Ahmed Mohamed Albehwar ? , Abeer Atia Tammam ? & Amr Ismail Hassan ? Abstract-Aim: Preparation of anti-rabies IgY in chicken egg yolk in addition to preparation of anti-rabies IgG in rabbits and evaluate their efficacy in protection against rabies infection in emergency cases simulated in mice.
Materials and Methods: Fifteen, 35week-old, Rhode Island Red (RIR) hens were used for preparation of anti-rabies IgY from egg yolk. It was found that the obtained IgY had a concentration of 2.32 g/dl. In addition, ten Bosket rabbits of about 3kg bodyweight were used for preparation of anti-rabies IgG which was found to have a concentration of 3.42g/dl. Both of chicken egg yolk IgY and rabbit IgG were found to be safe when they are inoculated intraperitoneally in mice.
Results: Serum neutralization test revealed that IgY and IgG had rabies antibody titers of 64 and 128 respectively. The tow preparations were tested for determination of their potency in experimentally infected mice with rabies virus on daily intervals post infection. It was found that intraperitoneal injection of infected mice with 0.5ml containing 116 mg of IgY and 171 mg of IgG were effective to prevent and overcome the progress of rabies signs when administrated on the 0; 1 st ; 2 nd and 3 rd day post exposure to the virus infection and not after that where treated mice on the 4 th to 7 th day post infection were unable to withstand the virus infection. The use of IgY and IgG with inactivated rabies vaccine showed the same results using IgY and IgG alone while the use of rabies vaccine alone did not provide efficient protection for the treated mice.
The use of anti-rabies IgY and IgG could be recommended as post exposure intervention providing a suitable period of protection until stimulation of the active immunity induced by rabies vaccine. In addition, the preparation of chicken IgY in a non-specific host provides safe, high potent product of lower cost than that prepared in rabbits or other mammals.
abies is a zoonotic disease that affects the central nervous system (CNS), provokes acute and fatal encephalitis in its mammal hosts. The disease etiologic agent is the rabies virus which is a neurotropic, RNA virus belonging to the order Mononegavirales, family Rhabdoviridae, genus Lyssavirus (1). Transmission of rabies infection usually occurs when infected saliva reaches a bite wound or skin scratches, or breaches mucous membranes. A rare route of rabies infection transmission include aerosol infection as in bat caves, ingestion of an infected carrier and transplacental infection. Transmission has occurred in man following transplants of corneas taken from infected patients. Not all animals or human bitten contract the infection. The severity, location, and multiplicity of bites inflicted on the victim, biotype of the virus and the susceptibility of the recipient influence the outcome of potential exposure to infection. Bites on the head and neck are associated with the shortest incubation period (2).
Rabies is a serious public health problem in developing countries, especially in Asia. Approximately 35000 to 50000 human deaths occur due to rabies each year (3). Administration of rabies vaccine along with antirabies immunoglobulin is known to prevent development of rabies; however, prompt and precise diagnosis is essential for rabies diagnosis, direct immunofluorescence detection of rabies virus antigens has been used worldwide as a rapid and reliable method.
Most rabies-specific antibodies used for diagnosis are made from sera of immunized mammals such as mice, rabbits and goats. However, producing a large amount of specific antibodies from these animals is time-consuming and labor intensive. There is a concern that handling live and large amounts of rabies virus to produce antigen may pose a potential risk of infection to laboratory personnel (4).
Recent advances in molecular biology together with newly invented methods of producing antigenspecific antibodies in egg yolk (IgY) have created new opportunities to develop a safe, convenient and inexpensive way of manufacturing various immunodiagnostics (5.6).
The IgY project which developed uncomplicated techniques for immunization of hens, isolation of egg yolk antibodies, and their applicability in various test systems creates continuous considerations in regard of changing from mammalian derived antibodies to egg yolk antibodies for both, (The National Laboratory for Immunology and diagnostics and the Internationally Orientated Service Laboratory).
Regarding commercially available polyclonal antibodies being produced worldwide it was found that 297 from almost 16,000 which is less than 2% of polyclonal antibodies deriving from chicken, and only three from these have been prepared from egg yolk, the rest apparently from chicken sera, it may be concluded that in general, chicken are potent antibody producers. Obviously, the IgY-technology needs further propagation (7).
The present work aims to prepare anti-rabies IgY in chicken egg yolk in addition to preparation of antirabies IgG in rabbits and evaluate their efficacy in protection against rabies infection in emergency cases simulated in mice.
Ethics approval: The experiments were carried out according to the protocol of Institutional Animal Ethics Committee and the authors had a permission of the animal owners at the private farms.
BHK21 was supplied by DPAVR, VSVRI and used in application of SNT to estimate rabies neutralizing antibody titers in the obtained anti-rabies IgY and IgG preparations.
Evelyn Rokitnicki Abelseth (ERA) strain of rabies virus adapted to BHK-21 cell line with a titer of 7 log 10 TCID 50 /ml was supplied by Department of Pet Animal Vaccine Research (DPAVR), Veterinary Serum and Vaccine Research Institute (VSVRI), Abbasia, Cairo, Egypt. It was used in serum neutralization test to estimate the induced antibody titer in immunized chicken.
Mice brain adapted rabies virus with a titer of 6.5 log 10 MLD 50 /ml was obtained from the DPAVR-VSVRI and used for experimental infection of mice.
Ten Bosket rabbits of about 3kg body weight were used for preparation of anti-rabies IgG after their immunization with the local rabies vaccine in a dose of 0.5 /rabbit inoculated subcutaneously on a week intervals for 5 successive weeks according to (8).
Weekly blood samples were collected from chicken and rabbits for 6 weeks for monitoring the levels of induced rabies antibodies in their sera.
iii. Mice Two hundreds and five weaned Swiss Albino mice (3-4 weeks old) were experimentally infected with 0.03 ml of CVS /mouse intramuscularly while five mice are kept without infection.
Inactivated cell culture rabies vaccine (ERA strain) was obtained from DPAVR-VSVRI and used for inoculation of chicken and rabbits to prepare anti-rabies IgY from chicken egg yolk and IgG from rabbit sera.
IgY was separated from egg yolk with PEG-6000 as described by (9). and its protein content was measured as described by (10). Where it was 2.32 g /dl.
Rabies IgG was precipitated from rabbit serum using ammonium sulphate according to (11). And its protein content was estimated according to (10), and it was 3.42 g /dl.
Quality control testing of the obtained preparations were carried out following the directions of (3). Including sterility, safety and potency tests.
SNT was carried out to estimate rabies neutralizing antibodies in test chickens and rabbits as described by (12), and the antibody titer was determined as the reciprocal of the final serum or immunoglobulin preparations dilution which neutralized and inhibited the appearance of the cytopathic effect (CPE) of 100 TCID 50 of rabies virus.
The previously mentioned infected mice were divided into seven groups where each of the first 5 groups included 40 mice, where the 1 st and 2 nd groups received anti-rabies IgY and IgG using a dose of 0.5 ml/mouse inoculated intraperitoneally containing 116 mg of IgY and 171 mg of IgG respectively on daily intervals started from the day of experimental infection up to 7 days post infection. The 3 rd and 4 th groups were treated with IgY with rabies vaccine and IgG with rabies vaccine respectively using the same mentioned doses (As in groups 1and 2) where rabies vaccine was inoculated once in a dose of 0.5ml /mouse inoculated intraperitoneally on the same day of infection. The 5 th group was treated with the rabies vaccine alone with the same dose.
The 6 th group of 5mice was kept infected without any treatment (Control +ve). In addition the Fifteen, 35 week-old Rhode Island Red (RIR) hens, were used for egg yolk IgY preparation after their immunization with local rabies vaccine in a dose of 0.5 ml/hen inoculated subcutaneously on a week intervals for 5 successive weeks according to (8). Eggs were collected weekly for 6 weeks from these hens where the anti-rabies IgY were separated and titrated. f) 7 th group of 5 mice was kept without infection as (Control -ve).
The present results showed that the prepared anti-rabies IgY and IgG were free from foreign contaminants (aerobic and anaerobic bacteria, fungi and mycoplasma) and safe, inducing no abnormal signs in inoculated mice either generally or at the site of inoculation as shown in Table (1). These findings come in agreement with the recommendations of (13). In addition, estimation of the protein contents in the prepared anti-rabies chicken egg yolk IgY and rabbit IgG revealed that they had levels of 2.32g/dl and 3.42g/dl respectively Table (1). Such high levels of protein contents could be attributed to the formation of antibodies which mainly consisted of globulins as stated by ( 14), ( 15), ( 16) and (17).
Table (2) demonstrated that rabies serum neutralizing antibody titers began to appear in the sera of chickens and rabbits by the 1 st week post immunization recorded their highest levels (64 and 128 respectively) by the 5 th week. These findings showed that rabbits have higher antibody titers than that of chickens, the thing which could be attributed to the host susceptibility as rabies is a mammal host specific virus (4). It was found that the levels of rabies antibodies in chicken and rabbit sera were increased gradually started from the 1 st week post immunization recording their peak by the 5 th week. In this respect similar findings were obtained by ( 18), (19)., (20) and (21). who considered such sera as hyper-immune preparations depending on their high antibody titers where the protective rabies antibody titer is 0.5 IU (about titer of 32). Treatment of experimentally rabies infected mice with the prepared anti-rabies IgY and IgG showed that the best time for administration of anti-rabies treatment is from 0 time to 2 days post exposure to virus infection providing 100% protection. This protection rate decreased to 40, 20 and 0% for treatment with IgY and 60, 30 and 0% for treatment with IgG on the 3, 4 and 5 days later as shown in Table (4). Non-treated infected mice showed typical rabies signs represented by paralysis of the hind limbs and tail by the 4 th day post infection ended with death while non-infected non-treated mice remained healthy allover the experimental period. These findings come in complete agreement with what reported by (21)., (22). And (17). who concluded that post exposure treatment through passive immunization of the victim should be carried out as soon as possible post exposure to viral infection recommended the same present recorded times. Similar results were obtained in case of infected mice treated with either of IgY or IgG with rabies vaccine while treatment with rabies vaccine alone was unable to protect mice against rabies virus infection the thing Rabies vaccine alone These mice were unable to withstand the virus infection where the active immunity required longer time to be effective (0%) 6
Infected ¬ treated mice showed typical rabies signs stared by the 4 th day post infection (control +ve) (0%)
Non infected &non treated mice remained healthy allover the experimental period (control -ve)
Depending on the obtained results through the present work, it could be concluded that both of antirabies chicken egg yolk IgY and rabbit serum IgG are able to withstand rabies infection when they are administrated on the optimum time post exposure as simulated in mice and further studies are in need to evaluate such preparations in farm animals. In addition, the preparation of chicken IgY in a non-specific host provides safe, high potent product of lower cost than that prepared in rabbits or other mammals where a huge amount of IgY could be obtained through the egg production life of hens with easily housing and simple management requirements.
Ahmed Mohamed Albehwar immunized hens and rabbits with rabies vaccine and collected egg of hens and serum of the rabbits and prepared the IgY and IgG solutions, applied SNT on the obtained preparations to determine the antibody titers, evaluated the results and revised the data and write the research.
Abeer Atia Tammam estimated the IgY and IgG content in the obtained preparations and made the quality control testing on both of them besides sharing in application of SNT and writing the research.
Amr Ismael Hassan made experimental Infection of mice then immunized them with the obtained preparations to determine their protection rates and analyzed and tabulated the obtained data. All authors read and approved the final manuscript.
| Tested preparation | Protein content | Sterility | Safety | Potency |
| Anti-rabies IgY | 2.32g/dl | Sterile | Safe | Potent |
| Anti-rabies IgG | 3.42g/dl |
| Tested serum | Mean serum rabies neutralizing antibody titer*/WPI** | |||||
| 1WPI | 2WPI | 3WPI | 4WPI | 5WPI | 6WPI | |
| Chicken | 8 | 16 | 32 | 32 | 64 | 64 |
| Rabbit | 8 | 16 | 32 | 64 | 128 | 128 |
| *Antibody titer = the reciprocal of the final serum dilution which neutralized and inhibited the CPE of 100TCID 50 of rabies virus | ||||||
| **WPI= week post immunization | ||||||
| On the other hand, rabies neutralizing antibody | parallel manner confirming that such preparation could | |||||
| titers were found to be 64 and 128 in chicken egg yolk | be considered as hyperimmune products against rabies | |||||
| IgY and rabbit serum IgG respectively Table (3) in a | virus. | |||||
| Tested preparation | Mean rabies neutralizing antibody titer*/WPI** | |||||
| 1WPI | 2WPI | 3WPI | 4WPI | 5WPI | 6WPI | |
| Chicken egg yolk IgY | 0 | 16 | 32 | 64 | 64 | 64 |
| Rabbit IgG | 4 | 8 | 16 | 32 | 64 | 128 |
| *Antibody titer = the reciprocal of the final immunoglobulin preparations dilution which neutralized and inhibited the CPE of | ||||||
| 100TCID 50 of rabies virus | ||||||
| **WPI= week post immunization | ||||||
| Number of survived mice/number of treated mice = protection% | |||||||||
| Group | Received | 0 DOT* | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| Treatment | DOT | DOT | DOT | DOT | DOT | DOT | DOT | ||
| 1 | IgY | 100 | 100 | 100 | 40 | 20 | 0 | 0 | 0 |
| 2 | IgG | 100 | 100 | 100 | 60 | 30 | 0 | 0 | 0 |
| 3 | IgY& rabies vaccine | 100 | 100 | 100 | 40 | 20 | 0 | 0 | 0 |
| 4 | IgG &rabies vaccine | 100 | 100 | 100 | 60 | 30 | 0 | 0 | 0 |
| 5 | |||||||||
| Efficacy of Antirabies IgG and IgY on Protection of Mice Against Experimental Viral Infection as a Model | ||||||||||||||||||||||
| for Emergency Intervention | ||||||||||||||||||||||
| Year 2016 | Year 2016 | |||||||||||||||||||||
| Volume XVI Issue I Version I | Volume XVI Issue I Version I | |||||||||||||||||||||
| D D D D ) | D D D D ) | |||||||||||||||||||||
| ( | ( G | |||||||||||||||||||||
| ????????? / ?????? | 2.32 | ???? ????????????? ??????? ???? ????? ????? ???????? ?????? ???????? ????? ???? ?ï»?"?? ???????? ??ï»?"????? ???????? | IgG | ??? | IgY | ??????? ??????? ????? ????? ???? | ||||||||||||||||
| ????????? ????? | IgG | ??? | IgY | ?�"?? | 128 | ??? | 64 | ?????????(? ??????? ???????? ?)????????? ???????�"? ????????�"? ???????? ??????? ??????? ?????? ????????? ????? ????????? ?????/? | 3.42 | ??? | ||||||||||||
| ???????????? ????? ??????? ?�"?? ?????????�"? ????????? ???????? ??????? ?�"?? ?????????????? | ||||||||||||||||||||||
| ???? | 30 | ? | 60 | ?????? | IgY | ?????????? ??????? ????? % | 0 | ???? | 20 | ? | 40 | ????? ??????�"? ????? ????? ??????? % | 100 | |||||||||
| ???????? ???????? ????? | 5 | ? | ? 4 | 3 | ??????? ?ï»?"?? | IgG | ????????? ????? % | 0 | ||||||||||||||
This work was funded by Veterinary Serum and Vaccine Research Institute, Abbasia, Cario, Egypt.
Competing interests: The authors declare that they have no competing interests.
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