The exposure of domestic farm animals like cattle, buffaloes, horses, goats and sheep to bites of rabid dogs is of great socio-economic concern especially in rural areas where the cattle are the backbone of family economy and a dependable livelihood. In Shimla Municipality in 2016, seven of 21 jersey cows died due to clinical rabies despite having been given post-exposure prophylaxis (PEP) with recommended IM injections of rabies vaccine on day 0, 3, 7, 14 and 28. One of the adult cows died after 20th day of exposure and another after 35th day of exposure. This may have occurred due to a short incubation period consequent to bite wounds mostly near brain (on head and neck region) or non-administration of rabies immunoglobulin (RIG) into wounds or failure of IM vaccination to induce early protective response. IM rabies vaccine in animals has shown protective effect  but still PEP failures in cattle have been reported from all parts of the Globe  . Even vaccine antigen titers when raised to 4.0 IU or even higher resulted in failure  underlining the inappropriateness of rabies vaccine alone in animal post exposure prophylaxis.
Intra-dermal rabies vaccination (IDRV) trials have shown their efficacy in limited studies in cattle   . IDRV rabies vaccination has proved to be more immunogenic than IM administration but IDRV protocols have not been formulated till date and no studies are there with IDRV Protocols tested on lab confirmed rabid dog bitten cattle; hence this study was undertaken. The efficacy of IDRV is well documented and practiced in human PEP in many countries including India and in our state of Himachal Pradesh  . Since the rabies immunoglobulins given based on body weight in animals have proven their efficacy in saving the life of animals like Sheep  (26 IU per Kg on the day of infection), they cannot be practiced in larger animals as the costs are overwhelming. Human rabies immunoglobulins (HRIG), as tried in sheep are @ $100 per 2 ml vial. A recent study by Bharti and Madhusudana et al. has shown that local infiltration of rabies immunoglobulins (RIGs) in and around wounds  is effective in preventing rabies even in lab confirmed rabid dog bite human patients and prompted us to do this study in animals to save their lives.
24 of the 60 healthy bovine without any known exposure to rabid animals, were randomly allocated to various groups (Table 1) by draw of lots and were given
Table 1. Phase I: exploring the feasibility of a new low cost intra-dermal pre & post exposure rabies prophylaxis protocol, the results are expressed as RVNA titers done through RFFIT in IU/ml of serum on different days (JC MEANS COWS & JB MEANS BUFFALOES).
GMT at Day 14 is 7.47 IU/ml; ANA means Animal not available.
0.2 ml ID on Day 0, 3 DAY GROUP-GROUP NO. 2
GMT at Day 14 is 7.5 IU/ml.
0.2 ml ID on Day 0, 3, 7 DAY GROUP-GROUP NO. 3
GMT at Day 14 is 5.2 IU/ml.
0.2 ml ID on Day 0, 3, 7, 14 DAY GROUP-GROUP NO. 4
GMT at Day 14 is 2.11 IU/ml.
0.2 ml ID on Day 0, 3, 7, 14, 28 DAY GROUP-GROUP NO. 5
GMT at Day 14 is 5.26 IU/ml.
INTRA MUSCULAR GROUP GROUP NO. 6
GMT at Day 14 is 0.38 IU/ml. N.B.: 5 doses of 1 ml rabies vaccine given IM is significantly less immunogenic than required on day 14 compare to 5 doses of IDRV given 0.2 ml. Test of Significance: GMTs ID Vs IM is 5.26 vs 0.38 (p = 0.0136).
Group 7: RFFIT RESULTS OF COWs in university Farm not given any rabies vaccination-SERUM SAMPLES
GMT: At day 0 are 0.22 IU/ml. NOTE: None of the farm kept cows had antibodies more than 0.5 IU/ml serum.
antirabies ID vaccination in varied schedules on day 0, 3, 7, 14 and 28 as 0.2 ml on Rt side of middle 1/3rd of neck. The volume and site were selected based on the study by Zdenek Beníšek  . To develop appropriate PEP protocol, serum samples were collected from all five groups on day 0, 14, 35, 90 and 365 and were tested for RVNA by RFFIT at National Institute of Mental Health and Neurosciences (NIMHANS), Bangalore. The experiment was approved by the ethical committee and animal owner were asked to give written informed consent for injection and blood testing. We did not draw blood from pregnant bovine. Data on animal variables was also collected. Rabivac vet, a Cell Culture Vaccine available in 1 ml per vial in government hospitals and manufactured indigenously by Brilliant Bio Pharma private limited, IDA Pashamytaram.Medak-502 307, T.S., India having potency of 2.5 IU/1ml Batch no 147/2015 Mfg Jan 2015 and Exp Dec 2017, was used. Equine rabies immunoglobulin (eRIG) procured from CRI Kasauli HP, India, having potency of 550 IU/ml was used.
2.1. Procedure/Technique of Intra-Dermal Vaccination
This involves injection of 0.2 ml of rabies vaccine ID on middle 1/3rd of neck of the bovine. Using aseptic technique, 1 ml insulin syringe 0.2 ml (up to 8 units in a 40 unit insulin syringe; i.e. 0.2 ml per ID site) of vaccine was injected into the dermal layer of middle 1/3rd of neck. A raised papule about 1 - 1.5 cm will appear immediately (Figure 1, Figure 2) causing a peau d’orange (orange peel) appearance.
2.2. Collection of Samples Post-Vaccination
Blood (Serum) samples for estimation of rabies Virus neutralizing antibodies (RVNA) by rapid fluorescent focus inhibition test (RFFIT) were collected on days 0, 14, 35 and 90 in vaccinated animals. 5 ml of venous blood was drawn with aseptic technique from the jugular vein of the animals. The blood samples were allowed to stand for about 30 minutes, centrifuged at 3000 rpm and the separated sera were collected and transported in cold chain to the Department of Neurovirology, NIMHANS, Bangalore.
Figure 1. Intra-dermal anti rabies vaccination done in a healthy cow in Jawali Clinic, HP, India.
Figure 2. Intradermal anti rabies vaccination done in healthy Buffalo in Jawali Clinic, HP, India.
2.3. Rapid Fluorescent Focus Inhibition Test (RFFIT)
RFFIT for estimation of rabies virus neutralizing antibodies (RVNA) was performed at the department of Neurovirology, NIMHANS using a WHO recommended procedure with minor modifications (Smith et al., 1996  ). BHK 21 cell line (ATCC CCL 10) and BHK 21 adapted CVS 11 strain of virus was used and the tests were performed in 96 well tissue culture plates. The antibody titers were expressed in International units (IU/ml) in comparison to an in house reference serum calibrated against 2nd International reference serum obtained from National Institute of Biological Standards, UK.
2.4. Safety Assessment
The adverse events (both local & systemic) were recorded by observing the subjects for about 60 minutes after administration of vaccine and eRIG. Adverse events were evaluated using 4-point scale. 0―None; 1―Mild (an adverse event which has no effect on normal daily activities); 2―Moderate (an adverse event which interfere with normal daily activities); 3―Severe (an adverse event which prevent daily activities).
2.5. Inclusion and Exclusion Criteria
1) Inclusion criteria: Animals reporting within 72 hours of bite, aged 4 months to 15 years, owner willing to consent and animal available for one year to follow.
2) Exclusion criteria: Reluctant owner, animal has received rabies vaccine or RIG in past, Pregnancy or animal having helminth infection or malnutrition.
3. Animal Subject Protection
Ethical approval was obtained from the Institutional Ethical Committee for CPCSEA of Dr. G.C.Negi College of Veterinary and Animal Sciences, CSK, HPKVV, university Palampur, HP in its meeting held on dated Feb 5, 2016 and subsequent meeting held on dated 9-11-2016 project Sr.no.5/2016 wide letter no-781/2016.
4. Three Phases of the Study Protocol
Phase I: We took 24 domestic bovine out of 60 recruited, in first phase for varied schedule of vaccination to arrive at a suitable pre exposure and post exposure protocols through intradermal route. The animals were randomized into five groups, as follow:
・ Group 1: Rabivac vaccine ID was given intradermal 0.2 ml in middle 1/3rd of neck on day 0. Group II was given Rabivac on day 0, 3, Group III on day 0, 3, 7; Group IV: on day 0, 3, 7, 14 and Group V on day 0, 3, 7, 14, 28. Details are at Table 1.
・ RFFIT titers were measured at day 0, 14, 35, 90 and 365 to develop appropriate pre and post exposure prophylaxis (PEP) protocol.
Phase II: 150 bovine, bitten by lab confirmed rabid dogs or mongoose or clinically conformed rabid dogs, were given PEP based on PEP protocol developed in Phase I after RFFIT evaluation and animals followed for more than one year to observe the survival rate. Details are at Table 2.
Phase III: To develop a pre exposure protocol (PrEP) in Bovines a booster of single 0.2 ml ID to a sub-group of available bovine was given on days 365 and RFFIT titers tested seen again tested on day 7. Details are at Table 3.
5. Results and Discussion
Phase I: Out of 60 animals recruited for study protocol, 25 animals, 41.7% (17 cows and 8 buffalos), were having natural antibody titers without vaccination above 0.5 IU/ml in the range of 0.9 IU/ml - 15 IU/ml. Some of these animals were subsequently observed as to how they respond to regular course of antirabies vaccination compare to those having titres below 0.5 IU/ml on day zero.
While we observed that the response in all 5 groups to ID vaccine was robust on day 14 (Group 1 - 5) given 0.2 ml vaccine ID on days 0, 3, 7, 14 and 28. Only one animal could reach the desired titers (>0.5 IU/ml) on day 14 in IM group which is considered adequate. As per WHO guidelines, a minimum level of 0.5 IU/mL is used as a correlate of protection. In healthy vaccinees, this level should be achieved by day 14 of a post-exposure immunization regimen, with or without simultaneous administration of RIG and irrespective of age  . Test of Significance: GMTs ID Vs IM is: 5.26 vs 0.38 (p = 0.0136), IM vaccine had significantly low titers than IDRV on day 14 which can be life threatening in cases of wounds on head and neck and may have contributed to observed vaccine failures IM.
To understand this strange phenomenon of existence of natural antibodies without vaccination mentioned above, we took serum samples from 12 cows
Table 2. Phsse II: results of implementation of low cost five dose Inradermal vaccination with local RIG infiltration of wounds in Bovine in real field conditions in villages of Jawali, Himachal Pradesh, India.
Table 3. Exploring the feasibility of low cost Inradermal PrEP in bovine.
GMT at Day 365 is 0.74 IU/ml and GMT at Day 372 is 1.48 IU/ml.
0, 3 DAY GROUP II
GMT at Day 365 is 0.65 IU/ml and GMT at Day 372 is 1.87 IU/ml.
0, 3 & 7 DAY GROUP III
GMT at Day 365 is 0.93 IU/ml and GMT at Day 372 is 1.87 IU/ml.
kept in the isolation farm since birth at Agriculture University, Palampur, and all these cows (Table 1 Group 7) were having RFFIT titers <0.5 IU/ml. This clearly shows that cows and buffalos when left stray for grazing in the open pastures interact with the rabies virus carrier stray dogs   and get infected and have rabies antibodies in their blood. Such high antibody titers without any history of exposure were also observed in unvaccinated rag pickers engaged to lift dead bodies of dogs and monkeys in Shimla Municipality  .
Therefore we are of the view that validation of preventive vaccination schedule in field animals that are basically domesticated but left to graze as stray in rabies endemic countries like India, compare to captive farm animals in Europe/India is entirely different issue due to presence of natural rabies antibodies in domestic animals without any vaccination. These antibodies may interfere with the killed/attenuated antigens used in rabies vaccination and give variable results on RFFIT as is seen in Table 1.
We decided to adopt group V protocol for ID rabies vaccination in bovine for PEP (Phase II) with local infiltration of eRIG to cover the non response of one animal in Group IV as also Group IV had lower GMTs on day 14 than Group V.
Phase II: After assured protection correlates with five dose IDRV protocol in Phase I trials, we decided to test the protocol in real field conditions. In the second phase we started giving PEP with 5 day 0.2 ml IDRV and local wound infiltration with eRIG in animals bitten by suspected or lab confirmed rabid dogs. 150 bovine bitten by suspected or laboratory confirmed rabid dogs were included in Phase-2 of the study. Out of 150 bovine 18 were bitten by lab confirmed rabid dogs/ mongooses and followed for one year. Out of 18, serum samples were taken from 15 bovine on day 14 and tested for RVNA by RFFIT and all had desired titers above 0.5 IU/ml. (Table 2). After appropriate wound wash and application of local antiseptics, eRIG was infiltrated locally (Figure 3) into all category-III wounds inflicted by suspected or lab confirmed rabid dogs/mongoose. Those animals that were not having any visible wound were not given any eRIG but only vaccine as IDRV and all survived. Brain samples of two rabid dogs and two mongoose were sent for lab confirmation by Fluorescent Antibody Test (FAT) to Central Research Institute (CRI), Kasauli. None of the animal had severe drug reaction and all had only mild redness and some of them had itching sensation that faded away by next day.
Few of the bovine and even one equine (Horse. Figure 4) brought for PEP at some of nearby vet hospitals, who were given five shots rabies vaccine IM with local eRIG infiltration also survived. Local eRIG infiltration appeared to have covered the lacuna of longer window period required for sufficient indigenous antibodies production through IM route that are not sufficiently produced by day 14 as demonstrated by our study.
Pre-vaccination of bovine, Phase-III: Pre-exposure prophylaxis was found to be effective as 0.2 ml dose of vaccine on day 0, 3, 7 and all bovine had titers higher than desired by day seven after single 0.2 ml vaccine booster at one year.
Figure 3. Local eRIG infiltration in a Buffalo bitten by rabid dog on nose with insulin syringe.
Figure 4. Local eRIG infiltration in a horse bitten by dog having paralytic rabies (Figure 5) on lower lip with insulin syringe.
Figure 5. Dog clinically diagnosed as having paralytic rabies after the attack on the horse lip (observe the jaw drop).
We had serum samples tested for seven calves born to cows during the study period while cows were vaccinated before pregnancy and three of them had more than desired antibody levels at 3 - 4 months of age. It is recommended that we vaccinate calves when they are older than 4 months and then yearly   .
Considering the enormous burden of rabies, we were able to demonstrate clinical effectiveness of a novel low cost protocol based on our previous experience with human rabies vaccination and local infiltration of eRIG and we called this protocol to memorise our guru as “Madhusudana-Bharti-Uppinder” protocol. Failure to achieve desired titers above 0.5 IU/ml at day 14 with IM schedule of rabies vaccination in bovine was identified as one of the reasons of observed PEP failure in bovine vaccinated by IM route in our area. Our study points towards a possibility of having short schedules of three shots ID vaccination with or without local RIG infiltration of bite wound/s as PEP and single shot ID as PrEP but that would require further studies on a large number of animals.
We recommend vets to follow this low cost protocol of IDRV with local eRIG wound infiltration where required for post exposure prophylaxis of bovine or may be in equine bitten by rabid animals to save their lives. We also recommend vet vaccine companies to label rabies vaccines as for “ID/IM” use like human vaccines to facilitate this process for wider pre exposure use that would make them cost effective for poor farmers in rabies endemic countries of Asia and Africa. Another factor that goes in favour of IDRV is less or almost no pain to fine needle insulin syringe compare to stressful response to IM vaccination in cattle as demonstrated by Luis S et al.  .
We are sincerely grateful to National Institute of Mental Health & Neurosciences (NIMHANS) Bangalore, India for doing serum RFFIT tests free and CRI Kasauli, Himachal for doing FAT test on animals brain free. Thanks to the Institutional Ethical Committee for CPCSEA of Dr. G.C. Negi College of Veterinary and Animal Sciences, CSK, HPKVV, university Palampur for according permission for the study.
RVNA: Rabies Virus Neutralizing Antibody, RFFIT: Rabies Fluorescent Focus Inhibition Test, eRIG: Equine Rabies immunoglobulin, FAT: Fluorescent Antibody Test, CRI: Central Research Institute, Kasauli (HP).
 Basheer, A.M., Ramakrishna, J., Manickam, R., et al. (1997) Evaluation of Post-Exposure Vaccination against Rabies in Cattle. The New Microbiologica, 20, 289-294.
 Wilson, P.J., Clark, K.A., et al. (2001) Postexposure Rabies Prophylaxis Protocol for Domestic Animals and Epidemiologic Characteristics of Rabies Vaccination Failures in Texas: 1995-1999. Journal of the American Veterinary Medical Association, 218, 522-525.
 Zhang, Y., Zhang, S.F., Li, L.T., et al. (2016) Ineffectiveness of Rabies Vaccination Alone for Post-Exposure Protection against Rabies Infection in Animal Models. Antiviral Research, 135, 56-61.
 Benísek, Z., Süli, J., et al. (2006) Intradermal Anti-Rabies Immunization—Possibilities of Needleless Rabies Vaccine Administration. Bulletin—Veterinary Institute in Pulawy, 50, 137-142.
 Ashokkumar, M., Ganesan, P.I., et al. (2016) Vaccination Studies against Rabies in Farm and Pet Animals Using Different Immunization Routes. The Indian Veterinary Journal, 93, 33-36.
 Bharti, O., Madhusudana, S., Kale, A., Gaunta, P., Chaudhry, L., Kumar, J., Gupta, N. and Shyam, D. (2015) Success Story of a Low Cost Intra-Dermal Rabies Vaccination (IDRV) Clinic-Lessons Learnt over Five Years of 12,000 Patient Vaccinations “Without Failure” at DDU Hospital Shimla, Himachal Pradesh, India—“Saving a Drop of Rabies Vaccine and Immunoglobulins” 12 Innovations to Make Himachal Pradesh Rabies Free State by 2020. World Journal of Vaccines, 5, 129-139.
 Blancou, J., Baltazar, R.S., Molli, I., Stoltz, J.F., et al. (1991) Effective Postexposure Treatment of Rabies-Infected Sheep with Rabies Immune Globulin and Vaccine. Vaccine, 9, 432-437.
 Bharti, O.K., Madhusudana, S.N., Gaunta, P.L. and Belludi, A.Y. (2015) Local Infiltration of Rabies Immunoglobulins without Systemic Intramuscular Administration: An Alternative Cost Effective Approach for Passive Immunization against Rabies. Human Vaccines & Immunotherapeutics, 12, 837-842.
 Smith, J.S., Yager, P.A. and Baer, G.M. (1996) A Rapid Fluorescent Focus Inhibition Test (RFFIT) for Determining Rabies Virus Neutralizing Antibody. In: Meslin, F.X., Koprowsky, H. and Kaplan, M.M., Eds., Laboratory Techniques in Rabies, 4th Edition, WHO, Geneva, 181-187.
 Atuman, Y.J., Adawa, Y.A., Solomon, A., Mshelbwala, P.P. and Ogunkoya, A.B. (2014) Potential Risks for Rabies Spill-Over from Apparently Healthy Dogs to Wildlife in Bauchi State, Nigeria. Journal of Animal and Veterinary Advances, 4, 493-498.
 Bharti, O.K. (2015) Immunizing Vulnerable Populations Like Rag Pickers, Garbage Collectors, Municipality Workers and Newspaper Hawkers against Rabies in Shimla Municipality, HP, India. World Journal of Vaccines, 5, 19-24.
 Yakobson, B., et al. (2015) Cattle Rabies Vaccination—A Longitudinal Study of Rabies Antibody Titres in an Israeli Dairy Herd. Preventive Veterinary Medicine, 121, 170-175.
 Reis, L.S.L.S., et al. (2013) Effects of Primovaccination and Booster Vaccination on Serum Cortisol and Humoral Immune Response in Cattle. Advances in Bioscience and Biotechnology, 4, 607-611.