations for some but not necessarily all studies were fed ad libitum from date of chick arrival as follows: Starter—DOT 0 until DOT 14, grower DOT 14 to DOT 28, and finisher from DOT 28 to DOT 42 (study termination).
Diets were fed as crumbles in starter and pellets in grower and finisher. Experimental treatment feeds were prepared from a basal feed formulation. Quantities of all basal feed and test articles included in treatment group batches were documented and included with source data files. Treatment feeds were mixed prior to pelleting to assure uniform distribution of respective test articles.
Fifteen hundred birds (1,500) were assigned to six (6) treatment groups with ten (10) replicate pens per treatment and 25 birds per pen. The study began when birds were placed (day-of-hatch; DOT 0), at which time birds were allocated to experimental pens. Only healthy birds were selected. On DOT 0, group body weights were recorded by pen. No birds were replaced during the course of the study.
Analysis Methodology—First Study
Birds received treatment-appropriate feed from day 0 to day 42. On day 14, remaining starter feed was removed, weighed, and replaced with grower feed to day 28. On day 28, the remaining grower feed was removed, weighed back, and replaced with finisher feed to day 42.
On Day 20 of the study all birds received a mixed E. acervulina, E. maxima, and E. tenella coccidial inoculum (100,000 oocysts per bird of E. acervulina; 50,000 oocysts per bird of E. maxima; and 75,000 oocysts per bird of E. tenella). The inoculum was mixed into the feed found in the base of each pen's tube feeder.
On Day 26, three birds from each pen were selected, sacrificed, weighed, and examined for the degree of presence of coccidia lesions. The Johnson and Reid (1970) method of coccidiosis lesion scoring was used to score the infected region(s) of the intestine. The scoring was based on a 0 to 4 score, with 0 being normal and 4 being the most severe.
On Day 26, fresh fecal samples were collected from each pen. These representative samples were tested to determine the degree of oocysts shedding/cycling. Oocysts per gram feces were determined for each sample.
On Day 42, the trial was terminated.
The Feed Corrected Days 0 to 42 illustrated in
E.
E.
E.
acervulina
maxima
tenella
Results—First Study
The peak of coccidia vaccine reaction for treatments 3 and 6 was about 14 days. The vaccine with the inventive composition in the starter had significantly heavier body weight and numerically lower FCR than the vaccine only treatment at 14 days (Table 1). The coccidia challenge occurred on day 20 with peak damage day 25 to 27. The vaccine and inventive composition continued to have statistically heaviest body weight and lowest FCR (Table 2). The one pound/ton of inventive composition continuously had similar performance to the drug, Salinomycin. The 42 day results demonstrated the combination of vaccine with the inventive composition had body weights numerically heavier than the challenged control birds and 11 points heavier than the vaccine alone (Table 3). The vaccine with the inventive composition had significantly lower non-adjusted FCR to the no treatment (
As illustrated in
Second Study
Rations consisted of non-medicated commercial-type broiler starter, grower, and finisher diets compounded according to NRC guidelines and contain feedstuffs commonly used in the United States. Day-of-hatch male broiler chicks were utilized in the study. Rations were fed ad libitum from date of chick arrival as follows: Starter—day 0 until day 14, grower day 14 to day 28, and finisher from day 28 to day 42 (the end date of the study). Coccidia vaccination for treatments in Groups 2 and 4 through 7.
Two thousand one hundred birds (2,100) were assigned to seven (7) treatment groups with twelve (12) replicate pens per treatment and 25 birds per pen. The study began when birds are placed (day-of-hatch; DOT 0), at which time birds were allocated to experimental pens. Only healthy birds were selected. On DOT 0, group body weights were recorded by pen. No birds were replaced during the course of the study.
Analysis Methodology—Second Study
Birds received treatment appropriate feed from DOT 0 to DOT 42. All birds were weighed by pen on DOT 0, 13, 28, and 42. Feed added to each pen's feeder was weighed at the beginning of each formulation period on DOT 0 (starter), 13 (grower), and 28 (finisher). Any additional bags of feed were weighed (and documented) for each pen (as required) during each formulation period. Feed was distributed as needed to feeders from pre-weighed bags (assigned to each pen) throughout each period. Feed remaining in feeders (and feed bags if applicable) was weighed and disposed of on DOT 13, 28, and 42. Empty pan feeder weights were recorded prior to study initiation. The trial was terminated on DOT 42.
On Day 21 of the study, all birds except the negative control group received a coccidial inoculum containing: 100,000 oocysts per bird of E. acervulina; 50,000 oocysts per bird of E. maxima; and 75,000 oocysts per bird of E. tenella. The inoculum was mixed onto the feed found in the base of each pen's tube feeder.
On Day 26, three birds from each pen were selected, sacrificed, weighed, and examined for the degree of presence of coccidia lesions. The Johnson and Reid (1970) method of coccidiosis lesion scoring was used to score the infected region(s) of the intestine. The scoring was based on a 0 to 4 score, with 0 being normal and 4 being the most severe.
Also on Day 26, fresh fecal samples were collected from each pen. These representative samples were tested to determine the degree of oocysts shedding/cycling. Oocysts per gram were determined for each sample.
E.
E.
E.
acervulina
maxima
tenella
Results—Second Study
This study compared the inventive product with and without a live coccidia vaccine in broilers challenged at 21 days of age with Eimeria acervulina, E. maxima, and E. tenella. It should be noted that there was not a designed Clostridium perfringens challenge. However, due to cleaning prior to this study with a pressure washer, the hardy C. perfringens spores were still present resulting in a mild N.E. in the study.
At 13 days, effects of the coccidia vaccine cycling can be observed (Table 6). By day 28, the coccidia challenge has peaked and begun to significantly affect performance. Treatments with the inventive composition in the grower had nearly the same FCR as the no challenge control (Table 7). Treatments with the inventive composition in the grower also had the heaviest body weight of the challenged treatments except the 2.0 pound/ton in grower.
By 42 days, compensatory growth resulted in less significant feed conversion differences (Table 8). Treatments with the inventive composition in the starter and grower plus the 1.5 pound in the grower only had the heaviest body weights. These were nearly the same as the not challenged control. Necrotic enteritis occurred at the peak of coccidia vaccine cycling and was found in all treatments, including those with no vaccine. Treatments using the inventive composition appeared to reduce the N.E. except the treatment containing two pounds in the grower.
Five days post coccidia challenge, three birds per pen were lesion scored for E. acervulina, E. maxima, and E. tenella. All treatments lowered overall coccidia lesion scores (Table 9). This is readily observed in
Third Study
Rations consisted of non-medicated commercial-type broiler starter, grower, and finisher diets compounded according to NRC guidelines and contain feedstuffs commonly used in the United States. Day-of-hatch male broiler chicks were utilized in the study. Rations were fed ad libitum from date of chick arrival as follows: Starter—day 0 until day 14, grower day 14 to day 28, and finisher from day 28 to day 42 (the end date of the study). Coccidia vaccination for treatments 4 and 5 only.
One thousand five hundred birds (1,500) were randomly assigned to 5 treatment groups with 12 replicate pens per treatment and 25 birds per pen. The study began when birds were placed (day-of-hatch; DAY 0), at which time birds were allocated to experimental pens. On day 0, group body weights were recorded by pen. No birds were replaced during the course of the study.
Analysis Methodology—Third Study
All birds were weighed by pen on days 0, 14, 28, and 42. Feed added to each pen's feeder was weighed at the beginning of each formulation period on day 0 (starter), day 14 (grower), and day 28 (finisher). Feed was distributed as needed to feeders from pre-weighed bags assigned to each pen throughout each period. Feed remaining in feeders was weighed and disposed of on days 14, 28, and 42. The trial was terminated on day 42.
On day 21 of the study, all birds except the negative control group received a coccidial inoculum containing: 100,000 oocysts per bird of E. acervulina, 50,000 oocysts per bird of E. maxima, and 75,000 oocysts per bird of E. tenella.
On Day 26, three birds from each pen were selected, sacrificed, weighed, and examined for the degree of presence of coccidia lesions. Scoring was based on a 0 to 4 score, with 0 being normal and 4 being the most severe.
On Day 26, fresh fecal samples were collected from each pen. These representative samples were tested to determine the degree of oocysts shedding/cycling. Oocysts per gram were determined for each sample.
Results—Third Study
Prior to the challenge at day 14, there were small differences in performance parameters (Table 10). At the peak of the challenge on day 28, birds treated using the vaccine plus the disclosed inventive compound had a body weight gain similar to salinomycin (Table 11). On day 28, it was found that those birds treated with the vaccine plus the disclosed inventive compound demonstrated lower non-adjusted FCR and adjusted FCR than the vaccine only treatment, and demonstrated similar FCR to birds treated with salinomycin (Table 12).
The overall average coccidia lesion scores six days post challenge demonstrated that vaccine plus the disclosed inventive compound had either numerically or statistically significant lower coccidia lesions than the groups receiving the vaccine only or the challenged no feed additive control (Table 13). The oocysts per gram (OPG) of feces gave a similar trend as the lesion scores (Table 14). The peak of the challenge which should be on the day of feces collection (day 27) found the challenge control, salinomycin, had the highest count of oocysts at this time. (This result also confirms the loss of ionophore effectiveness noted above.)
For the entire 41-day study period, the group receiving the vaccine plus the disclosed inventive anticoccidial compound had improved FCR over the vaccine alone group, and results similar to salinomycin (Table 3). The heaviest weight gain of any challenged group was observed in the group receiving the vaccine plus the disclosed inventive compound (2.52 kgB). Only the birds not challenged were heavier at 2.65 kgA. The highest mortality (5.67%A) was in the vaccine only treatment and the lowest in the salinomycin only (1.33%B). It was found that the combination of the vaccine and the disclosed inventive compound had similar overall mortality to salinomycin (Table 12).
This application is a US. Non-provisional patent application of U.S. Provisional Patent Application No. 63/047,517, entitled “Enhancement of Vaccine Efficacy Via Biomass and/or Related Material In Animal Feed,” filed Jul. 2, 2020, which is herein incorporated by reference in its entirety for all purposes.
| Number | Date | Country | |
|---|---|---|---|
| 63047517 | Jul 2020 | US |
