APCRI JOURNAL Official Journal of the Association for Prevention and Control of Rabies in India (Regd.) Volume 2 Issues 1 & 2 January - July 2001
Foreword From editor's desk Review Article: Rabies Vaccines and Vaccination - Y. U. B. Rao Original Articles: 1) TRANSMISSION OF RABIES : AWARENESS INCREASING IN DEVELOPING COUNTRIES - J. K. Dutta 2) A CLINICAL ALGORITHM OF POST-EXPOSURE RABIES PROPHYLAXIS FOR CLINICIANS IN INDIA - M. K. SUDARSHAN 3) A CASE SERIES REPORT OF SUCCESSFUL POST-EXPOSURE TREATMENT OF PROVEN RABID ANIMAL BITES. - M. K. SUDARSHAN, B.J. MAHENDRA, D.H. ASHWATHNARAYAN & K ROHIT 4) COMBINED IMMUNIZATION WITH PURIFIED CHICK EMBRYO CELL VACCINE AND ALUMINIUM ADJUVANTED PURIFIED TETANUS TOXOID FOR RABIES PROPHALYXIS - S. N. MADHUSUDANA, M KAPOOR AND H SINGH 5) PRODUCTION OF RABIES GLYCOPROTEIN IN MUSK MELON PLANTS AND TESTING ITS IMMUNOGENICITY IN EXPERIMENTAL MICE - P. H. RAMANJINI GOWDA AND S. N. MADHUSUDANA 6) A SIMPLE INDIRECT IMMUNOFLOURESCENCE TEST FOR ASSAYING RABIES ANTIBODIES. - R. Shamasundar, K. Arvind, M.S. Suja, S Sarswati and S. N. Madhusudhana 7) A SIMPLE LATEX AGGLUTINATION TEST FOR RABIES ANTIBODIES. - S. Saraswati and S. N. Madhusudana 8) A PASSIVE HAEMAGLUTINATION FOR RABIES ANTIBODIES USING PURIFIED RABIES GLYCOPROTEIN COUPLED TO SHEEP ERYTHROCYTES. - S. saraswati, B.V.S. Raju, K. Chithra, R. Shamasundar and S. N. Madhusudhana 9. PURIFIED VEROCELL RABIES VACCINE : A CHOICE VACCINE FOR INFANTS AND CHILDREN. - J. K. Dutta 10. TOPOGRAPHICAL DISTRIBUTION OF RABIES VIRUS ANTIGEN IN DOG BRAIN - M. S. S uja, Anita Mahadevan, Madhusudhana S. N., Vijayasarathi and S. K. Shankar
INTRADERMAL IMMUNIZATION WITH CELL CULTURE VACCINES: A COST EFFECTIVE STRATEGY TO REPLACE NERVE TISSUE VACCINE
It is a matter of great concern and shame that even at the dawn of 21st century, we are still having thousands of human deaths due to rabies. We all know that reasons for the continued rabies problem in our country and we continue to repeat these at various international or national forums. We have conveyed our concerns to the health authorities individually and through the association. One major step in the right direction is to stop completely the use of sheep brain vaccine. The state of Kerala was the first to replace Semple vaccine with tissue culture vaccines. Recently I have been informed that the Government of Andhra Pradesh has decided to stop use of Semple vaccine in all government run hospitals and dispensaries. I wish such bold steps are taken by other state governments as well. Approximately about 1 million people take post- exposure treatment with Semple vaccine in our country. If all these people needs to be administered with 5 doses of a modern cell culture vaccine the government has to bear the burden of 5 million doses of these vaccines and spend approximately 125 crores. Considering the amount of financial allocations for the health services, this is a huge burden on the state or central government. Is there a way to reduce this burden on the government exchequer? We may find a way if we only look at our neighboring countries. For instance, countries like Thailand and Sri Lanka, which are in no way economically superior, have stopped use of Semple vaccine almost a decade ago and replaced with tissue culture vaccines like PCEC and PVRV. They could do it effectively and economically because they accepted and approved the use of cost-effective intradermal regimens with cell culture vaccines, which saves almost 60% on the quantum of vaccine required per patient. This is a huge saving for developing countries, which still depend on produced only by private sectors. As of today, thousands of people have been vaccinated in countries like Thailand and Sri Lanka with either the 2-site ID regimen or 8 site regimens. They have been systematically studied and followed for several years and not a single case of treatment failure has been reported so far. The technique of ID immunization is not new to our country as millions of babies born is administered BCG vaccination by ID technique by paramedical staff. The very idea of ID immunization emerged in order to economize the use of cell culture vaccines so that they can be routinely used in the developing countries. It is now scientifically proved that minute amounts of antigen if administered intradermally within the layers of skin, an effective and rapid immune response can be obtained as the antigen presenting cells present in the skin take up the antigen and present directly Warrel et al at Oxford University demonstrated the efficacy of a multi site ID regimen with human diploid cell vaccine. First reports on the efficacy of ID immunization for post-exposure prophylaxis came from India as back as 1987. In 1992 WHO conveyed several meetings with experts from different countries and finally approved 2 ID regiments to be followed with PVRV and PCEC respectively. Subsequent WHO consultations in the year 1996 and 2001 have only further corroborated and strengthened these previous recommendations. Keeping all these in mind, is it not the right time for our health authorities to look into the advantages, cost-effectiveness and practicability of these ID regimens and implement routinely in all government hospitals and phase out the use of a century old crude nerve tissue vaccine? By continuing the use of a highly reactogenic vaccine which is not even fit for animal use, are we not doing great injustice to our people? Paradoxically, the dogs, which bite thousand of people every year and spread the disease, receive a better vaccine than the people bitten by them do! When government can find means and ways to finance controversial animal birth control programmes (ABC) of unproven and questionable efficacy, why can’t taxpayer money be spent for a better vaccine to prevent rabies in humans, which they get, by bites from the very same animals? It is high time for the health authorities to realize that the common and poor people of India will not continue to bear the gross negligence and injustice inflicted on them for the past 5 decades. They are now sufficiently aware and educated to know the drawbacks of sheep brain vaccine. It is a matter of great shame that a country, which boasts every day of its achievements in IT industry, biotechnology and nuclear power, also boasts of contributing 80% of all rabies deaths in the world. This paradoxical situation should be averted in near future. We have all the infrastructure and technical know how to produce modern tissue culture vaccines. Only willingness on the part of the health authorities is lacking. Till such time, when we become self sufficient in production of cell culture vaccine for the entire country why not adapt the cost-effective strategy of ID immunization that has proved to be highly successful in other countries? We from the APCRI have not been idle in this regard. Time and again we have approached the Drugs Controller General of India on this issue and requested him to approve the WHO recommended ID schedule for routine use in India. The association is still waiting for a response. Meanwhile I appeal to all the Medical officers working in anti-rabies clinics attached to Medical colleges and large hospitals to come forward and conduct clinical trials with ID immunization, initially on people with low risk exposures and accumulate enough data to convince our health authorities and the Drugs Controller to approve these regimens for routine use in India. We can no longer be idle and we have to begin somewhere. And that is the need of the hour! Dr. S. N. Madhusudhana Editor Review Article : RABIES VACCINES AND VACCINATION Y. U. B. Rao Deputy Director, Pasteur Institute of India, Coonor, 643103, Tamilnadu ABSTRACT Rabies vaccines have come a long way ever since their development by Louis Pasteur in 1885. Remarkable improvisation took place over the years leading to the development of primary cell culture vaccine, diploid cell vaccines and finally continuous cell line vaccines. An effort has been made in this article to review the salient developments over the years in the field of rabies vaccination, and current concepts in rabies post-exposure prophylaxis. INTRODUCTION Rabies is a disease of major significance to human and veterinary medicine. Once the signs of disease appear, it is almost in-variably fatal. The use of rabies vaccinations is unique in that it has been primarily used as therapeutic rather than a prophylactic agent. Rabies is endemic in India and approximately 30,000 people die of the disease every year and more than 10,00,000 people undergo anti-rabies vaccination every year1 The principal vector transmitting the disease to man is dog through its infected saliva and 96% of people seeking anti-rabies treatment are exposed by dogs. The incubation period of rabies in humans generally ranges between 30-90 days but can be as short as <10 days or >1 year1. The incubation period depends on the virulence of virus strain, species of biting animal, the site and severity of bite and certain host factors like immunological status. During the early incubation period if vaccination with or without rabies immunoglobulins is given, the chances of saving the patient from developing rabies is very high. Twelve institutions are producing the nervous tissue vaccine for human (40 million ml) and animal use (90 million ml) annually in our country. VIRUS STRAINS USED IN VACCINE PRODUCTION Virus strains used for production of rabies vaccines must be carefully selected from the WHO approved list of strains. The vaccines produced from these strains should protect against local field rabies infection. The different fixed rabies strains used for the production of inactivated vaccine is given below 3: a) Paris Pasteur Strain of rabbit fixed rabies virus. b) PV –12 Strain of Pasteur Rabbit fixed rabies virus. c) Pitman – Moore (PM) strain of fixed rabies virus. d) CVS – 27 (Challenge Virus Standard) mouse brain strain of fixed rabies virus. e) CVS – 11 Kissling strain. f) LEP (40 – 50 Passages) Flury chick embryo adapted rabies virus. g) HEP (227 – 230 Passages) Flury chick embryo adapted rabies virus. h) Kelev (100 Passengers) chick embryo adapted rabies virus. i) SAD (Street – Alabama – Duffering) virus. j) ERA (Evelyn – Rokitniki – Abelseth) strain of SAD virus. k) Vnukovo-32 strain of SAD virus. l) Beijing strain of fixed rabies virus.
HISTORY OF RABIES VACCINATION The first vaccine developed by LOUIS PASTEUR was a live attenuated vaccine produced by air-drying of infected rabbit spinal cord in potassium hydroxide. The vaccine was administered to 9-year-old boy, JOSEPH MEISTER on July 5, 1885 that was bitten by a rabid dog. He was given 13 injections of rabies vaccine. The second patient was given the same vaccine on October 20, 1885 after 6days of bite. By April 1886 Pasteur treated more than 700 patients and majority of them survived. In the year 1887 rabies vaccine was officially recognized by the Medical Community as an effective post-exposure therapeutic measure. Later on, the vaccines were inactivated by heat, UV light, Phenol and other chemicals. Presently, Betap0ropiolactone (BPL) is used widely as an inactivating agent of rabies virus. The BPL is advantageous over Phenol is that a amore antigenic vaccine can be prepared.
RABIES VACCINES The rabies vaccines have been broadly classified into three types: a) Neural tissue vaccines. b) Non-neural tissue vaccines. c) Cell culture vaccines.
a) NEUTRAL TISSUE VACCINES The vaccines are produced from the brains of animals inoculated with rabies fixed virus strain. In our country, the animal of choice for production of rabies vaccine is sheep. The vaccines are effective in controlling rabies but they have drawbacks like post vaccinal nervous system reactions. These reactions range from mild and transient paralysis to severe paralysis due to the presence of myelinated tissue in the vaccine. Since the brains of young animals contain less myelin content, scientists started using the suckling animals for preparation of vaccines. The use of suckling animals significantly reduced the incidence of post-vaccinal reaction. The vaccines produced by using neural tissue are presented in Table 1.
b) NON-NEURAL TISSUE RABIES VACCINES The non-neural vaccines were introduced to benefit the people who are reactogenic to nervous tissue vaccines. The Duck embryos and chick embryos were used to prepare the vaccine. The Duck embryo vaccine is very simple to produce and economical but the disadvantage is high rate of adverse reactions. Also large number of doses are required ranging 14-21 doses to create sufficient immune response. The vaccines are presented in Table 2. Table 2: non-neural tissue rabies vaccines
c) CELL CULTURE VACCINES The vaccine of choice for rabies vaccination is a cell culture vaccine, which is very safe and highly immunogenic. The number and volume of doses have been reduced sufficiently. The main drawback is high cost of the vaccine. The various cell culture vaccines are presented in Table 3. Table 3. Cell culture vaccines
PRE-EXPOSURE IMMUNIZATION – HUMAN a) Pre-exposure immunization is recommended to persons at high risk of exposure like – laboratory staff working with rabies virus - Veterinarians - Animal handlers - Medical and para medical staff attending rabies cases b) It is preferable to use cell culture vaccines and the schedule is three doses on days 0, 7 and 28. c) Periodical booster injections are recommended if the virus neutralizing antibody titer falls below 0.5 IU/ ml. d) For administration of vaccine, use deltoid area of arm.
POST – EXPOSURE TREATMENT – IN ~ MAN In all cases of severe exposure to rabies, combination of local treatment of wound, passive immunization and vaccinations is recommended. Pregnancy is never a contraindication to post-exposure rabies vaccination. a) LOCAL TREATMENT OF WOUNDS - Flushing and washing of wound immediately with soap and water, detergent or other substances of had proven lethal effect on rabies virus. - Avoid suturing of wounds (if necessary anti-rabies immunoglobulin should be infiltrated around the wounds). - Inject Tetanus Toxoid. - Suitable course of antibiotic to prevent sepsis in wound. b) PASSIVE IMMUNIZATION - Rabies immunoglobulin should be given for all category III exposures. – Human rabies immunoglobulin – 20 IU/ Kg body weight. - Equine rabies immunoglobulin – 40 IU/ Kg weight. c) VACCINATION Table 4 Schedule of 5% sheep brain anti-rabies vaccine produced by Pasteur institute of India, Coonoor.
Table 5 Schedule of cell culture vaccines (HDCV OR PVRV or PCEC)
NEW POST-EXPOSURE VACCINATION SCHEDULE a) 3-1 Schedule On ‘0’ day Three vaccine doses applied in the deltoid muscle of the right And left arm.
On ‘7’ day One dose
This schedule produced an early and elevated cellular and humoral immune response. b) 2-1-1 Schedule On ‘0’ day Two vaccine doses On ‘7’ day One vaccine dose On ‘21’day One vaccine dose Early immunity was achieved by this schedule with a sero conversion rate of 78% from the 7-day and of 100% from the 14-day.
a) Intradermal schedules 1) The 2 site regimen (with PVRV) Days 0, 3 and 7 2 injections of 0.1 ml at two different sites 1 injection at one site Days 30 and 90 1 injection at 1 site 2) The 8 site regimen (with HDCV and PCECV) Day 0 8 injections of 0.1 ml at 8 sites Day 7 4 injection of 0.1 ml at 4 sites Day 29 and 90 1 injection of 0.1 ml at 1 site. Produces good results but can be applied only in specialized centers.
Table 6: Vaccination schedule – animal
VACCINATION PROCEDURE There are many vaccines available today that are efficacious when administered properly and two important factors to be considered are age and route of vaccination. a. Age: The dogs vaccinated at 11-16 weeks of age with FLURY LEP or HEP vaccines withstood the challenge better than dogs vaccinated at 5-10 weeks of age. Also it has been reported that six months old dogs responded better to vaccination that did younger dogs. b. Route of administration: Modified live virus vaccines are much more effective when given intramuscularly instead of subcutaneous. Always it is preferable to follow the instructions given by the manufacturer.
VACCINES FOR ANIMALS A. BRAIN TISSUE VACCINES Inactivated vaccines using brains of sheep are produced in our country. The Pasteur institute of India is producing the 5% sheep brain vaccine inactivated by beta-propiolactone (BPL). The experimental evidence indicates that BPL inactivated vaccines given in smaller doses is more effective than phenolized vaccine in post exposure treatment of animals challenged with rabies street virus. The schedule of treatment is presented in Table 7. Table 7: Schedule of treatment of animals
This schedule of treatment can be used for post exposure treatment as well as pre exposure immunization. B. CHICK EMBRYO VACCINES The vaccines are prepared from Flury Strain of rabies virus propagated in embryonated eggs. The strain lost its virulence for the dog and other animals at various passage levels. This is a live attenuated vaccine. 1) Low Egg Passaged (LEP) Flury Vaccine (40 – 50 passages) LEP Flury vaccine has been extensively used in the field to immunize dogs. A single intramuscular injection produces good immunity for 3 years. This vaccine is recommended to use only for dogs and not for cats and cattle. The vaccine became less popular in control programmes as it produced numerous cases of vaccine induced rabies and it is no longer recommended to use in many countries. 2)High Egg Passaged (HEP) Flury Vaccine (176 – 182 passages) This vaccine is recommended to use in dogs, cats and cattle. Puppies less than three months old can be vaccinated safely with HEP Flury vaccine but not with LEP Flury vaccine. In view of the vaccine induced rabies cases with LEP Flury vaccines, the use of HEP Flury vaccine is recommended for the prophylactic immunization of dogs. A single intramuscular injection of HEP Flury vaccine protects dogs for two years. It is recommended that puppies immunized before three months of age are revaccinated after they are 12 weeks old but allowing an interval of 3-4 weeks between injection.
C. CELL CULTURE VACCINES Inactivated and modified live virus vaccines are produced in cell culture using either primary or continuous cell line. The following cell culture vaccines are available in our country, which are inactivated. a) Rabies vaccine, Inactivated (adsorbed) produced by Pasteur Institute of India, Coonor for dogs only. b) ‘Raksharb’ cell culture vaccine produced by Indian Immunologicals, Hyderabad. c) DELCAVAC-r for dogs and cats d) RABISIN for dogs, cats and cattle. e) NOVIVAC for dogs, cats and cattle. A primary vaccination can be administered at the early age of 4 weeks in dogs and cats, 2 months in horses and cattle using cell culture vaccines very safely. If this is dose, a repeated vaccination must be given at 3 months or 6 months respectively according to species. The other cell culture vaccines used in other countries is presented in Table 8 Table 8: Utilization of vaccines for veterinary use (Other than carnivores)
CONCLUSIONS 1) Death in human should no longer occur by rabies infection in this century as we have efficient rabies vaccines, which can be applied properly at appropriate time to prevent rabies virus from reaching the central nervous system. 2) Increase the public awareness by intensive, frequent educational campaigns regarding the vaccination of domestic animals, first aid to be done in case of animal bites and danger of rabies infection. 3) Safe, potent and inexpensive vaccines must be developed and made available for both human and veterinary use.
REFERENCES Report of the symposium on Rabies Control in Asian Countries. WHO/Rab, Res/93, 44, 1993. Plotking SA, Rupprecht LE and Koprowski HWA (.Eds) Vaccines. Philadelphia: WB Saunders 1999 pp 7430-766 WHO Expert Committee on Rabies. WHO Tech Rep Ser 824, 1992 Pierre P, and Jean – Paul S Vaccines for domestic animals. In: George M Baer (.Ed). The Natural History of Rabies. Boston: CRC Press, Inc. 1991 pp 445-459 TRANSMISSION OF RABIES : AWARENESS INCREASING IN DEVELOPING COUNTRIES J. K. Dutta Senior Consultant Physician, Amarnath Polyclinic, Balasore 756001, Orissa INTRODUCTION Several mammals like dog, cat, monkey, and mongoose live in close proximity to human population in developing countries. Hence animal bites, particularly dog bites are very common in these regions1. In olden days animal bites were often ignored or treated by indigenous methods. For dog bites, cauterization with red-hot copper or iron, herbal medicine, mantras by charmers were often used2. Facilities for immunization with rabies vaccine were almost unavailable except in few metropolitan cities in India. In recent years number of clinics attending to animal bite victims have increased considerably. Simultaneously the public awareness about the disease has also increased and often treating physician comes across unusual types of exposures. This paper describes some such case histories. STUDY METHODS During the period of 1996 through 1998,33 persons sought advice for different types of exposures other than exposure to mammals. Epidemiological history was ascertained in each case and relevant clinical examination was carried out. After assessment of risk involved, patients were advised immunization with a tissue culture vaccine when considered necessary. In-group A, 15 patients had consumed milk from cows who later died of rabies. (Table-1). In-group B,11 patients had shared food with rabid men and children who also died of rabies. (Table-2). In-group C,08 person had different types of exposures(Table-3). In no group a patient died of rabies during the follow-up period varying from the 2-3 years. ILLUSTRATIVE CASES CASE NO-1 : A young girl attended an emergency clinic for injuries on legs, which were reported to have been caused by fall from bicycle. The wounds were dressed and anti-tetanus therapy was advised. After two months later she developed paralysis of limbs. Examination of CSF revealed no abnormality. At this point diagnosis could not be established. She died about 6 days after the onset of illness. Postmortem confirmed rabies in that patient though history of dog bite was never available. After her death her boyfriend disclosed to one of his close relatives that he had sexual intercourse with the girl few days prior to her illness. . Even though the transmission by sexual act is extremely rare, he was advised 5 doses of PVRV as he was extremely anxious.
CASE NO-2: A person had a domestic boy servant aged about 14 years. One day a street dog bit the boy. The dog was not available for observation. So he was advised 5 doses of PVRV for post exposure treatment. During the course of treatment the servant had a fight with the 8-yr. old son of the master. During the fight the child had bitten the servant causing bleeding injury on his forearm. There after the parents became worried and came for consultation. After listening to the history clinical examination was carried out. Since the servant had no manifestation of rabies and was under regular PET, no immunization was advised. The child has remained will for 3 years.
CASE-3: A lady aged 59 years had some chickens in her residential compound. One day her pet unimmunised dog had bitten one of the chickens. Later the bitten chicken was killed and curry was prepared which she had taken. Few days later the dog died and rabies antigen was detected by FAT in a laboratory nearby. So the lady who had taken the chicken curry became worried on the assumption that the poison (virus) might have been transmitted from the dog to the chicken and then to her. After ascertaining the history clinical examination was carried out. Since the chicken had been boiled during the preparation of the curry, the virus if any in the chicken must have been destroyed. So no PET was advised. She has remained well for last 2 ½ years.
DISCUSSION The educated population of India and the other developing countries are now aware that rabies can be prevented by timely immunization. In government run animal bit clinics, nerve tissue vaccine (Semple vaccine) is supplied free but neuroparalytic complications are frequent. Even the people of low-income status usually avail the facility under compelling circumstances. Now doubt, replacement of nerve tissue vaccine by tissue culture vaccine is an urgent need. Affluent persons prefer TCV like Human diploid cell vaccine (HDCV) purified chick embryo cell vaccine (PCEC) or purified vero cell rabies vaccine, which are currently available in India. In recent years due to increased awareness regarding modes of transmission and fatal outcome of disease, advice is often sought even after trivial exposures involving doubtful risk, which would have been otherwise ignored. In-group A patients came for consultation when the cow died of rabies after bite by a rabid vector but they had consumed milk without suspicion in early part of illness. In-group B people had shared food with children and adults in early stage of rabies that died of rabies few days after the incident. In these two groups, principal issue was whether rabies virus could be transmitted by oral route. It is generally believed that transmission by oral route is a rare phenomenon. However evidence of successful experimental infection by oral route is available. In an experimental study, infected mother sheep transmitted virus to her lamb that had suckled for 4 days3. Fixed strain of virus was recovered from both animals after death. Soave recorded rabies in mice after ingestion of infected mouse brain4. In another experimental study rabies virus was transmitted orally to mice, guinea pigs and hamsters using challenge virus (CVS) and 2 strains of street virus5. The investigators observed that the virus dose required for oral infection was very high. So the possibility of oral transmission by oral route by consumption of raw milk, improperly cooked meat or other vehicles cannot be totally ignored. Consumption of raw (unboiled) or improperly boiled milk at low temperature is a common practice in many households. Further more wrestlers usually use unboiled milk. Raw milk is also used for worship purpose as a religious custom. In group-c patients had miscellaneous types of exposures. Some of the interesting cases have been discussed in detail. Detail history taking and proper assessment of risk involved in each case are essential steps6.
Table 1 (GROUP-A) Analysis of cases who consumed milk from rabid cows
Table 2 (GROUP-B) Analysis of case who shared food with patients of human rabies
Table 3 (GROUP-C) Analysis or cases with uncommon modes of exposures.
REFERENCES: Dutta JK, Dutta TK Rabies in endemic countries (editorial) BM J 1994;308: 488-489. Dutta JK Human rabies in India: Epidemiological features, management and current methods of prevention. Trop Doct 199; 29: 196-201. Schneider JE McGoarty BJ Transmission of experimental rabies from mother to young. J Am Vet Med. Assoc. 1933; 82:627-630. Soave OA Transmission of rabies to mice by ingestion of infected tissues. Am J Vet Res. 1996, 27:44-46. Madhusudhana S N, Tripathy K K. Oral infectivity of street and fixed rabies virus strains in laboratory animals. Ind. J Expt. Biol.1990; 28:497-499. Dutta JK Oral transmission of rabies in cow: Milk consumers protected by immunization. J. Assoc. Physicians. Ind. 1996;584. A CLINICAL ALGORITHM OF POST EXPOSURE RABIES PROPHYLAXIS FOR PHYSICIANS IN INDIA M. K. Sudarshan Department of Community Medicine, Kempegowda Institute of Medical Sciences Bangalore ABSTRACT Rabies prophylaxis is lifesaving in rabid animal bites and decision to treat such cases is complex. A clinical algorithm of decision chart comprising of three steps viz. Examination of wound (s), assessment of the biting animal and advocating most appropriate treatment plan is presented to help treating physicians for making judicious decisions. It is hoped that this is suitably adapted by pharmaceutical industry and to popularize its use by physicians in their clinics/ hospitals in India. INTRODUCTION According to World Health Organisation (1990) annually 30,000 persons die of rabies in India which constitutes 60% of the global report of 50,000 deaths1. One of the reasons for these deaths is the improper, inadequate and incomplete post exposure rabies prophylaxis by medical professionals 2,3. This is attributed to low level of awareness about anti-rabies treatment amongst medical doctors more so for WHO recommendations on modern rabies prophylaxis4. The medical doctors, particularly the general practitioners / family physicians who treat the majority of dog bites are often unclear and confused and hence provide inappropriate advice or treatment, which has sometimes resulted in development of rabies an death of the patients2, 3 Rabies prophylaxis, which is lifesaving in rabid animal bites as such is "Clinical speciality area" where treatment decisions, more so for the use of rabies immunoglobulin vary on the merit or description of each case and exposure (bite). Though in the past "decision tree" have been evolved 5, 6 still their scope and application is limited for several reasons. In this background the author has developed a clinical algorithm as a "Decision chart" for rabies prophylaxis for use by medical practitioners in India.
METHODOLOGY The author based on his experience of running an Institutional anti-rabies clinic for over 15 years and also taking it into account the interactions with the doctors in various seminars / CME programs all over India has developed a simple decision chart keeping in mind the following overbearing influences. a) The common animal reservoirs of rabies, nature and magnitude of the problem. b) The vaccinations status of dogs and cats, their availability for ten days observation and plausibility of ascertaining their condition of sickness. c) Current recommendations of WHO, its adaptations to rabies endemic India, and a modification of it in special / exceptional circumstances. d) Practical considerations of availability of physicians, vaccines and rabies immunoglobulins (RIGS), socio-economic factors of the patients, transport and communication systems etc. e) The simple golden rule of "Better and safer to over treat than under treat" in view of risk benefit analysis of safer modern vaccines vis-à-vis the risk of rabies death in endemic India. Based on these criteria, the decision chart is made as simple as possible to make it physician (user) friendly who otherwise is busy treating various other common medical problems also. Simultaneously, due care and caution has been taken to ensure the decision chart as comprehensive and as precise as possible. Because post exposure rabies prophylaxis in rabid animal bites is definitely a life saving treatment and any failure in this regard by the physician would make him vulnerable for litigation under consumer protection act of the country for providing deficient / negligent service to his patients.
DISCUSSION The three step decision chart (vide annexure) is a simple decision making tool for physicians to provide most appropriate modern rabies prophylaxis to their patients. It has primarily 3 steps of approach viz. Firstly, to examine the wounds (step – 1) Secondly, to ascertain some details of biting animals (mostly the dog or cat) (step – 2). Thirdly, to assess the danger /risk (category) based on the information available and to offer post exposure rabies prophylaxis (code plan) (Step-3). In this context the physician must remember that use of this decision chart is an intelligent affair and is possible only when the patient / attendant provides information, which is authentic, valid and reliable. In many such instances /circumstances the decision chart can even be sued for "Tele advice" to provide guidance to the bite victim over telephone or Internet and this can also be experienced and seasoned physician. However the author would caution and advice the physicians to exercise discretion to use this guide with great care and diligence as this is not cent percent foolproof but would certainly benefit majority in most of the common cases or situations. Lastly, the author hopes that this simple educational tool is popularized and propagated through wider dissemination's amongst medical professionals by the pharmaceutical industry that produces vaccines and sera. This simple 3 step approach information may be presented on or as a simple hand held device or a wall chart or in any other innovative way to the otherwise busy physician in his clinic / hospital to enable him to make best decisions for post exposure rabies prophylaxis.
REFERENCE: World Health Organization. World survey of Rabies, 34, 1998, Geneva. Paramashiviah G B, A note on the Prevention and Management of Hydrophobia. Karnataka Journal of Community Health, 1995; 2: 66-72. Mahendra BJ, Sujitkumar, Kiran S Rao, Sudarshan M K and Gangaboriah: Clinico- epidemiological study of human rabies cases at Epidemic Diseases Hospital, Bangalore, Journal of APCRI; 2000;1& 2,: 43-48. Sudarshan MK A study of anti-rabic treatment practice by private medical practitioners in Bangalore City, Indian Journal of Preventive and Social Medicine 1995; 26: 45-48. Jacob John T an ethical dilemma in rabies immunization. Vaccine 1997;15: S12-15. Bhatia R, Ichhpujani RL Rabies – The killer disease, first edition, Jaypee Brother Medical publishers (P) Ltd., New Delhi 1994. World Health Organization (WHO): Expert Committee on Rabies. 8th Report, WHO Tech Rep Ser 824 Geneva, 1992.
ANNEXURE A 3 STEP APPROACH TO DECISION MAKING FOR POST-EXPOSURE RABIES PROPHYLAXIS IN INDIA: A CLINICAL ALGORITHM OF THE DECISION CHART. STEP-1 ALL WOUNDS: To see in broad daylight / bright torchlight in darkness. ADAPTED WHO CLASSFICATION7 Category I — Now wound(s) / Marks at all II — Wounds (s) without blood III — Wounds (s) with blood. Note: Gentle washing of all wounds using a detergent soap preferably under running tap water for at least five minutes. Application of veridical agents like povidone iodine, tincture iodine or other house hold antiseptics like dettol, savlon etc.
STEP-II THE DOG (OR CAT)+: ask the responsible owner for reliable information.
+ Wherever feasible the biting animal must be observed everyday from the day of biting for the next 10 days for signs of rabies and on suspicion to get it examined by veterinarian. ++ Received at least two doses of modern rabies vaccine in the last 2 years from a veterinary doctor and having an immunization card / record with the veterinarian / pet owner.
STEP-III The Treatment Plan.
Note: 1. In case of doubt/ ambiguity institute a higher code of treatment plan. 2. Give an extra dose of vaccine (or in severe exposure even 2 extra doses), if the patient reports late viz. Beyond 24 hrs of bite or RIGs not injected simultaneously with first dose of vaccine or RIGs administration delayed beyond 72hrs of bite / first dose of vaccine. 3. If a patient has received minimum 3 doses of modern vaccine (any type) in the last I yr. then he / she needs only 2 doses (on days 0 & 3) of vaccine in category II wounds and 3 doses (on days 0, 3 & 7) in category III wounds. 4. There is no contraindication to post exposure rabies prophylaxis including pregnancy and lactation. 5. In immuno-compromised individuals or for those on immuno-suppressant drugs, double or even treble the first dose of vaccine. 6. All modern vaccines shall strictly be injected intramuscularly in deltoid (in older children and adults) and in infants and in younger children in the anterolateral aspect of thigh. They shall never be injected into gluteal region. 7. (a) Inject the equine RIGs (serum)( around all the wounds (after a skin test for safety) and if the quantity is insufficient dilute it up to 3 times with equal volume s of sterile normal saline. (b) Inject the human RIGs (serum) around all the wounds (skin test not required) and if the quantity is insufficient dilute it upto 3 times with equal volume of Human normal immunoglobulin. © Any left over RIGs (equine / Human) after infiltrating all wounds shall be injected deep intramuscularly into gluteal region (s) (upper outer quadrant (s) only).
A CASE SERIES REPORT OF SUCCESSFUL POSTEXPOSURE TREATMENT OF PROVEN RABID ANIMAL BITES+ M. K. Sudarshan, B. J. Mahendra, D. H. Ashwathnarayan and K. Rohith Department of Community Medicine, Kempegowda Institute of Medical Sciences. Bangalore 560004 ABSTRACT Nearly 50% of people bitten by rabid animals develop rabies if effective post-exposure treatment is not administered. The treating physicians generally lack proper understanding and necessary confidence in providing this life saving treatment to animal bite victims, thus becoming responsible for many otherwise preventable deaths due to rabies. In this paper seven recent successfully treated cases of proven rabid animal bites are reported which should inform the appropriate method of post-exposure treatment and instill confidence in the treating physicians in the country.
INTRODUCTION According to World Health Organization (1998) annually 30,000 persons die of rabies in India which constitutes 60% of the global report of 50,000 deaths1. One of the reasons for these deaths is the improper, inadequate and incomplete post-exposure rabies prophylaxis by medical professionals2, 3. This is attributed to low level of awareness about anti-rabies treatment amongst medical doctors, more so for WHO recommendations on modern rabies prophylaxis4. The medical doctors particularly the general practitioners / family physicians who treat the majority of dog bites are often unclear and confused and hence provide inappropriate advice / treatment, which has even resulted in development of rabies and death of the patient2 & 3. Post-Exposure treatment (PET) in proven rabid animal bites is a life saving measure and it includes 3 important components of proper and immediate wound treatment, modern rabies vaccine within 24 hours of bite and in class III exposures infiltration of all bleeding wounds with rabies immunoglobulins (RIGs) at the earliest, preferably before 72 hours of bite. This treatment is now possible in all the nursing homes / polyclinics / hospitals of the state capitals and many of the district and Taluk headquarters in the country. But due to lack of facilities / practical difficulties for laboratory confirmation of rabies in the biting animal all stray and most of the pet animal / dog bites are to be considered as suspect / presumed rabid animal bites and treated accordingly. In this context, we report our recent successful treatment of seven confirmed rabies exposures recently in Bangalore and with their post treatment survival of 11/2 years approximately.
Case 1: VB, 31/2 year-old boy, on September 7, 1998 was bitten by a stray dog on the anterior abdominal wall (over clothing) with about 8 extensive and deep wounds with bleeding (WHO class III). The wounds were washed with water and soap after half-an-hour of bite and dettol was applied. The patient was started on PCEC vaccine following Essen regimen. The dog was killed by the people (as it was biting other people) and after postmortem at the Veterinary College it was positive for Negri bodies. Subsequently, on September 12 (five days after bite) the child received HRIG (20 IU / Kg body weight) infiltration into wounds with an additional dose of PCEC vaccine. In all the child received 7 doses of PCEC vaccine (on days 0, 3, 7, 14, 30 and 90) with an extra dose of vaccine on day 5 along with delayed HRIG administration. The child is alive and well. Case 2: CBS, 17-year-old woman, a German National, resident of Bangalore, in October 1998, was bitten by a stray pup over the lower lip, right arm and fingers with slight ooze of blood (WHO class III). The lip was washed with water only immediately, two hours later the wounds on fingers with soap and water and after 24 hours the wounds on forearm with soap and water. After 24 hours of the bite, the pup died and the postmortem at the local Veterinary College revealed it was positive for Negri bodies. She had received a 3 does pre-exposure modern rabies vaccination (vaccine not known) one year back in Germany (days 0,5 and 22). Due to her late reporting after 9 days of this proven rabid exposure in Bangalore, she was started on HDECV by Essen regimen on days 9,3,7,14,30 and 90. An additional extra dose of had to be given on day one as the first dose of HDCV was wrongly given in gluteal region at another institution. So, in all, she received 7 doses of HDCV. She also received HRIG (201U / kg body weight) into gluteal region (full dose) as no local infiltration was possible. Her day zero blood sample revealed rabies neutralizing antibody titer 0 .5 IU / ml at Institute of Tropical Medicine, Germany; by RFFIT at Pasteur Institute of India, Coonoor, Tamilnadu, 1.0 IU / ml; and by Serum Mouse Neutralization test (SMNT) at NIMHANS, 1:16. The lady is alive and well.
Case 3: MK, 8 year old boy, of February 2, 1999, was bitten by a stray dog on the right thumb and right foot with about 8 deep lacerated wounds with bleeding (WHO Class III) the wounds were immediately washed with soap and water and povidone iodine was applied. The wounds were infiltrated with HRIG (20 IU / kg of the body weight dose) after 12 hours of bite. The child received 6 doses of PCEC vaccine as per Essen regimen of days 9,3,7,14,30 and even 90. An extra dose of vaccine was given on day 0 as the referring private doctor had administered first dose of PCEC vaccine wrongly in the gluteal region. The biting dog was immediately captured and following postmortem was found positive for Negri bodies at the local Veterinary College. The child is alive and well.
Case 4: AJ, 31/2 year – old boy, on April 6, 1999, was bitten by a stray pup on the leg. The injury was superficial with two wo7unds on the leg, with slight oozing of blood (Who Class III). The wounds were immediately washed with water, soap and dettol was applied. After 12 hours o the bite the wounds were infiltrated with ERG (@ 40 IU / kg body weight dosage) and the left over quantity was administered in the gluteal region (upper outer quadrant). The child received 6 doses of PCEC vaccine as per Essen regimen (on days 0,3,7,14,30 and even 90). An extra dose of vaccine was given on day 0 as the referring private doctor had administered first dose of PCEC vaccine wrongly in the gluteal region. The biting dog was immediately captured and following postmortem it was found positive for Negri bodies at the local Veterinary College. The child is well and alive.
Case 5: BN, a 43 year-old woman, on July 12, 1999, was bitten by a stray dog on the fingers with deep four and extensive wounds with water immediately and some household applicant was applied to the wounds. Subsequently, she received sheep brain (sample) vaccine for 7 days regularly. In the mean while the dog died and following postmortem at Veterinary College, it was found positive for Negri bodies. Then on the 8th day the patient received ERIG (@ 40 IU / kg weight dose) infiltration to all (healing) wounds and 5 doses of PCEC vaccine as per Essen regimen and an additional optional dose on day 90 also. The woman is well and alive.
Case 6: SM, 40 year-old woman, on September 28, 1999, was bitten by a domestic dog on the pulp of the left middle finger. The wound was superficial with little bleeding (WHO Class III). The wounds was immediately washed with water, soap and dettol applied. After 2 days the dog died and following postmortem at local Veterinary College, it tested positive for Negri bodies. Then the patient (21/2 days after the bite) received ERIG (@40 IU / kg of the weight dosage) infiltration to wound and the left over ERIG given in gluteal region. The patient received 5 dose of PCEC vaccine as per Essen regimen. The woman is well and alive.
Case 7: FR, 48-year-old man, on September 27, 1999, was bitten by a domestic dog on the fingers with 5 extensive bleeding wounds (WHO class III). The wound \s were washed with soap and water for 15 minutes after bite. On September 28 (12 hours after the bite) the wounds were infiltrated with ERIG (@ 40 IU / kg body weight dosage). The patient was given 6 doses of PCEC vaccine by Essen regiment (days 0, 3, 7, 14, 30 and 90) with the first dose given on the day of the bite itself. The dog was positive for Negri bodies by postmortem done on September 27 at the local Veterinary College. The person is alive and well. All the seven patients treated well tolerated the vaccines and the RIGs including the purified equine rabies immunoglobulin (serum) without any notable adverse effects. In conclusion, these case treatment reports should build confidence in treating physicians an their timely and proper post-exposure rabies prophylaxis to animal bite victims should go a long away in saving live s these patients from this dreadful disease. Lastly, it must be noted that the types of modern vaccines used in this case series report are purely coincidental and don not imply superiority in whatsoever manner one over the other Who approved modern rabies vaccines available in the country. Likewise, the decision to use HRIG or ERIG was mostly influenced by the economic status of the patient and extent and severity of the wounds. This study was possible due to the facilities and co-operation of the Veterinary College, Bangalore. REFERENCE: World Health Organization. World Survey of Rabies, No, 34, 1998, Geneva Paramashiviah G B, A note on the Prevention and Management of Hydrophobia. Karnataka Journal of Community Health, 1995; 2: 66-72. Mahendra BJ, Sujitkumar, Kiran S Rao, Sudarshan M K and Gangaboriah: Clinico- epidemiological study of human rabies cases at Epidemic Diseases Hospital, Bangalore, Journal of APCRI; 2000;1& 2,: 43-48. Sudarshan MK A study of anti-rabic treatment practice by private medical practitioners in Bangalore City, Indian Journal of Preventive and Social Medicine 1995; 26: 45-48. COMBINED IMMUNIZATION WITH PURIFIED CHICK EMBRYO CELL VACCINE AND ALUMINIUM ADJUVANTED PURIFIED TETANUS TOXOID FOR RABIES PROPHYLAXIS Madhusudhana S. N.*, Kapoor M. & Singh H Central Research Institute, Kasauli, 173204 * Presently NIMHANS, Bangalore 560002 ABSTRACT In the present study we have investigated the adjuvant effect of aluminium phosphate adjuvented purified tetanus Toxoid (APTT) on the neutralizing antibody response to chick embryo cell vaccine (PCECV) for post exposure prophylaxis in 146 low risk category patients. By combining the dose of PCECV and with APTT and given as a single shot on day 0 and 21 of a 1-1-1 regimen to 78 patients with category I exposure to rabies, a significantly enhanced neutralizing antibody response (0.05) was seen in all age groups from day 14 onwards, as compared to the response in 68 patients who were administered PCECV alone on the same days. The protective titers persisted for more than 1-year in-patients who took the combined regimen. There were minimal side effects to both the regimens but their incidence did not differ significantly. As the combined regimen was found to be cost effective and well tolerated with minimal side effects, the feasibility of advocating this regimen to certain category of rabies post exposure cases needs to further evaluated.
INTRODUCTION Rabies is a fatal viral encephalitis, which is still a serious health problem in most of the developing countries including India where an annual human death rate of 30,000 is reported to world health organization (WHO) 1. Presently, effective post exposure immunization is the only means to prevent and control this dreaded disease. In the developed countries of the west, the earlier nerve tissue vaccines like the semples vaccine have been completely replaced by highly potent and safe all culture vaccines (CCVs) like human diploid cell vaccine (HDCV), purified chick embryo cell vaccine (PCECV) and the latest purified Vero cell rabies vaccine (PVRV). However, one major constraint with these vaccines, particularly in the developing countries is their cost and with conventional intramuscular dose advocated by WHO, only the rich and affluent people will be able to afford the cost of treatment. The PCEC vaccine is now manufactured in India by private sectors and costs around 300 per dose taking the cost of treatment to more than Rs 1500 which is an economic burden to majority of people. Recently the WHO has recommended the use of the cost-effective economical regimens with a view to completely abolish the use of nerve tissue derived vaccines. The regimens, which are now accepted by WHO include the 2-1-1 intramuscular regimen and the intradermal regimens2. Another way to reduce the treatment cost is to use adjuvant, which can augment immune response with less quantity of vaccine One such study was reported by Phanuphak et al 3 in which they demonstrated immune enhancement when PVRV was mixed with aluminium adjuvented tetanus Toxoid in the 2-1-1 regimen. In the present study we investigated the effect of the combined regimen with PCEC vaccine which is now freely available in India.
MATERIALS AND METHODS Subjects: One hundred and forty-six patients who attended anti-rabies treatment center attached to central research institute (CRI), Kasauli over a period of 8 months were recruited. They all had category I exposure with negligible risk but preferred to take cell culture vaccine. Among these 59 were adult males, 50 were adult females and 37 were children between 8-13 years age. These patients were neither exposed to rabies nor had taken any rabies vaccine in the past. Vaccines and Regimens: The vaccines used were PCEC (Rabipur) from Hoechst pharmaceuticals (India) belonging to a single batch with the potency of 7.4 IU / ml and aluminium phosphate adjuvented purified tetanus Toxoid (APTT) produced at CRI, Kasauli with an Lf value 5 per 0.5 ml. Sixty-eight patients (25 adult males, 22 adult females and 21 children) were administered with1 ml of vaccine i.m. on days 0,7 and 21 whereas 78 patients (24 adult males 28 adult females and 26 children) were given the combined regimen in which the dose of PCEC on days 0 and 21 were reconstituted with 0.5 ml of diluent provided by the manufacturer and 0.5 ml of APTT and inoculated intramuscularly. The study was approved by the research and ethical committee of CRI, Kasauli. The patients was followed for one year and at each visit they were enquired for and also physically examined for many adverse side effects to the two regimens. Estimation of Antibody titers: Blood samples from these patients were collected on day 0(before vaccination), 14,90,180 and 365. The immune response in these vaccines was evaluated by estimating the neutralizing antibody titers in their sera by performing standard mouse neutralization test (MNT) advocated by WHO 4. The titers were expressed in IU / ml of serum in comparison to a reference immunoglobulin (RIG) previously calibrated against the WHO international reference preparation with a potency of 59IU / ml. The results were statistically analyzed using student’s ‘t’ test.
RESULTS The rabies neutralizing antibody titers obtained with both the regimens is depicted in Table – 1. The minimum antibody titers detectable by the mouse neutralization test is 0.04 IU / ml and the WHO has advocated a minimum protective levels of 0.5 IU units / ml of the serum2. None of the subjects had detectable levels of antibodies on day 0.More than adequate levels of antibodies were present from day 14 onwards and with the regimens though significantly higher titers were seen with the combined regimen in both the sexes and all age groups (p<0.005). it is to be noted that protective titers were present in-patients given the combined regimen even at the end of one year. Some patients reported minor side effects like pain, myalgia, fever but their incidence did not differ significantly in the two groups.
DISCUSSION In a rabies endemic country like India where more than 0.5 million people undergo the unpleasant experience of taking multiple and painful injections of Semple vaccine subjecting themselves to severe local and neuroparalytic complications5, there is a scope for advocating some cost effective abbreviated regimens with safe and potent but expensive CCVs. The regular i.m dosage schedule with HDCV, PCECV and PVRV will cost around price 3280,Rs 1075 and Rs1075 and R 1155 respectively which is an economic burden for most of the low and middle-income groups6. In the present study we have taken the advantage of the adjuvant effect of aluminium adjuvented PTT to see if we can reduce the dose of PCECV with APTT and given as a single shot i.m and enhance the immune responses to rabies component. This enhanced effect was obvious on day 14 itself and maintained through out the study period. Our results are in accordance with those reported by Phanuphak et al 3. With PVRV, though we have used only a single dose of PCEC on day 0. Their study has also shown that rabies component did not interfere with the immune response to PTT. However this aspect was not studied in the present work. The main advantage with this regimen is considerable reduction in the cost of post exposure treatment. Presently CCVs are not produced in government establishments and hence not available for general use. Patients have to pay heavily if they opt for any CCVs Considering this, regimen may prove useful to certain category of patients who come under low risk group and like to avoid painful course of Semple vaccination but cannot afford the cost of regular CCV schedule. Further, there were no serious side effects to this regimen and we found excellent tolerance and good acceptability. Another advantage is the reduction in total number of injections and hence visits to the medical practitioner. Most of the victims of animal bites also require a dose of tetanus Toxoid, which can be combined with rabies vaccine and given as single vaccine on day 0. The results of the present study with PCECV and previous studies with PVRV suggest that CCVs may become more cost effective if adjuvented with aluminium salts, as lyophilisation is not indicated with aluminium adjuvented vaccines, avoiding this procedure may further economize the manufacture of vaccine. Indeed, a aluminium adjuvented liquid rhesus diploid cell Vaccine is already produced in USA by the biologic products division of the Michigan department of public health and is licensed for both pre and post exposure treatment. 7. REFERENCES
7. Barth R, Franke V Foetal rhesus monkey lung diploid cells vaccine for humans. In: Meslin FX, Kaplan MM, Koprowsky H (eds). Laboratory techniques in Rabies, 4th ed. Geneva, World health Organization 1996 pp 297-300.
Table 1: Rabies neutralizing antibody titers in-patient’s administered with single or combined regimen with PCEC vaccine
PCECV = Purified chick embryo cell vaccine PTT = Purified tetanus toxoid *See text for detailed ** Geometric mean titers in UI / ml
PRODUCTION OF RABIES GLYCOPROTEIN IN MUSK MELON (VAR.NS-ABHIJIT) THROUGH AGROBACTERIUM MEDIATED TRANSFORMATION AND TESTING FOR IT’S EFFICACY IN EXPERIMENTAL MICE P. H. Ramanjini Gowda* and Madhusudhana S. N. Department of Biotechnology, University of Agricultural Sceinces, Bangalore and Department of Neurovirology, NIMHANS, Bangalore
ABSTRACT In an effort to produce an edible vaccine for rabies, the rabies glycoprotein gene of the ERA strain of rabies virus along with the npt II marker were transformed into cucumis melo Var. NS — Abhijit through agrobactrioum mediated transformation. The presence of the transgene was confirmed through PCR analysis. The stable integration and expression of the rabies glycoprotein gene was detected by SDS-PAGE. The presence of a 66kDa glycoprotein in the transgenic muskmelon plants as compared to the absence of this protein in the untransformed plant confirmed expression of the transgene. Western blots confirmed the specificity of this foreign protein. The extract of the fruits when inoculated intraperitoneally in to mice produced Substantial levels of neutralizing antibodies. Further studies on its efficacy by oral route are in progress.
INTRODUCTION Oral vaccination against rabies has received considerable attention during recent years. These vaccines have been mainly based on the immunogenecity of the rabies virus glycoprotein. The use of transgenic plants as source of an edible rabies vaccine is a tantalizing possibility that has been pursued with varying degrees of success for half a decade now. Although an early attempt to produce a rabies vaccine in tomato by Mc Garvey et al1. was unsuccessful, recent efforts have met with more success. The fact is buttressed by the productions of high levels of rabies neutralizing antibodies in mice using rabies g-protein expressed in tobacco plants by Gowda et al 2 . However the impracticability of the tobacco plant expressed G protein as an edible vaccine for humans has thrown up the need for producing the vaccine in other edible plants like Musk melon plant (cucumis melo L), which is a popular fruit crop with suitable post harvest characters that can serve as an ideal crop for vaccine production. The present paper is a preliminary report of the ongoing work of expression of glycoprotein gene in muskmelon plant.
MATERIALS AND METHODS In the present study Cucumis melo L. var. NS- Abhijit, has been transformed with the same rabies G protein gene used in the earlier stages using npt II gene (neomycin transferase II) as selectable marker through Agro bacterium mediated transformation. The binary Agro bacterium tumificans vector RG-2 used here is a derivative of Bin 19 and is similar to the pBI 121-vector family. It contains the complete unmodified cDNA of the ERA strain rabies virus G protein gene along with the nptII gene as a selectable marker. These genes are under the control of the a hybrid CaMV 35S promoter built by combining a triple repeat of the octopine synthase (ocs) activator sequence along with the mannopine synthase (mas) activator elements fused tom the mas promoter called as the super promoter.3 . This construct, named as the pRGSPRgpKDEL, is borne in the disarmed Agro bacterium tumificans strain EHA 105 and was a gift from Hill ary Koprowsky, Thomas Jefferson University, USA. The bacteria bearing the pRGSPRgpKDEL construct were cultured overnight at 28 m C in YEP broth (Yeast extract 10g / 1, peptone 10g / 1 NaC1 5g / 1, pH 7.0) supplemented with 50 mg / 1 Kanamycin. The bacteria were then centrifuged at 5500 rpm, suspended in hormone free, half strength MS medium and its density was adjusted to A600=0.9. The cotylednory explants of muskmelon were suspended in the bacterial inoculam for 3 min, blotted dry on sterile blotting paper, and placed over the regeneration medium without any selection agents in it. After co-cultivation with Agro bacterium for 72 hours, the explants were washed with sterile water, and placed over fresh regeneration medium containing 75mg / 1 Kanamycin and 500mg. / cefotaxime. Plants from these explants, which grew healthy on the selection media, were chosen as putative transformants. The green and healthy rooted plants were transferred into 3:1 peat sand mix for acclimatization. These plants were initially covered with a polythene bag for two weeks. The peat – sand mix was kept moist wit half strength liquid MS medium, diluted hundred times with water, during this period. They were later transferred into larger pots and maintained at 25 C. . Observations were noted down on the growth characteristics of five confirmed transgenic plants and statistically analyzed. The presence of the nptII gene was detected through PCR analysis, as primers specific to rabies G protein were not available. Plant DNA was extracted using CTAB buffer from five putative transgenic plants and one control (untransformed) plant according to the method of Murray and Thompson 4. The final pellet was treated with RNAase, repurified by phenol chloroform method and dissolved in Tris – EDTA buffer. A positive control in the form of 100ng of pRGSPRgpKDEL plasmid DNA was also included in this experiment. Primers specific to the nptII gene (Forward 5’ GAAGGCGATAGAAGGCG 3’ from Ban galore genie LTD, India) were used here. The PCR mixture contained 2.5mM of each DNTP, 1mM of each oligonucleotide primer, 150 Ng of template DNA, 1X TAQ polymerase assay buffer with 15mM MgC12 and 3 units of Taq DNA polymerase microlitres overlaid with mineral oil. PCR was performed at 94m C for initial denaturation of the genomic DNA. Subsequently 35 thermal cycles, each cycle consisting of denaturation (1 min at 94m C, annealing (1 min at 58m C) and polymerization (1.5 min at 72m C) were performed. The 35th cycle was modified so that the polymerization at 72m C was carried out for 6 min. The PCR products were then analyzed by electrophoresis in 0.8% (W/V) agarose gels. In order to simultaneously confirm the presence and expression of the rabies-G protein gene in the transgenic muskmelon plants, total soluble protein was extracted from the leaves of these explants and analyzed by SDS-PAGE. This was to detect the presence of the 66kDa sized rabies glycoprotein that has been reported by Wunner et al 5 in the ERA rabies strain. The presence of rabies glycoprotein in the fruit extract was confirmed by doing a Western blot using polyclonal anti glycoprotein antibody.
Immunogenecity studies in mice: The saline extracts from the transgenic fruits and normal control fruits were inoculated intraperitoneally (0.5 ml per mice) to groups of 10 mice each on days 0,7, 21, and 90. The mice were bled by retro-orbital route a week later. The serum from 3 mice each were pooled, inactivated and tested for presence of neutralizing antibodies by mouse neutralization test. (MNT) as advocated by WHO6.
RESULTS AND DISCUSSION Plants tested through PCR analysis showed the presence of the nptII gene. This was shown by the appearance of an approximately 800bp-sized band in agarose gel, which is the size of the nptII gene. The plasmid used as a positive control also showed an exactly similar band while the control-untransformed plants did not have any band. Observations on the growth parameters of the transgenic plants showed a marked decrease in their height and number of leaves as compared to the untransformed plant. This could be due to the inhibitory effect of the foreign gene integration and expression. The SDS-PAGE of the total muskmelon leaf proteins showed the presence of a large, dark 66 KD a band only in the transgenic plants. The control-untransformed plant did not have this band. This confirmed the presence and expression of the rabies G protein gene in the transformed plants. This is in contrast to the results reported by Mc Garvey et al 1 who reported the production of a 62kDa glycoprotein in tomato transformed with the same gene. This difference can be attributed to the varying glycosylation patterns of the two plant systems. The cultivation of the transgenic musk melon plants at 25m C could have also had an effect on the glycosylation pattern of the rabies glycoprotein, which has been reported to be temperature sensitive by Deitzschold.7 The fruit extract tested from 3 transgenic plants produced a significant levels of rabies antibodies in the mice inoculated. The results are shown in Table 1. The protective levels of rabies neutralizing antibodies is reported to be 0.5 IU / ml. In this study all the fruits tested had induced levels greater than 9.5 IU / ml after 5 doses of extracts. This report of transformation of a rabies vaccine gene into a popular local muskmelon variety and its stable integration and expression is an important step towards developing an edible plant vaccine against rabies. The standardization of an efficient, fast, and cost-effective regeneration protocol for muskmelon also provides a stepping-stone for future transformations with other vaccine genes in this important food crop. Future immunogenecity studies on the rabies G protein produced in muskmelon here could finally establish the feasibility of an edible plant vaccine against rabies.
REFERENCES
3. Prakash CS, ISB news reports 1996,5-7. 4. Murray MG and Thompson WF, Nucleic Acid Res., 1993, 8:4321-4325. 5. Wunner WH, Larson, JK, Dietzshold, B and Smith CL, Reviews of infectious diseases, 1988, Vol19; (4): S771-S783. 6. Atanasiu P Quantitative assay and potency test of anti-rabies serum and immunoglobulin. In: Kaplan MM, Koprowsky H (Eds) The Laboratory Techniques in Rabies, 3rd ed. Geneva, WHO. 1973 pp 314-318.
Table 1 Results of immunogenecity testing in mice
A SIMPLE INDIRECT IMMUNOFLUORESCENCE TEST FOR ASSAYING RABIES ANTIBODIES Shamasundar S., Arvind K., Suja M. S., Saraswati S., & Madhusudahana S. N. Deapartment of Neurovirology, NIMHANS Bangalore ABSTRACT In this study, we have developed and evaluated a simple indirect immunofluorescence test (IIFT) to detect rabies antibodies in a two-step immunofluorescence assay. Eighty five serum samples from people who had taken different rabies vaccines and 6 pairs of serum and CSF samples from confirmed paralytic rabies cases were tested by IIFT and results evaluated in comparison to standard mouse neutralization test (MNT). Though the titters of rabies antibodies obtained with IIFT were 2-4 correlation was seen between the two tests (r = 9.883). the specificity of this IIFT was found to be 97.9% and the sensitivity was 97.2%. These results indicate that this simple and rapid IIFT can be used to screen large number of serum samples to monitor sero-conversion after pre or post exposure vaccination and may also assist in rapid ante-mortem diagnosis of atypical human rabies. INTRODUCTION One of the common indications for estimating rabies specific antibodies is to monitor sero-conversion after pre or post-exposure vaccination. Evaluation of protective status by estimating antibody titters after vaccination and timely advice for booster doses is necessary, particularly in category III exposure. The other situation where estimation of rabies antibodies may be helpful is for ant-mortem diagnosis of human rabies, particularly atypical paralytic rabies 1. Several tests have been developed for estimation of rabies antibodies, but WHO generally recommends only two tests. Of this the mouse neutralization test (MNT) is laborious, time consuming and involves use of large number of mice2. On the other hand RFFIT requires facilities for cell culture and constant supply of rabies conjugate3 and these tests are performed generally in reference laboratories. Recently ELISA kits have been commercially introduced but they are very expensive for general use. There is a need to develop a simple and inexpensive test, which can be performed in any laboratory. In this paper we describe a simple indirect immunofluorescence test for estimation of rabies antibodies. The results of this test have been evaluated in comparison to the standard MNT.
MATERIALS AND METHODS Clinical samples: Blood samples were collected from 85 patients who attended our department for post exposure advice and treatment. They were advised to take either PCECV (Rabipur) or PVRV (Verorab) on day 0,3,7,14. Blood samples were collected on day 0 (Before vaccination) and 7-10 days after full course of vaccination. Out of these 32 patients had a complete course of PVRV and 53 had complete course of PCECV Apart from these. The negative controls included 25 serum samples from healthy people with no history of dog bite or prior rabies vaccination. The positive control serum used in each test was the commercially available Human Rabies immunoglobulin (HRIG, Berirab, and 1:5000 dilution) Preparation of antigen smears: Four to six weeks old Swiss albino mice were infected with 100 LD50 of challenge virus standard (CVS, from Central research institute, Kasauli) intracerebrally. Brains were harvested from these mice when they were completely moribund with the disease. Thin and uniform smears were made on clean, microscopic glass slides and fixed in cold acetone at – 75oC overnight. Presence and uniform distribution of rabies antigen in these smears were confirmed by performing standard direct immunofluorescence on some randomly selected smears. The rabies FITC conjugate used was obtained from Central Research Institute, Kasauli. Indirect Immunofluorescence tests (IIFT): The sera to be tested were first inactivated at 56oC for 30 minutes in a water bath. The smears were washed twice in sterile PBS. Different dilutions of serum and CSF were applied on to smears and incubated at 37oC for 2 hours. The lowest dilution of the sample tested was also applied on to smears from brain of normal mice as controls. After incubation the slides were washed thrice with PBS, dried and then treated with anti-human IgG-FITC conjugate (Bangalore Genei, 1:80 dilution) for 30 minutes at 37oC. the slides were again washed thrice with PBS, dried, mounted and examined under a fluorescence microscope (Leitz, 40 X 10). Uniformly distributed apple green fluorescence particles were present in positive samples in CVS infected brain smears but not in normal brain smears (Fig 1) The highest dilution of serum or CSF, which gave positive fluorescence was taken as the end point dilution. Mouse neutralization tests (MNT): This was performed as per WHO recommended procedure2. Briefly, different dilutions of inactivated serum samples were mixed with 100 LD50 of CVS and incubated at 37oC for 1 hour in water bath along with serum and virus controls. The serum virus mixtures were then inoculated intra-cerebrally into 4-6 week old mice that were kept under observation for 14 days. Any death occurring within 6 days were ignored and depending on the survival mortality between 6-14 days, end point dilutions were calculated using Reed and Muench method.4 Statistical analysis: The antibody titters obtained with these tests were statistically evaluated using Pearson’s product movement correlation analysis.
RESULTS Antibody titters in the vaccines: Barring 4 samples all post-vaccination serum samples which were positive by MNT were also positive by IIFT. In general, the titters observed with IIFT were 2-4 fold lower than those with MNT (Table 1). However, a significant correlation was found between the titters when analyzed by Pearson’s product movement correlation (r=0.883). The four cases, which were negative by IIFT, had very low MNT titters (< 1:4). Two of the pre-immunization samples from people administered with PVRV were found positive by IIFT in low titters (1:2) but were negative by MNT. None of the pre-immunization samples from people vaccinated with PCECV reacted in IIFT. Among the healthy controls, two out of 25 was also found reactive in very low dilutions by IIFT but negative by MNT. In all, non-specific fluorescence was found in 4 cases making IIFT 97.9% specific in comparison to MNT. The sensitivity was found to be 97.2% in comparison to MNT. Antibody titters in rabies patients: Twelve paired serum and CSF samples from 6 cases of paralytic rabies confirmed on autopsy were tested. Among these 4 patients had taken partial treatment with Semple vaccine after the exposure. Antibodies could be detected in serum and CSF of vaccinated patients with significant increase in the titers observed with bot MNT and IIFT (table 2). The other two patients had not taken any rabies vaccine after the bite and both MNT and IIFT detected antibody in the second samples of these patients.
DISCUSSION Use of Indirect Immunofluorescence technique for assaying antiviral antibodies is well established for many viruses like herpes simplex, measles, rubella etc using established cell lines infected with the virus as a source of antigen in the present study we have avoided the use of cell culture and used instead, rabies infected mouse brain smears as a source of antigen. The main aim of this present study was to evaluate the feasibility of using this simple indirect immunofluorescence assay for detection and quantification of rabies antibodies. We were particularly interested to apply such techniques for the rapid antemortem diagnosis of atypical paralytic rabies cases admitted in our institute. Keeping this in mind, initially we evaluated post-vaccination serum samples known to contain adequate levels of we tested serum and CSF samples from rabies cases. The technique described here is rapid (results can be given within 4 hours) and cost effective. About 10-12 uniform smears can be made from one infected mouse brain and can be stored at –75oC for several months. One constraint is the use of experimental mice and live virus and hence pre-exposure rabies vaccination becomes necessary before using this technique. The smears fixed in acetone may still be infectious and all precautions are to be followed while doing the test and slides to be safely discarded. The titers obtained with this technique were lower than those with MNT This was expected as IIFT detects only IgG antibodies whereas MNT detects neutralizing antibodies of all types. However as there was a significant correlation between the two, one can safely presume the protective status after immunization, by initial screening of the samples by doing an IIFT. The minimum protective titer recommended by WHO is 1: 16 (0.5 IU / ml) by both MNT and RFFIT6. After evaluation of large number of samples by IIFT, it may be possible to predict a comparative protective titer by IIFT. People with low antibody titers need to be advised booster doses of vaccine and rapid tests like IIFT may be of great help in such situations. Samples negative by IIFT our study with very low MNT (<1:8) titers were negative by IIFT. Ante-mortem diagnosis of rabies by laboratory tests is not always positive. The much-talked about corneal test is not very sensitive and sometimes non-specific fluorescence gives false positives (personnel experience). Antibodies to rabies virus generally appear late in the disease, and may not be detectable in classical hydrophobia cases with 2-4 days illness followed by death. Laboratory assistance may not be necessary in such cases, as clinical manifestations are typical. However, cases of rabies, presenting with ascending paralysis like Gullian-Barre syndrome may be difficult to diagnose clinically. Rabies specific antibodies appear in such cases as the duration of illness is generally longer and patients are kept alive for many days in intensive care units1. Detection of antibodies by rapid techniques, particularly in CSF may point to the diagnosis of rabies. This will help medical and paramedical staff to take adequate precautions to prevent personal infection. Though the number of paralytic rabies cases tested is small, the results are encouraging and a rapid ante-mortem diagnosis by IIFT will be of great help to clinicians. We did observe some non-specific fluorescence in very low dilutions of confirmed negative samples. However, this was overcome by prior adsorption with suspension of normal mouse brain (date not shown). In all, the specificity and sensitivity of this IIFT was quite high when evaluated against standard MNT. To conclude, the IIFT described here is a rapid and cost effective technique for assaying rabies antibodies and may be useful for screening a large number of samples of monitor protective status after vaccination. It may also help in rapid ante-mortem diagnosis of paralytic rabies cases.
ACKNOWLEDGEMENTS The authors are grateful to Dr. M Subbakrishna, Addl. Professor of Bio-Statistics, NIMHANS for assisting in statistical evaluation of results and Dr. Suresh Chandra, veterinary officer, NIMHANS for supplying experimental animals. REFERENCES
Table 1 Mean Antibody titers observed with IIFT and MNT in people vaccinated with PCECV and PVRV.
Table 2 Antibody titers in first and second samples of serum and CSF. In-patients with paralytic rabies.
*These two cases had taken incomplete course of Semple vaccine.
A SIMPLE LATEX AGGLUTINATION TEST FOR DETECTING RABIES ANTIBODIES Saraswati S. and Madhusudhana S. N. Department of Neurovirology, NIMHANS Bangalore ABSTRACT The Presently recommended tests for assaying rabies antibodies like mouse neutralization test (MNT) and rapid fluorescent focus inhibition test (RFFIT) are either time consuming or expensive and are generally performed in reference laboratories. There is a need to develop simple and rapid method for detection of rabies antibodies, which can be used to monitor sero-conversion after pre or post exposure vaccination. In this study, we have developed a latex agglutination test using purified rabies virus glycoprotein coupled latex particles. We tested 65 serum samples from people vaccinated with different rabies vaccines and results were evaluated with standard MNT or RFFIT Fifty-two out of 65 samples wee positive by both the tests. However it was found that serum samples having a minimum titer of 2.5 IU / ml or greater only showed clear-cut agglutination in the test. All the 15 negative controls tested showed no agglutination thus showing 100% screening for sero-conversion after a full course of any cell culture vaccine likely to produce high antibody titers.
INTRODUCTION Rabies is a major public health problem in many developing countries including India where an annual incidence of 30,000 human deaths is reported1. Presently the only means available to control this disease is effective post-exposure vaccination. In addition to Semple vaccine, which forms the mainstay of post-exposure treatment in India, three types of cell culture vaccines such as human diploid cell vaccine (HDCV), purified chick embryo cell vaccine (PCECV) and purified verocell rabies vaccine (PVRV) are also available2. These vaccines prevent development of the disease by inducing the formation of neutralizing antibodies. The role of cell mediated immune responses if any is still not clear3 The evaluation of the protective status of vaccines by estimating antibody levels becomes necessary for timely recommendation of booster doses, particularly in the case of severe category III exposure, in immunocompromised and malnourished people. Another situation where the estimation of rabies antibodies could be helpful is in the ante-mortem diagnosis of rabies, particularly paralytic rabies4, 5 The WHO recommends two tests for the estimation of antibodies to rabies, viz., the Mouse Neutralization Test (MNT) and the Rapid Fluorescent Focus Inhibition Test (RFFIT)6 Of these the former is laborious, time-consuming and needs the use of a large number of experimental mice.7 a regular supply of rabies conjugate and costly equipment like a fluorescent microscope8. These tests are usually performed in reference laboratories. ELISA kits are commercially available but are very expensive for routine use. Thus there is a need for development of simple, inexpensive and rapid test which can be performed in any laboratory. Earlier, Perrin et al reported a latex agglutination test using purified rabies virus8. Glycoprotein G of the rabies virus is the major protein, which induces neutralizing antibodies that offers protection against rabies. Keeping this in view, we have developed and evaluated a latex agglutination test. We report our preliminary findings. MATERIALS AND METHODS Clinical Samples: Sixty-five serum samples from people vaccinated with either PCECV (Rabipur, n=20) or rhesus diploid cell vaccine (RDRV, n=45) were tested. The serum samples collected 7-10 days after a full course of vaccination. We also tested 15 serum samples from healthy people who had neither taken rabies vaccine nor bitten by any animal in the past as negative controls. Growth and concentration of rabies virus: The challenge virus strain (CVS) obtained from Central Research Institute, Kasauli as a freeze dried mouse brain homogenate, having a titer of 107.0 MICLD50( Mice intracerebral lethal dose 50) per 0.03 ml was adapted to grow in Vero cell line (ATCC CCL 81, obtained from National Center for cell Science, Pune) by following established procedure9. The virus harvest at passage number 33 had titer of 10 6.5 MICLD 50 per 0.03 ml when titrated intracerebrally in young mice. This was used as a seed virus for further large-scale cultivation in Roux bottles Viral harvests were collected every third day and pooled. The pooled harvest was 10 5..5 MICLD50. Per 0.03 ml The harvest was concentrated by zinc acetate earlier10. The infectivity of the concentrated virus was 10 7.5 MICLD50 per 0.03 ml. Purification of rabies glycoprotein: This was carried out by affinity column chromatography using concanavalin —A sepharose column as described earlier11 Briefly one ml of concentrated virus was incubated with an equal volume of virus-solubilizing buffer (20mM Tris HC1, 800 mM NaC1, 0.5mM phenyl methy1 sulfony1 fluoride (PMSF), 20% glycerol and 0.5% TritonX-100) for one hour at 4 oC. It was then sonicated in a Sonicator at 20 kHz frequency ( Misonix, USA) for three minutes and the lysate dialyzed overnight against normal saline at 4oC. The lysate was then loaded on a 4 ml packed Con-A sepharose column ( Sigma) pre-equilibrated with 1M methy1-D-mannopyranoside (Sigma) in phosphate buffered saline (PBS .pH 7.6) After washing with PBS, the glycoprotein was eluted with 1M methy1-D-mannopyranoside in PBS. Ten 1ml fractions were collected at a flow rate of 9.1ml / min. The absorbance of these fractions was read at 280nm. The fractions with high protein were pooled and dialyzed. The protein content of the pooled fractions was 0.48mg / ml as determined by Lowry’s method. The purity of the potein was confirmed by SDS PAGE when a single distinct band of 65 kD was observed. Further, identity was established by doing a Western blot using anti-glycoprotein polyclonal antibody ( a gift from P. Perinea, Rabies Unit, Institute Pasteur, Paris, France ) The glycoprotein was aliquoted and stored at – 70 oC. Sensitization of latex beads with glycoprotein: One ml of 10% latex beads were washed three times in 5ml of carbonate buffer, pH 9.6. They were then centrifuged at 2500g for 30 minutes. The sediment was resuspended in 3ml of carbonate buffer and 1.75ml of glycoprotein (concentration 1mg / ml) and incubated for 3 hours at 37 C then overnight at 4 C. The sensitized beads were centrifuged and resuspended in 5ml of carbonate buffer containing 5% sucrose and 0.3% BSA. The beads were then incubated for 30minutes at 37 C. The beads were centrifuged and then washed twice in phosphate-buffered saline (PBS). They were then re suspended in 3 ml PBS. Latex agglutination Test: One drop of 10 ul of inactivated serum was deposited on a glass slide. 15 ul of the sensitized beads were added onto the serum. The slide was rotated for 15 minutes at 37 C. The slide was then observed for agglutination of the sensitized beads. Mouse Neutralization test (MNT): The serum samples from vaccinated people was tested by MNT as per WHO recommended procedure. Rapid Fluorescent focus inhibition test (RFFIT): This was performed at Pasteur Institute, Coonoor and results obtained from Dr. M.K. Sudarshan, KIMS, Bangalore.
RESULTS Antibody titers in vaccines: Out of 20 serum samples from PCEC vaccines tested 12 were positive by both MNT and LAT. Out of 45 serum samples from people vaccinated with RDRV 40 were positive by both RFFIT and LAT. Remaining samples were positive only by MNT and RFFIT respectively. It was also found that samples having a titer greater than 2 .5 IU / ml with either MNT or RFFIT were found positive by LAT. Samples having titers less than 2.5 IU / ml showed either negative or equivocal results. None of the fifteen control samples showed agglutination.
DISCUSSION Use of antigen coupled latex particles for detection of specific antibodies is a well-established procedure in clinical microbiology. Earlier the utility of this procedure has been demonstrated for detection of antibodies to varicella zoster12, rubella13 and HIV14. As mentioned earlier, neutralizing antibodies play an important role in conferring protection against rabies. The rabies virus envelope protein (G protein) is the major protein known to induce the development of neutralizing antibodies. In our study, we have purified the rabies G protein by a simple affinity column chromatography, confirmed its purity and then used for coupling to latex so that only anti-glycoprotein antibodies are detected. This has the additional advantage of increasing the specificity of the test. In general, sera with high antibody titters ( >2.5 IU / ml) only reacted in this agglutination test. Therefore this test cannot be used to quantify or titrate rabies antibodies but instead can be used to screen the presence or absence of antibodies after vaccination. One reason for discrepancy between MNT and LAT may be that whereas MNT detects all types of neutralizing antibodies, the LAT detects only agglutinating antibodies. To conclude, the LAT test developed in this study is a simple and economical procedure that can be easily adapted to any laboratory. However, basic tissue culture facilities are required. The column chromatography described here is a simple procedure and the yield of glycoprotein is quite good. The sensitized latex beads can be stored for a long time without loss of activity. The test can be completed in 30 minutes compared to 48 hours for RFFIT and 14 days for MNT. Another major advantage of this test is that a large number of samples can be screened at a time. Thus it can be used for rapid screening of serum samples of vaccines for presence of high titters to rabies antibodies.
ACKNOWLEDGEMENT The authors are grateful to Dr. M.K. Sudarshan, Professor and Head, Department of Community Medicine, Kempegowda Institute of Medical Science, Bangalore for providing serum samples from people vaccinated with RDRV. REFERENCES
A PASSIVE HAEMAGGLUTINATION TEST FOR RABIES ANTIBODIES USING PURIFIED RABIES GLYCOPROTEIN COUPLED TO SHEEP ERYTHROCYTES Saraswati S., Raju B.V.S., Chitra K., Shamsundar R., and Madhusudhana S. N., Department of Neurovirology, NIMHANS, Bangalore ABSTRACT The Presently recommended tests for assaying rabies antibodies like mouse neutralization test (MNT) and rapid fluorescent focus inhibition test are either time consuming or expensive and are generally performed in reference laboratories. There is a need to develop simple and rapid method for detection of rabies antibodies which can be used to monitor sero-conversion after pre or post exposure vaccination. In this study, we have developed a simple passive haemagglutination (PHA) using purified rabies virus glycoprotein coupled to sheep erythrocytes using chromium chloride (09.04%) as a coupling agent. One hundred and twenty samples from people vaccinated with different rabies vaccines, 6 paired serum and CSF samples from autopsy confirmed cases of paralytic rabies, and serum samples from 30 normal healthy controls were tested and evaluated in comparison to standard MNT. One hundred and fourteen samples were negative by PHA but positive by MNT. The titers obtained by PHA were lower compared to MNT, but there was significant correlation between the two (r=0.885). the specificity of the test was 98.6% and sensitivity was 98.4 % as compared to MNT.
INTRODUCTION Rabies is a major public health problem in many developing countries including India where an annual incidence of 30,000 human deaths is reported1. Presently the only means available to control this disease is effective post-exposure vaccination. In addition to Semple vaccine, which forms the mainstay of post-exposure treatment in India, three types of cell culture vaccines such as human diploid cell vaccine (HDCV), purified chick embryo cell vaccine (PCECV) and purified verocell rabies vaccine (PVRV) are also available2. These vaccines prevent development of the disease by inducing the formation of neutralizing antibodies. The role of cell mediated immune responses if any is still not clear3. The evaluation of the protective status of vaccines by estimating antibody levels becomes necessary for timely recommendation of booster doses, particularly in the case of severe category III exposures, in immunocompromised and malnourished people. Another situation where the estimation of rabies antibodies could be helpful is in the antemortem diagnosis of rabies, particularly paralytic rabies4, 5. The WHO recommends two tests for the estimation of antibodies to rabies, viz., the Mouse Neutralization Test (MNT) and the Rapid Fluorescent Focus Inhibition Test (RFFIT)6 Of these the former is laborious, time-consuming and needs the use of a large number of experimental mic.7 a regular supply of rabies conjugate and costly equipment like a fluorescent microscope8. These tests are usually performed in reference laboratories. ELISA kits are commercially available but are very expensive for routine use. Thus there is a need for development of simple, inexpensive and rapid test which can be performed in any laboratory. Earlier Gouge et al reported a simple passive haemagglutinaiton test (PHA) using unpurified but concentrated virus9 glycoprotein G of the rabies virus is the major protein which induces neutralizing antibodies that offers protection against rabies. Keeping this in view, we have developed and evaluated a PHA test using sheep erythrocytes coupled to purified rabies virus glycoprotein.
MATERIALS AND METHODS Clinical Samples: The study was conducted between January 1999 and June 1999 and serum samples available in the department( stored at – 70 oC) were used. One hundred and twenty serum samples from people vaccinated with either PCECV (Rabipur, n=74) or PVRV (Verorab, n=30) were tested. The serum samples collected on day 0 before vaccination and 7-10 days after a full course of vaccination. Twelve paired CSF and serum samples were collected from 6 cases of paralytic rabies (after confirmed by autopsy) admitted to Neurological services of NIMHANS. Thirty serum samples were collected from normal age and sex matched healthy individuals without history of previous rabies vaccination or dog bite. Growth and concentration of rabies virus: the challenge virus strain (CVS) obtained from Central Research Institute, Kasauli as a freeze dried mouse brain homogenate, having a titer of 107.0 MICLD50(Mice intracerebral lethal dose 50) per 0.03 ml was adapted to grow in Vero cell line (ATCC CCL 81, obtained from National Center for cell Science, Pune) by following established procedure10. The virus harvest at passage number 33 had titer of 10 6.5 MICLD 50 per 0.03 ml when titrated intracerebrally in young mice. This was used as a seed virus for further large-scale cultivation in Roux bottles Viral harvests were collected every third day and pooled. The pooled harvest was titrated by intracerebral inoculation of young mice. The infectivity titer of pooled harvest was 10 5.5 MICLD50. Per 0.03 ml The harvest was concentrated by zinc acetate precipitation followed by dialysis as described earlier10. The infectivity of the concentrated virus was 10 7.5 MICLD 50 per 0.03 ml. Purification of rabies glycoprotein: this was carried out by affinity column chromatography using concanavalin –A sepharose column as described earlier11 Briefly one ml of concentrated virus was incubated with an equal volume of virus-solubilizing buffer (20mM Tris HCl, 800 mM NaC1, 0.5mM pheny1 methy1 sulfony1 fluoride (PMSF), 20% glycerol and 0.5% TritonX-100) for one hour at 4 oC. It was then sonicated in a Sonicator at 20 kHz frequency ( Misonix, USA) for three minutes and the lysate dialyzed overnight against normal saline at 4 oC. The lysate was then loaded on a 4 ml packed Con-A sepharose column (Sigma) pre-equilibrated with 1M methy1 –D-mannopyranoside (Sigma) in phosphate buffered saline (PBS. pH 7.6) After washing with PBS, the glycoprotein was eluted with 1M mathy1-D-mannopyranoside in PBS. Ten 1ml fractions were collected at a flow rate of 0.1ml / min. the absorbance of these fractions was read at 280nm. The fractions with high protein were pooled and dialyzed. The protein content of the pooled fractions was 0.48mg / ml as determined by Lowry’s method. The purity of the protein was confirmed by SDS PAGE when a single distinct band of 65 kD was observed. Further, identity was established by doing a Western blot using anti-glycoprotein polyclonal antibody( a gift from P. Perin, Rabies Unit, Institute Pasteur, Paris, France ) The glycoprotein was aliquoted and stored at – 70 oC. Coupling of glycoprotein to sheep erythrocytes: Blood was collected from healthy sheep reared at Central Animal house, NIMHANS in Alsever solution and kept at 4 C for 4-6 days for aging. The coupling of glycoprotein to trypsin treated sheep erythrocytes was carried out using chromium chloride as coupling agent as described earlier12. Briefly, 10% of washed sheep erythrocytes was treated with chymotrypsin (0.05%) at 37 oC for 15min followed by trypsin inhibitor for 10 min at room temperature. Rabies. Glycoprotein was coupled to sheep erythrocytes at an optimal concentration of 200úg / ml in the presence of 0.04 % chromium chloride(Sigma). After incubation for one hour, the coupled erythrocytes were washed thrice with PBS and stabilized by overnight treatment with 0.1% glutaraldehyde. After stabilization, erythrocytes were washed thrice with 0.1% BSA-PBS and then suspended to a concentration of 1.25% in BSA-PBS and stored at 4 C. Passive haemagglutinaiton Test: The sera to be tested were along with the reference serum were first inactivated at 56o C for 30 min. Samples of CSF were tested as such. The sera, CSF and reference rabies immunoglobulin (RIG, described under MNT) were pre adsorbed with normal sheep erythrocytes to remove nonspecific and heterophile antibodies. The samples were then serially diluted in round-bottomed 96 well microtier plates (Nunc) using 0.1% BSA-PBS pH 7.2. To 25 ul of each dilution 25 ul of coupled erythrocytes were added and the plate kept at room temperature for 30 min and readings were taken. Clear cut mat formation indicating agglutination was observed with positive samples and the reference serum. Clear button formed with all negative samples and cell control( Fig 1). The titers observed with test samples were converted to IU / ml in comparison to values observed with reference serum. Mouse Neutralization Test: This was performed as per WHO recommended procedure7. Briefly, different dilutions of inactivated serum samples were mixed with 100LD50 of CVS and incubated at 37 C in a water bath for 90min along with serum and virus controls. The serum-virus mixtures were then inoculated intracerebrally into 4 – 6 weeks old Swiss albino mice, which were kept under observation for 14 days. Any death occurring within 6 days was ignored and depending on the survival / mortality between 6-14 days, end point dilutions were calculated by the Reed-Meunch method. The titters were expressed in IU / ml in comparison to a reference preparation of RIG (obtained form Central Research Institute, Kasauli) having a potency of 85 IU / ml. Statistical Analysis: the titers obtained by the two tests were statistically evaluated by Pearson’s product movement correlation test. A correlation coefficient greater than 0.7 was considered significant. The differences in titers observed in serial samples after a PVRV vaccination was also analyzed by a Student’s test.
RESULTS Antibody titers in vaccines: Out of 120 serum samples tested, 114 were positive by both PHA and MNT, 6 samples were positive by MNT but negative by PHA. Two samples on day 0 before vaccination were positive by PHA but negative by MNT. Two of the 30 negative controls were also found to be positive by PHA and negative by MNT. Though the titters obtained with PHA were lower than those obtained with MNT (Table 1), significant correlation was observed between them(r=0.885). Thus the overall false positivity of this PHA in comparison to MNT was 4 out of 240 making this PHA 98.6 % specific. The sensitivity is 97.3%. Antibody titers in rabies patients: We also tested paired serum and CSF sample from 6 cases of paralytic rabies later confirmed by autopsy. Out of these, 2 cases had not taken any previous vaccination but the remaining had taken incomplete treatment with Semple vaccine. Both PHA and MNT detected antibodies in 2nd serum sample of all vaccinated cases with significant increase in titers between the first and second samples. Antibodies were also detected in first CSF sample of 4 vaccinated cases by both MNT and PHA and in one case by MNT only. (Table 4). There was rise in titer by both tests in the corresponding second samples. Among the unvaccinated cases, 2 had antibody titers in the first serum by both tests but first CSF sample was positive by both tests in one case only. In one case antibodies were negative by both tests in the first sample but positive in the second samples.
DISCUSSION Use of antigen coupled erythrocytes for detection of specific antibodies is a well-established procedure in clinical microbiology. Earlier the utility of this procedure has been demonstrated for detection of antibodies to herpes viruses13, hepatitis viruses14 etc. In an earlier study, the value of this procedure was demonstrated for detection of rabies antibodies by Gouge et al9 who used concentrated but un purified virus grown in cell culture, as a source of antigen for coupling to sheep erythrocytes. As mentioned earlier, neutralizing antibodies play an important role in conferring protection against rabies. The rabies virus envelope protein (G protein) is the major protein known to induce the development of neutralizing antibodies. In our study, we have purified the rabies G protein by a simple affinity column chromatography, confirmed its purity and then used for coupling. To erythrocytes so that only anti-glycoprotein antibodies are detected. This has the additional advantage of increasing the specificity of the test. In general, there was good agreement between the result of MNT and PHA. Barring 4 samples, all the samples which were positive by MNT were also positive by PHA making this test 98.4% sensitive. The lower titers observed with PHA is in agreement with earlier report of Gough et al who showed that PHA is more sensitive to pick up 1gM antibodies than IgG antibodies. The blood samples from most of our patients were collected 7-10 days after a complete course of vaccination by which time it is expected that the antibody response would be more of IgG type. In those cases where we could obtain serial samples beginning day 14 post-vaccination it is evident that the rise or fall in antibody titer paralleled MNT titers and more importantly, there is no significant difference in titers between the tests on samples obtained on day 14 when the antibody response could still be of IgM type ( p= >0.05). Though this simple test is useful to detect and quantify rabies antibodies it is to be kept ion mind that both neutralizing and other antibodies (some epitopes of glycoprotein may not induce neutralizing antibodies) may be detected in total. The WHO recommends a titer of 0.5 IU / ml obtained with MNT as indicative of protection6 It may be possible to determine and suggest a protective titer with PHA after testing large number of samples with consistent results. However for antemortem diagnosis of rabies, this aspect may not be applicable. As any amount of rabies antibody detected in an unvaccinated patient indicates a diagnosis of rabies. In rabies encephalitis, antibodies are not usually present in the earlier stages of the disease in serum and CSF but may develop later, particularly in paralytic rabies. Thus this test could be used for the detection of antibodies in patient sera or CSF. In this study, we tested 8 paired serum and CSF samples from autopsy confirmed paralytic rabies cases. We could detect antibodies in first CSF sample of 7 cases. Interestingly, 2 un vaccinated cases showed antibody titer in the first serum sample. A rise in the antibody titer was observed in second serum and CSF in all cases. Therefore antemortem diagnosis was possible in these paralytic rabies cases. However, utility of this PHA for antemortem diagnosis of rabies needs to be investigated further. To conclude the PHA test developed in this study is a simple and economical procedure that can be easily adapted to any laboratory. However, basic tissue culture facilities are required. The column chromatography described here is a simple chromatography described here is a simple procedure and the yield of glycoprotein is quite good. The sensitized and glutaraldehyde stabilized coupled erythrocytes can be stored for a long time without loss of activity. The test can be completed in 3 hours, compared to 48 hours for RFFIT and 14 days for MNT. Another major advantage of this test is that a large number of samples can be screened at a time. Thus it can be used for rapid screening of serum samples of vaccines for presence of adequate titers of rabies antibodies. This is of crucial importance in situation where booster doses are required, due to low antibody titer. This test may also be useful for rapid diagnosis of paralytic rabies which is difficult diagnosis based on clinical presentation alone.
ACKNOWLEDGEMENTS The authors are grateful to Dr. M. Subbakrishna, Additional Professor of Biostatistics, NIMHANS for helping in statistical evaluation, to Dr. Sureshchandra, senior veterinary officer, NIMHANS for supplying experimental mice. REFERENCES
Table I Antibody titers (IU/m)*observed with PHA and MNT in people vaccinated with PCEV and PVRV vaccine:
PCECV= Purified Chick embryo cell vaccine (Rabipur) PHA= passive haemagglutination test MNT= Mouse neutralization test ND= not detected except **non specific agglutination in 2 cases in 1:2 dilution *Mean titers with range in parenthesis.
Table II Antibody titers in paired serum and CSF samples ( 1st, 2nd) of paralytic rabies patients.
PHA= Passive haemagglutination test MNT= Mouse neutralization test *These cases had not taken previous vaccination ND= not detected.
PURIFIED VEROCELL RABIES VACCINE: A CHOICE VACCINE FOR INFANTS AND CHILDREN J. K. Dutta Senior Consulting Physician, Amaranath Clinic, Balasore 756001, Orissa
ABSTRACT Purified vero cell rabies vaccine (PVRV) is the latest cell culture vaccine marketed in India. Many studies in India and abroad have already confirmed its safety and efficacy in all groups of people. This vaccine is highly concentrated and is effective in 0.5-ml quantities. Thus it is more acceptable to people specially in small children and infants. This paper describes the use, safety and efficacy of PVRV in children attending an anti-rabies clinic in Orissa.
INTRODUCTION Rabies is a common health hazard in most developing countries. Mammal vectors are frequently responsible for transmission, which includes dog, cat, jackal, monkey, mongoose etc. Dogs are the principal agents of transmission of human rabies. They are observed to be responsible for transmission in 93.4% of cases as observed in another study 2) Rabies transmission by chronic canine excreta is a rare event. 3) There are some specific reasons why children become the common victims of mammal bite 4) They are very fond of pups and kittens with whom they play, pet, nuzzle and sometimes tease them. During the process pups, kitten or their mothers may attack them when they fail to protect themselves. Secondly, because of small stature, underdeveloped skull and large size of head compared to body they are more vulnerable to aggressive attacks by different mammals. Hence prevention of rabies in this vulnerable group is of utmost importance. So the present study was designed to assess the efficiency and tolerance of purified Vero cell rabies vaccine (PVRV) against rabies.
MATERIALS AND METHODS During the period 1996 through 1998 the study was carried out in infants and children who came for consultation regarding post exposure treatment (PET) following exposure to animal bites. Out of 254 infants and children in the group 12 (4.72%) patients were below one year of age while 242 were in the age group of 1 to 14 years (Table –1). Some of the patients had received indigenous treatment prior to consultation. None had received any type of tissue culture vaccine or Semple vaccine. Epidemiological and clinical history was collected in each case. After instant clinical examination, patients were administered Vero cell rabies vaccines by intramuscular route in deltoid region or antero-lateral aspect of the thigh. Most of the patients were administered the vaccine in clinic. Few others coming from a distance had the vaccine administered by the local medical or the Para medical staff. Strict instruction was issued to get the vaccine injected in deltoid region or anterolateral aspect of thigh in thin children. In 157 (61.8%) patients the exposure was noted in lower extremity (Table-4). Other findings have also been tabulated (Tables-2, 3,5,6).
RESULTS & DISCUSSION During consultation it is often observed that parents and guardians feel reluctant to get their children vaccinated lest any serious reaction may develop if there was no poison (virus). Furthermore, they also express reluctance when for children adult dose of vaccine is prepared unlike other injectables and medicines, which are usually prescribed in lower doses. In the present study parents were fully explained the details of therapy by which anxiety was relieved and they consent for vaccination. Purified Vero cell rabies vaccine was used in each patient by intramuscular route. Because of small quantity (0.5 ml) of vaccine administered each time pain at the site of inoculation was not a problem even in young children. Many of them congratulated the vaccine and clinician for not causing the pain during and after vaccine administration. All the patients were fully protected and there was no death from rabies in this group. Unfortunately 2 patients were vaccinated in gluteal region due to ignorance of paramedical staff who administered the vaccine. An optional dose was advised to compensate for the lapse. As there was no death from rabies during follows up period of 2 years or more the vaccine was considered to be very effective. Tolerance was excellent in 252 (99.21%) patients (Table-6). PVRV has been used in previous studies to access tolerance and efficacy in young children. In one study 566 children under 15 years of age received PVRV by intramuscularly or intradermal route. All the vaccines tolerated the drug well5. In another study6 administration of PVRV in 6 doses by deep subcutaneous routes induced adequate neutralizing antibody. The level was 1.57 IU or higher in all the patients on day 180 but the level declined in some children when tested on day364. In this study side effects were seen in 2 patients (0.78%). One patient’s complained that the child had developed anorexia. He was not concentrating properly in study. Another child developed pruritis on elbow and around anal region. As pruritis was severe the parents applied kerosene oil over the affected region with which he did not improve. On consultation he was advised cetrizine orally with which he had satisfactory improvement. Tolerance was excellent in 252 (99.21%) children. In view of repeated success indifferent studies including the present one, PVRV is recommended as a choice vaccine for prophylaxis against rabies in children. The cost is nearly one third of HDCV and is freely available. Because of these advantages the vaccine is almost invariably used in the clinic where the study was carried out.
Table-1 Age distribution of patients. Age No % Under one year of age 12 4.72 1-3 years 42 16.53 4-6 years 60 23.62 7-10 years 110 43.30 11-14 years 30 11.81 Table –2 Sex of patients in study group Sex No % Male 182 71.65 Female 72 28.35 Table-3 Nature of exposures Nature of exposure No % Dog bite 116 45.66 Scratch by dog 36 14.17 Pup bite 24 9.44 Scratch by pup 20 7.87 Cat bite 24 3.14 Scratch by cat 8 3.14 Monkey bite 26 10.23 Table-4 site of bite or scratch by mammals Site No % Head and neck 14 5.51 Upper extremity 54 21.25 Palm and finger 20 7.87 Lower extremity 149 58.66 Toe 8 3.14 Abdomen 3 1.18 Buttock 5 1.96 Penis 1 0.39
Table-5 Number and percentage of patients who received PVRV and PVRV with HRIG. Vaccine No % *PVRV 248 97.63 **PVRV & HRIG 6 2.36 *PVRV – purified Vero cell rabies vaccine (verorab) **HRIG-Human rabies immunoglobulin.
Table-6 Tolerance of patients to PVEV. Tolerance No % Excellent 252 99.21 Good 2 0.78
REFERENCES:
TOPOGRAPHIC DISTRIBUTION OF RABIES VIRAL ANTIGEN IN DOG BRAIN – ITS IMPLICATIONS Suja M. S.*, Anita Mahadevan**, Madhusudhana S. N.$, Vijayasarathi S***, Shankar S. K.**. Departments of Neurovirology* and Neuropathology** NIMHANS Department of Veterinary Pathology ***, Veterinary College Hebbal, Bangalore $ corresponding author
ABSTRACT Till date, there is no study from Asian countries describing the pathology and topographic distribution of virulent, street’ rabies viral strain in canine brain. As a preliminary study, five brains of street dogs from urban areas of Bangalore infected with rabies were collected. The diagnosis was confirmed by immunofluorescent study. The patho-morphological feature and the anatomic distribution of the viral antigen by immunohistochemistry were studied. The vital antigen was mostly localized to the neuronal perikaryon extending along the dendrites, while occasional astrocytes were also labeled. In the brain, the limbic areas, thalamus and the reticular formation of the brain stem, the trigeminal and vagal nuclei were involved, corresponding to areas of cholinergic innovation. It’s proposed that the preferential involvement of these cholinergic zones could explain some of the clinical features of rabies in canines. The involvement of thalamus and immuno localization of the rabies viral antigen in the axons are the unusual features noted in dog brains in contrast to murine experimental studies with ‘fixed’ virus.
INTRODUCTION The incidence of rabies in free living wild animals has been well recognized. Foxes, Jackals, wolves, hyenas, raccoons; skunks, wild cats and mongoose have been recognized as potential sources that harbor the rabies virus. Freely roaming unvaccinated dogs and cats in many countries in Asia, Africa and South America present a serious health hazard to humans. In fact according to the WHO survey of 1996, 35,000 to 50,000 people worldwide die of rabies every year. Of these, 32,772 deaths are reported from Asia of which 30,000 deaths are recorded from India alone1. The domestic dog is the principal reservoir of rabies infection and plays an important role in transmission of this dreaded disease. The rabies virus in almost always transmitted by a bite of an infected animal and rarely by exposure of mucous membranes to the infected saliva or other secretions for example, bat urine for grazing calves. Similar to bimodal clinical presentation of rabies in humans as the encephalitic / hydrophobic and paralytic forms, even dogs and cats have either classical symptoms of "furious" rabies such as irritability, aggressiveness and injured gums and teeth or manifest with flaccid paralysis. Occasionally the infected animal may even look apparently normal. Rarely the dogs recover from rabies but continue to secrete viable virus in saliva, thereby remaining a carrier of infection. Definitive pathological diagnosis of rabies infection is based on demonstration of pathognomonic intracytoplasimic eosinophilic inclusions in the nerve cells and the dendrites extending from them. Unlike most viral infections, the frequent absence of meningeal reaction, minimal perivascular inflammation, and the paucity of cellular reaction in the tissues that surround the neurons containing Negri bodies raises many questions on the pathogenesis of the clinical manifestations that have remained unanswered. Rabies is a strict neurotropic viral infection, nicotinic acety1-choline receptor being the receptor that facilitates the entry of the virus into the nervous system and subsequent transport along the axons. Based on the knowledge that different species have different levels of susceptibility, latency and infectivity to the rabies virus, it was believed that the type of animal host and the viral strain were the major determinant for the two major types of clinical rabies. But characterization of the rabies virus isolates that caused furious and dumb rabies in Thai dogs using a panel of nucleoprotein (N) and glycoprotein (G) – specific monoclonal antibodies and molecular genetic analysis revealed identical forms2. It has been also been recorded that the same dog that transmitted paralytic rabies to one patient also caused classical hydrophobic rabies in another 3. This suggests that the clinical presentation of the illness may be dependent on the areas of the brain affected by the virus rather than the strain of the viral isolate or the host factors. In this study, a preliminary attempt is made to map the regional distribution of the viral antigen in the brains of five street dogs confirmed to have contracted rabies infection using histopathological and immunohistochemical techniques. As the study is retrospective, detailed antemortem clinical features like the incubation period, the duration of illness, clinical presentation etc. Were not available. However, an attempt is made to correlate the anatomical areas involved with the most common clinical symptoms seen in dogs. While the issue of the regions involved by the virus has been previously addressed both in humans and laboratory animals that employed fixed rabies strains, detailed description of the specific nuclei involved was not documented nor was correlation of clinical symptoms with the regions involved attempted. The only study that has documented work on striped skunks inoculated with street rabies virus. Till date, to the best of our knowledge no study has addressed the question of topographic distribution of rabies viral antigen in the dog brain with specific reference to the street dog and attempted to correlate with the clinical manifestations of the infection.
MATERIALS AND METHODS Formalin fixed brains from five dogs, as one sagital half or the whole brain were obtained from the Department of Veterinary Pathology, Veterinary College, Hebbal, Bangalore. The dogs were street dogs from the urban areas of Bangalore City. These animals had been sacrificed based on clinical suspicion of rabies and submitted to the reference laboratory for confirmation where the brain was collected following autopsy. A small bit of fresh brain tissue from the medical temporal lobe was collected at the time of autopsy and frozen. He smears made from the fresh tissue were fixed in cold acetone. The smears were stained with FITC-conjugated hyperimmune Equine anti-rabies serum (Source: Central Research Institute, Kasauli, Himachal Pradesh, dilution 1:32) and mounted in glycerol. Appropriate positive and negative controls were incorporated. The sections were screened under fluorescent microscope for the presence of inra-and extra cellular fluorescent Negri bodies. The brains after Formalin fixation were sliced in the coronal plane, and processed for paraffin embedding. Sections were taken from various anatomical areas. Five-micron thick paraffin section were stained with haematoxylin-eosin stain for routine histological evaluation. Sections were also immuno stained by the indirect immunoperoxidase method, following microwave treatment for unmasking the antigen, Briefly, the sections after stabilizing in phosphate buffer saline (pH 7.4) were treated with 9.3% Methanol-H2O2 to block endogenous peroxidase and 3% delipidised milk powder solution to block nonspecific labeling. The sections were incubated for 1 hr with anti-rabies antibody prepared in horses (Source: Central Research Institute, Kasauli, Himachal Pradesh) at a dilution of 1:100. Following washes thrice in buffer, the sections were treated for 30 min with goat antihorse antibody (Bangalore Genei, 1:20) tagged with horseradish peroxidase. After rinsing thrice in buffer, the immuno reaction was visualized by the chromogen DAB / H2O2. The sections were counterstained with Harris’ haematoxylin. Sections treated identically, omitting the primary antibody served as negative control, while a section from confirmed case of rabies containing numerous Negri bodies was used as positive control to monitor the efficacy of the immunostaining. The patho morphological features including microglial proliferation, perivascular inflammation, neuronophagia, and presence of intraneuronal eosinophilic Negri bodies were evaluated. The distribution of antigen positive cells in various anatomical areas, the pattern of neuronal / glial / white matter tracts labeling were mapped on brain tracing at different levels. The number of antigen positive cells in different regions were visually graded from + to ++++.
Results: All the five dog brains revealed presence of diagnostic fluorescent Negri bodies in neurons on smears from the hippocampal region, confirming rabies infection. Detailed clinical history and findings were not available in all the cases, except for the mention of excessive salivation and aggressive behavior. On examination using routine haematoxylin & Eosin stains, the most consistent finding was a diffuse microglial proliferation of varying intensity in different regions. In the cortex, microglial proliferation was consistently most marked in the temporal cortex followed by the cingulate gyrus-Microglial nodules were seen in only one case, which also showed destruction of neurons by microglial cells. This was most evident in the frontal and temporal cortices. The changes in the parietal cortex was minimal in comparison with other regions. Proliferation of microglial cells both in diffuse and nodular fashion was also noted in the thalamus and brainstem in 4 / 5 cases. Neuronophagia was more often found in the brain stem reticular neurons. Meningeal inflammation indicative of a menigitic process was seen in only 2 / 5 cases, though dense perivascular mononuclear infiltrates were seen in 4, especially in the brainstem. Changes in the neurons paralleled the degree of inflammation. The single case with mild / no inflammation showed well-preserved neurons while in the others, the neurons showed evidence of degeneration / ischemia with condensed, eosinophilic perikarya, condensed nuclei, and loss of Nissl substance. A peculiar change noted in the large neurons of the midline reticular formation was swelling and pallor of the cytoplasm with chromatolysis. The white matter in most cases appeared uninvolved except in one case, which showed focal demyelinating lesions with attendant gliosis in the internal capsule extending laterally. Dense subpial carpet of gliosis was seen in the frontal cortex and rostral colliculus of the midbrain in 2 cases. Presence of reactive astrocytes was restricted to these subpial zones but not found in the white matter despite the encephalitic process. Occasional round to oval discrete eosinophilic Negri bodies were identified in the large reticular neurons and Purkinje cells of the cerebellum. However, immunostaining highlighted presence of overwhelmingly large number of intracytoplasimic as well as dendritic and axonal Negri bodies representing the distribution of the viral antigen within the cell. By immunohistochemistry, antigen containing neurons were found in almost all the regions examined but differed in the location, number and morphology. The overall distribution was similar in all 5 cases with greater involvement of the brain stem in comparison with the supra-tentorial structures. In all except one case, the involvement was essentially bilateral. The pattern of antigen distribution in the five cases was intriguing appearing to be confined largely to areas with a rich cholinergic innovation (Table 1). In the cortex, maximal involvement was found in the temporal and entorrhinal cortex followed by the cingulate gyrus, frontal and lastly the parietal cortex. The distribution closely seemed to parallel the microglial proliferation. In the frontal and parietal cortex, the lower layers were selectively involved sparing the upper layers in 3 / 5 animals examined. In 2 of the animals, all layers of the cortex were involved and in addition showed subpial astrocytosis and gliosis. Presence of the antigen was detectable in the cytoplasm of the reactive astrocytes in these cases both in the molecular layer as well as the deep white matter tracts. Distinct and intense staining of the small neurons of the indiseum griseum was noted in all 5 animals. By immunohistochemistry, the pattern of antigen deposition varied from diffuse cytiokasnuc staining to stippled granular deposits in the neuronal cytoplasm with extension along the apical dendrites, some forming discrete, small Negri bodies (Fig.1). intracytoplasimic Negri bodies were seen as large, round dark brown masses within the perikaryon of the neuron. Although all three patterns of staining were demonstrable in four cases, in one of the animals, ‘Negri bodies’ was the only type of antigen deposition seen in all the infected neurons. Hippocampal involvement was seen in all animals but to varying degrees. Interestingly, the pyramidal neurons of the Ammon’s horn was uniformly involved in all 5 animals (Fig. 2B) while the small granule neurons of the dentate gyrus showed presence of the viral antigen in only 3 cases (Fig.2A). The neurons of the CA4-CA5 and CA1 – subiculum as well as the entorrhinal cortex showed maximal involvement with numerous Negri bodies in the cytoplasm. Extension of viral antigen into the axons of the stratum radiation and lacunosum was prominent (Fig.2B). In two of the cases where the amygdala was available for study, numerous neurons were noted to have intense cytoplasmic staining or multiple, small Negri bodies. Therefore, involvement of the limbic system including the hippocampus, amygdala, cingulate gyrus and the indiseum griseum was a prominent finding in this study. Antigen deposition in the cerebellum was found in the Purkinje cells (3 cases) as a diffuse cytoplasm staining or multiple, small Negri bodies in the perikaryon with extension upwards along the apical dendrites into the molecular layer (Fig.3). The granule neurons of the internal granular layer were spared but focal positivity of the synaptic glomeruli was noted in one case. Intriguingly the intensity of immunoreactivity in the cerebellum was lesser in comparison with the cortex and brainstem. In the basal ganglia, the caudate nucleus, putamen and globus pallidus (2 cases) showed abundant antigen deposition within the neurons. The thalamic neurons showed intense immunoreactivity in all five animals. The distribution of the viral antigen was uniform in all. The reticular nucleus showed maximal immunoreactivity followed by the lateral group and the ventral posteromedial and ventral posterolateral muclei. The subthalamic and hypothalamic muclei were involved in only 2 cases each. In the midbrain, the neurons of the periaqueductal Grey, the large neurons of the midline and lateral reticular formation, bilateral oculomotor and red mucleus, Substantia nigra and the mesencephalic mucleus of the trigeminal showed presence of viral antigen in all cases. The dilated and tortuous axons in the central segmental fasciculus also showed immunoreactivity. Interestingly in 3 cased, the brachium of the caudal colliculus (white fiber tract that connects to the medial geniculate body associated with the auditory pathway) showed intense immunoreactivity, with asymmetric involvement in only one of the cases. The pons was available in only 3 cases, all of which showed intense immunoreactivity of the large neurons of the reticular formation as well as the trigeminal nucleus. In the medulla oblongata (2 cases), the midline reticular formation neurons were involved (fig.4) as well as the hypoglossal and vagal cranial nerve nuclei in two. The neurons of the inferior olivary nucleus in one case showed presence of the viral antigen. Neurons were the most common cell type to be involved but in the cortex occasional astrocytes and oligodendroglia were found to contain the antigen. In all five animals, the brainstem as well as the supratentorial structures were involved, varying only in the intensity and pattern of immunoreactivity (Fig.5). The regions involved roughly corresponded to areas rich in cholinergic innovation.
DISCUSSION: In literature, most studies have focused on involvement of regions of the brain in general such as cortex, cerebellum, medulla, pons etc 4, 5 but detailed studies that have delineated specific nuclear involvement are few6. These include an experimental study following intracerebral or subcutaneous inoculation into adult mice, wherein the presence of the viral antigen was recorded in neurons of rhinencephalic structures, recorded in neurons of rhinencephalic structure, brainstem nuclei, dorsal root ganglion, anterior horn neurons and cerebellar purkinje cells 5. However no correlation to the clinical features was undertaken. Tiravantupong et al 4 studied the regional distribution of rabies viral antigen in central nervous system of human encephalitic and paralytic rabies and opined that there were no differences in virus localization between the hydrophobic and paralytic type of rabies. Hence patterns of viral localization in the brain could not explain the this study, the broad regional areas have been examined, detailed examination of individual nuclear groups is lacking. The only study that has recorded in detail specific nuclear groups involved, is an experimental study on skunks that were inoculated intramuscularly with ‘street virus’ in the pelvic area 6. The sequence of events in the early stages and the transit ways of the virus to the brain were traced from the regional areas involved. The authors surmise that once the virus enters the spinal cord, infection spreads locally via the propriospinal neurons and upwards via long descending and ascending tracts to the brain to involve the reticular formation, motor cortex, the ventral postero-lateral and reticular nucleus of thalamus, the vestibular nucleus, and the nucleus interpositus. The authors however have not attempted correlation of clinical symptoms based on the topographic distribution of the viral antigen. In this study, the antigen distribution was uniform in all cases examined although the density varied. The principal regions affected included the reticular formation of the brain stem, bilateral trigeminal, facial, vagal and hypoglossal cranial nerve nuclei, the inferior olivary nucleus in the medulla oblongata, bilateral oculomotor and red nuclei, Substantia nigra and the periaqueductal Grey matter in the midbrain. Of the supratentorial structures, there was striking involvement of the lateral and ventral group of thalamic nuclei as well as caudate and putaminal neurons in addition to the limbic structures. By and large these areas of the brain have Acetylcholine (Ach) as one of the neurotransmitters or receive a prominent cholinergic input from the basal forebrain and brainstem. Involvement of these regions as well as the trigeminal, facial and vagal nerves which carry the preganglionic parasympathetic afferents reflect the affinity of these viruses for the Ach receptor. This is further strengthened by the involvement of the neurons of the Substantia nigra which contain Ach in addition to dopamine. The initial symptoms of rabies in dogs such as ruffling of fur, excessive salivation, paralysis of the muscles of mastication (dropped jaw syndrome), pupillary dilation, altered phonation, piloerection, and tremors could be explained as a generalized activation of the parasympathetic system via the V, VII, IX, and X cranial nerves which are secretomotor to the lacrimal and salivary glands, and supply the muscles of mastication, the pharyngeal and laryngeal muscles and the pupillary muscles of the eye. In fact, transaxonal spread of the virus to the salivary gland was considered by Tordo et al (1998) 7 who electromicroscopically demonstrated the presence of the viral particles within the lumen of the glands. The earliest manifestation of the paralytic rabies in dogs is as a wobbly, labored gait. It is tempting to speculate that cerebellar involvement is the basis for this, while involvement of the Substantia nigral neurons could be the cause of tremors in these animals. But this can only remain as speculation as detailed record of clinical manifestations in these dogs were lacking thereby precluding a meaningful structure-function correlation. The involvement of the limbic system including the cingulate gyrus, amygdala and hippocampus have previously been implicated to explain the altered behaviour and aggressiveness in the animals. It is well known that the amygdala, pyramidal neurons of the Ammon’s horn of the hippocampus and the Purkinje cells of the cerebellum have GABA as the main inhibitory neurotransmitter, in addition to their cholinergic inputs. As seen in this study, the rabies virus preferentially involves these sites. This could result in loss of this inhibitory mechanism, resulting in excessive excitation. Based on the preliminary findings in this study, although a possible pathogenesis for some of the common clinical symptoms in dogs is suggested, the lack of detailed clinical records including incubation period constitutes a major drawback in this study that precluded a meaningful structure – function correlation. However, the overall pattern of antigen distribution found in the study of street dog brain suggests preferential involvement of the parasympathetic and cholinergic regions of the brain. For confirmation, comprehensive studies that correlate precisely the anatomic areas involved with accurate clinical records are essential. The evaluation of the nervous system of the street canine infected by the ‘street’ virus, unlike the experimental studies that employ ‘fixed’ virus strains, is more likely to provide realistic information to understand the biology and natural history of the rabies infection, so that in course of time, effective therapy maybe found to combat this dreaded disease.
REFERENCES
Table 5: Anatomic Distribution of Rabies Viral Antigen in Five Street Dogs
NA = NOT AVAILABLE
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||