Abstract:
The invention refers to anticoccidial compositions consisting mainly of yolk immunoglobulins derived from eggs of hens immunized with one or more  Eimeria  species. The invention refers also to the use of said anticoccidial compositions for prevention or treatment of coccidiosis. The administration of immunoglobulins, both in liquid as well as in powder or pellet presentation, decreases mortality, lesions, oocysts counts and increases weight gain of receiving animals.

Description:
FIELD OF THE INVENTION  
       [0001]     The present invention consists in offering new compositions for treatment and prevention of infections caused by  Eimeria  parasites in birds principally based on an oral administration of such compositions, which such compositions are suitable to be used as part of food compositions.  
       BACKGROUND OF THE INVENTION  
       [0002]     There are two ways of protecting animals against infectious agents: they can be exposed to antigens derived from an infectious agent to stimulate a protective immune reaction or they can receive a preformed antibody obtained from an immunized subject.  
         [0003]     The first way is conducted through different types of vaccines: freeze-dried live viruses or bacteria, through dead viruses or bacteria in oily emulsions; and recently through the creation of cloned and recombinant vaccines. Each of them presents advantages and drawbacks with regard to protection, immune response and protection duration. Besides, in some cases, the first general approach may lead to undesirable complications in the host due to the vaccination (Tizard, I. R. 1998)  
         [0004]     The second form of protection, also called passive immunity, includes the transference of specific antibodies against infectious agents into A susceptible subject.  
         [0005]     Traditionally, at the research level, antibodies are mainly obtained from mammals and less frequently from birds. The types of antibodies obtained are monoclonal and polyclonal antibodies in mammals, and polyclonal antibodies in birds (Larsson, et al. 1993).  
         [0006]     In the case of birds, the chicken is the only species from which antibodies are obtained in a most accessible and highly defined form. The main serum antibody present in the chicken is IgG, even though IgG is transported to the egg in a similar way to the transfer of mammal IgG through the placenta.  
         [0007]     In the egg, IgG is found in higher concentrations in the yolk although it is also found in small concentrations in the white; it is even found in larger quantities in the yolk than in the hen serum (Larsson, et al. 1993).  
         [0008]     To have an idea of the quantity of antibodies made in the hen, we must take into account that an egg-laying hen produces approximately 5 to 6 eggs per week with a yolk volume of about 15 ml. Thus, in a week, a hen produces antibodies in yolk equivalent to 90-100 ml of serum or 180-200 ml of whole blood. This is to be compared with the 20 ml of whole blood given per week by an immunized rabbit. Obviously if we use animals such as horses or cows, the quantity of serum and antibodies is larger than in the egg but it is more expensive and more painful for the animals.  
         [0009]     Egg yolk antibodies (immunoglobulins-Ig&#39;s) have been employed as tools for diagnosis and therapy (Schmidt, et al. 1989). Thus, taking advantage of its phylogenetic difference with mammal immunoglobulins, the Ig&#39;s have presented several advantages when used in immune diagnosis. For example, yolk Ig&#39;s has been used to detect several viruses through ELISA, immunodiffusion, and immunofluorescence. Because of their low isoelectric point, compared to human IgG, they are employed in electrophoresis assays for the quantification of immunoglobulins in the serum of several animals (Altschuh, D. 1984, Larsson, et al. 1988, Larsson, et al. 1992, Larsson, et al. 1993, Schade, R. 1996). With regard to their therapeutic application, the Ig&#39;s have been used as immunotherapy in some scientific fields. For example, the administration of egg yolk immunoglobulins orally has prevented rotavirus infections in mice, bovines, and pigs, among others (Ikemori, et al 1992, Kuroki, et al 1994, Marquardt, et al 1998). Moreover, they have been used as antivenins against viper and scorpions that can be injected to neutralize the toxins without the risk of anaphylactic reactions, such as those reactions commonly caused by antivenins obtained from immunized horses (Larsson, et al. 1993). A further application has been to prevent caries caused by  Streptococcus mutans  in humans (Hatta, H. et al 1984).  
         [0010]     In the past, several methods have been used in attempts to control coccidiosis including the use of chemical drugs and vaccines including live and recombinant vaccines. However, there are problems with existing coccidiosis vaccines, such as reduced efficacy, cross-infection with other parasites (e.g.,  Clostridium  spp.) and poor avian performance. Monoclonal and recombinant antibodies are still under experimentation. Vaccines would have some positive impact in preventing infections but they are not suitable for reducing ongoing infections.  
         [0011]     At present the use of antibodies obtained from either mammals or birds for treatment or prevention of coccidia infections cause by  Eimeria  species has not been reported. Reynolds, in U.S. Pat. No. 5,807,551, describes a method for inducing long-term passive immunity in birds but research was limited to Newcastle disease virus and infectious bursal disease virus. Sterling et al., in U.S. Pat. No. 5,753,228, discloses a method for treating infections caused by  Cryptosporidium parvum  in mammals. Thus, there exists a need for efficacious coccidiosis and coccidiosis control methods.  
       SUMMARY OF THE INVENTION  
       [0012]     An object of at least one of the preferred embodiments disclosed herein is to offer prevention and treatment methods of coccidia infections in birds with the use of compositions suitable for the oral administration through drinking water or mixed with food or through the use of anticoccidial food.  
         [0013]     The invention also provides new compositions from anticoccidial immunoglobulins obtained from the egg of hyperimmunized hens, and more specific from the yolk of such eggs. The new compositions consist of one or more anticoccidial immunoglobulins.  
         [0014]     The invention also prevents weight loss to the animals treated with a composition of anticoccidial immunoglobulins specifically directed against  Eimeria  type coccidia parasites.  
         [0015]     Moreover, within the scope of the present invention, the use of egg yolk anticoccidial immunoglobulin compositions against parasites is claimed to eliminate or substantially reduce the symptoms, mortality and ill transmission in the treated animals.  
         [0016]     Finally, the invention discloses a process to prepare a composition or a food containing such immunoglobulins specifically directed against coccidia parasites of chicken obtained from the egg yolk of hyperimmunized hens.  
         [0017]     Through the present invention, in the case of animals, the quantity of oocysts of protozoans in the digestive tract decreases, and the productive parameters of the animals improve. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0018]     The detailed characteristics of the novel invention disclosed herein are clearly shown in the following description and in the attached figures.  
         [0019]     The present invention is based on the fact that the immunoglobulins extracted from the aqueous phase of egg yolk offer protection against parasitic illnesses or infection.  
         [0020]     To obtain the immunoglobulins (Igs) specifically directed against animal parasites, it is necessary to have a vaccination schedule in a flock of SPF (Specific Pathogens Free) birds.  
         [0021]     The vaccination schedule can include the administration, orally, subcutaneously or through any other way, of an effective amount of the antigen to reduce infection or its symptoms. The effective amount is calculated to induce an effective response without causing infection.  
         [0022]     The parasite could be administered live or dead. Ways to kill parasites are through chemical or physical methods known to those skilled in the art.  
         [0023]     The immunization program includes inoculations of each particular coccidia-causing parasites in order to development specific immune response against such parasites.  
         [0024]     In the preferred embodiments of the invention the antigen administered could be the parasite in any stage of its lifecycle, including oocyst, sporozoites, merozoites or whole parasites from the family of  Eimerias  consisting of  E. tenella, E. acervulina, E. maxima, E. necatrix, E. brunetti, E. mitis, E. praecox  or  E. hagani.    
         [0025]     Through extensive experimentation we have found that the use of a mix of two or more species such as  E. tenella, E. acervulina, E. maxima, E. necatrix, E. brunetti, E. mitis, E. praecox  or  E. hagani  provides better results than using only one particular species. The main reason is that each  Eimeria  species has a different mechanism of action due to little difference in structure between them.  
         [0026]     To obtain the anticoccidial immunoglobulins, live or dead parasites are administered orally in an antigen suspension combined with parenteral administration of an oily or semi-oily vehicle or within vehicle of any other type in such a way as to ensure an immune response in the hen.  
         [0027]     The parasites administered are selected from the following  Eimeria &#39; species:  E. acervulina, E. maxima, E. tenella, E. necatrix, E. brunetti, E. mitis, E. praecox, E. hagani  or combination of two or more species.  
         [0028]     The recommended parasites dose is 4,000 to 20,000 parasites in any stage of its lifecycle contained in 0.3-0.8 ml of the antigen solution used for immunized laying hens in the growing stage at 8, 12 and 16 weeks of age. A booster vaccination could be carried out every 3-8 weeks.  
         [0029]     Once the eggs from the immunized laying hens show anticoccidial immunoglobulins, measured by microneutralization test (MNT), the eggs are collected.  
         [0030]     The yolk from the eggs containing anti- Eimeria  immunoglobulins is collected, separated and purified from the white by well known methods by a person skilled in the art and diluted 1:2-1:8 with a 0.005-0.01% sodium azide solution or any other preservative solution to obtain liquid anticoccidial composition. The purification step consists optionally of lipoprotein removal.  
         [0031]     It is preferred the use of specific liquid anticoccidial composition with titles of 1:16-1:128.  
         [0032]     The anti- Eimeria  immunoglobulin yolk contains one or more specific anti- Eimeria  immunoglobulins, preferable 0-35% of  E. acervulina  specific immunoglobulin, 0-20% of  E. brunetti  specific immunoglobulin, 0-20% of  E. maxima  specific immunoglobulin and 25-90% of E. tenella specific immunoglobulin.  
         [0033]     The anti- Eimeria  immunoglobulin yolk could be obtained by immunization of the hen with an effective amount of a mix of two or more antigens consisting of the different  Eimeria  species.  
         [0034]     In another preferred embodiment of the invention the anti- Eimeria  immunoglobulins yolk is prepared by obtaining each one of the specific anti- Eimeria  immunoglobulins and mixing in correct quantities each specific anti- Eimeria  immunoglobulin yolk.  
         [0035]     In a preferred embodiment, a liquid anticoccidial composition is obtained by mixing anti- Eimeria  immunoglobulins yolk. Liquid anticoccidial composition has the following specification Anti- Eimerias  immunoglobulins yolk 15-30%, water 70-85% and preservative 0.001-0.03%.  
         [0036]     The liquid composition is suitable for both coccidia infection treatment and therapeutic methods. Liquid anticoccidial composition could be administered in broiler birds from 2 weeks of age to eight weeks as preventive method at a dose of 0.5-2 ml per bird per oral route daily during 1-2 weeks and as therapeutic at a rate of 2-4 ml per bird by oral route daily during 2-3 days.  
         [0037]     For coccidia infection prevention, it was found through several experiments that good results are also encountered if the anticoccidial immuglobulins composition is added to bird&#39;s food.  
         [0038]     In order to add to the food, the anti- Eimeria  immunoglobulin yolk obtained after yolk purification could be dehydrated through the Spray Dried method.  
         [0039]     The powder anticoccidial immunoglobulins yolk is mixed with any suitable food, by any well known methods of food producing, at a rate of 0.1-1 kg of powder anticoccidial yolk per ton. Bird food with anticoccidial immunoglobulins yolk could also being formulated in pellets.  
         [0040]     Anticoccidial food could be administered during the whole growing period of animals to prevent infections caused by coccidia parasites.  
         [0041]     As could be inferred from the present invention disclosure, specific  Eimeria  specie or species to produce the anticoccidial composition could be selected depending on that specific species found in the geographical area where the animals are growing.  
         [0042]     The quality control tests of anticoccidial immunoglobulins include:  
         [0043]     1.—Sterility test to check if the product is free from contamination by bacteria, fungi and yeast according to the Code of Federal Regulations of the United States of America.  
         [0044]     2.—Antibody quantification. ELISA techniques or any other method like microneutralization test in chicken cell cultures were used to detect specific immunoglobulins for different coccidia species.  
         [0045]     The yolk and serum titers by MNT were expressed as the reciprocal of the highest dilution in which citopathic effect was observed, the Igs titer obtained must be 1:4-1:128.  
         [0046]     3.—Innocuous assay in order to detect physiologic alterations or injury in the immunoglobulins recipient.  
         [0047]     Hereinafter tests are presented as non-limiting examples showing the use of immunoglobulins against Coccidia in broiler chicken, although other birds may be used.  
       EXAMPLE 1  
       [0048]     A liquid anticoccidial composition was prepared according to the detailed invention using oocyst of  E. tenella  parasite having 17% of Anti- Eimeria  immunoglobulins yolk with a titer of 1:8, 0.001% of sodium azide and water. 40 broiler chickens 3 weeks-old were fed without any anticoccidial drugs during ten day and then 5 groups were formed. Group 1 remained as Control Group without anticoccidial composition treatment.  
         [0049]     Group 2, 3, 4 and 5 received respectively 0.5, 1, 2 and 4 ml of liquid immunoglobulins composition by oral route, one dose at day 1 and a second dose 8 days later. At 31 days old, birds were challenged with 200,000 sporulated oocysts of  E. tenella  by oral route. The animals were sacrificed 7 days after the challenge to determine lesions according to well known Johnson and Reid method, based on severity of lesion in a scale ranged from 0 to 4, where 0 mean no lesion and +4 sever lessions, and caecal weight. It is well known that animals with  E. tenella  infection increase their caecal weights because there is an inflammatory process resulting in a swelling of the caecal wall. The results are shown in Table 1. It can be observed that the all groups with treatment maintain lower caecal weight compared to caecal weight of control group. The lesions of treated groups were lower than in the control group.  
                               TABLE 1                               Liquid       Lesion               immunoglobulins   Caecal   score           No of   composition   weight   Johnson-       Group   birds   Volume ml   g   Reid Scale                   1   40   0.0   24.3   +4       2   40   0.5   22.9   +2       3   40   1.0   21.0   +2       4   40   2.0   15.0   +2       5   40   4.0   13.6   +3                  
 
       EXAMPLE 2  
       [0050]     Two groups of 25 broiler birds were formed: Group 1 received 1 ml of liquid immunoglobulins composition, prepared as described in Example 1, on daily basis through drinking water during two weeks. The control group did not receive any treatment. After two weeks of treatment, both groups were challenged with 150,000 sporulated oocysts of  E. tenella  per ml by oral route.  
         [0051]     All the animals were sacrificed 7 days later and the caecal lesions were qualified according to Johnson and Reid method. Mortality was also recorded. Results are given in Table 2. In the treated group mortality, caecal weight and lesion score according to Johnson-Reid scale were lower than the control group.  
                                                     TABLE 2                                       Lesion                   Caecal   score           No of   Mortality   weight   Johnson-       Group   birds   %   (g)   Reid Scale                                1   25   55   16.2   +2       Control   25   83.3   35.0   +4                  
 
       EXAMPLE 3  
       [0052]     Three groups of 30 one-week old broiler birds were formed. The first group received 2 ml of liquid immunoglobulins composition against coccidia, consisting of 17% of anti- Eimeria  immunoglobulins yolk suspension with titer of 1:32 obtained through hen immunization with 800 oocysts from  E. acervulina,  450 oocysts from  E. brunetti,  450 oocysts from  E. maxima  and 1100 oocysts from  E. tenella , through drinking water on a daily basis during 14 days and they were fed with food without anticoccidial drugs. The second group was fed with a commercial polyether anticoccidial drug and the last group remained as a control group.  
         [0053]     After the treatment period with liquid immunoglobulins composition, all groups were challenged by oral route with 200,000 sporulated oocysts per ml of a mixture of  E. acervulina  (72.5%),  E. brunetti  (12.5%),  E. maxima  (12.5%) and  E. tenella  (2.5%). The parameters to evaluate were: weight gain, oocysts counts in caecal content and conversion index (CI), calculated as the ratio weight feed to weight gain. Results are presented in Table 3. The treated group showed 68.5% weight gain compared to the control group. With regard to oocysts recovery, in the treated group there was no recovery while in the group that received food with anticcoccidial drug, the average count was 192,000 and 288,000 oocysts in the control group. The better conversion index (CI=2.23) was observed in the treated group compared with group 2 (CI=3.22) and group 3 (CI=3.90).  
                                                             TABLE 3                               Final   Oocysts   Oocysts in               No of   weight   in caeca   intestinal   Conversión       Group   Birds   gain (g)   contents   contents   Index                                1   30   337.0   0.0   0.0   2.33       2   30   234.0   288,000   192,000   3.22       3   30   200   576,000   288,000   3.90                  
 
       EXAMPLE 4  
       [0054]     75 two-weeks old age broiler chickens were divided in two groups of 30 and one group of 15 birds. The groups were identified as Group A, B and C. During the whole experiment the birds consumed food without anticoccidial drug. Group A received 2 ml of liquid immunoglobulins composition mentioned in Example 3 on a daily basis by drinking water during 4 weeks. After 4 weeks the bird weights were recorded. Group A and B were challenged with 150,000 sporulated oocysts of  E. tenella  per ml by oral route. Group B received food with anticoccidial drug after the challenge. Group C remained as a control group. One week later all birds were sacrificed and were scored the following data weight gain, lesions scored by Johnson and Reid method. Results are showed on Table 4 where it can observed a better gain weight (208.2 g) in Group A treated with immunoglobulins compared with Group B (46.7 g) and control group (6.7 g). Also Group A showed less lesions (scale +2) than Group B (score+4) and control group (score+4).  
                                                                               TABLE 4                                           Lesion           Weight (g)   Score                No of   Before   After       Johnson-       Group   Birds   Challenge   challenge   Difference   Reid Scale                    A   15   636.6   844.8   208.2   +2       B   15   626.6   673.3   46.7   +4       C   15   613.3   620.0   6.7   +4                  
 
       EXAMPLE 5  
       [0055]     Three groups of 10 3-weeks old age broiler chickens were formed and maintained on wire cages. All birds were challenged with 100,000 sporulated oocysts of  E. tenella  per ml by oral route. After 4-5 days, Group A of 10 birds were treated with 2 ml of liquid immunoglobulins composition of Example 3 on a daily basis during 2 days; Group B of 10 birds were treated with a commercial anticoccidial drug according to manufacturer instructions (1 ml of anticoccidial drug per 1000 ml of water on daily basis during 2 days). Group C remained as the Control Group without treatment.  
         [0056]     All birds were fed with a food without anticoccidial drug or growing additive during whole experiment.  
         [0057]     Four days later all birds were weighted and sacrificed, in order to establish weight gain and lesions on caecal according to Johnson and Reid scale.  
         [0058]     Results are showing on Table 5. Group A showed a little better weight gain than birds treated with anticoccidial drug but group A treated with liquid immunoglobulins composition showed less lesions (average lesion of 2.1) compared with lesions of anticoccidial drug treated group (3.0 average). The Control Group showed an average score of 3.5 and there was no practical weight gained. In normal conditions birds infected with coccidian lose weight and have high mortality.  
                                                         TABLE 5                                           Average   Average               No of   Weight   Score           Group   Birds   Gain (g)   lesion                                        A   10   107.5   2.1           B   10   103.9   3.0           C   10   5.5   3.5                      
 
       EXAMPLE 6  
       [0059]     Five groups of 10 2-weeks old broiler chickens were formed. Four types of food were manufactured; three of them were in powder presentation containing 200 g/ton of food. The three foods contained a titer of immunoglobulins of 1:4, 1:64 and 1:128 measured previously by a microneutralization test in kidney chicken cell culture; the last one food was manufactured without immunoglobulins and without growth additive.  
         [0060]     Group 1, Group 2 and 3 were treated during a week with a anticoccidial food with a titer of 1:4, 1:64 and 1:128 respectively; Groups 4 (Positive Control) and 5 (Negative Control) were fed with a food without anticoccidial drug nor anti- Eimeria  immunoglobulin yolk powder. A week later all birds except the Negative Control were challenged with 100,000 sporulated oocysts of  E. tenella  by oral route. The feed schedule was maintained during entire experiment. One week after the  E. tenella  challenge all birds were sacrificed and necropsied to score lesions by the Johnson and Reid method. A sample of feaces was taken per group to determine counts of oocysts per weigth. Results are shown on Table 6 where it can be observed that animals fed with anticoccidial food with a titer of 1:4 showed lesions of +3 scale similar to the control group. However, animals fed with anticoccidial food with a titer with 1:64 and 1:128 showed lower score lesion +2 considering only a week of treatment. As same way it could observed a decrease in count of oocysts per gram of faeces while titer or immunoglobulins was increased.  
                                                         TABLE 6                                           Average   Count of               No of   Score   oocysts per g           Group   birds   lesion   of faeces                                        1   10   3   765,000           2   10   2   615,000           3   10   2   407,000           4   10   3.5   805,000           5   10   0.5   0                      
 
       EXAMPLE 7  
       [0061]     Two groups of 25 two-weeks old broiler chickens were formed. The groups were identified as Group A and B. During three weeks the birds consumed food without anticoccidial drug. During the next week, Group A was fed with food added with 500 g/ton of food of powder immunoglobulins composition obtained by spray dried from yolks of Example 3, and the same dried yolks were used to form pelleted food with same rate of immunoglobulins per food and it was administered to Group B. After one week that the bird were fed with anticoccidial food, Group A and B were challenged with 100,000 sporulated oocysts of  E. tenella  by oral route. Both groups received the same food as previous week with anticoccidial immunoglobulins after the challenge. One week later all birds were sacrificed and were scored the following data: weight gain and lesions scored by Johnson and Reid method. Results are showed on Table 7 where it can observed similar results in gain weight and lesions.  
                                   TABLE 7                                               Lesion                       Score               No of   Weight   Johnson-           Group   Birds   gain(g)   Reid Scale                           A   25   427   +1.1           B   25   483   +1.4                      
 
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