Exploring the Feasibility of a New Low Cost Intra-Dermal Pre & Post Exposure Rabies Prophylaxis Protocol in Domestic Bovine in Jawali Veterinary Hospital, District Kangra, Himachal Pradesh, India

Cattle are the backbone of household economy in rural areas of India and many of them die after bites by potentially rabid dogs, despite being given currently recommended five shots of intramuscular (IM) rabies vaccination as Post Exposure Prophylaxis (PEP). In 2016, seven of 21 bovine bitten by rabid dogs given IM rabies vaccination died due to rabies in Shimla Municipality. This scenario prompted the authors to look for a suitable protocol, based on human studies, to save animals. We tested various schedules of IDRV in bovine and found that a schedule of 0.2 ml given in middle 1/3 of neck on day 0, 3, 7, 14 and 28 along with local wound infiltration of eRIG is sufficiently immunogenic and life saving in all of them, even if bitten by lab confirmed rabid dogs/mongoose as tested by CRI. Rabivac Vet, a Cell Culture Rabies Vaccine, available as 1 ml per vial was used off level for IDRV. While injecting the vaccine, a raised papule of ≥1 cm will appear slowly causing a peau d’orange appearance. All 60 bovine serum samples tested by RFFIT after IDRV, had titers more than 0.5 IU/ml on day 14. Thereafter, a total of 150 animals were given five doses of IDRV as PEP, with or without RIG, after their exposure to clinically or lab confirmed rabid dogs/mongoose and all surHow to cite this paper: Bharti, O.K., Sharma, U.K., Kumar, A. and Phull, A. (2018) Exploring the Feasibility of a New Low Cost Intra-Dermal Pre & Post Exposure Rabies Prophylaxis Protocol in Domestic Bovine in Jawali Veterinary Hospital, District Kangra, Himachal Pradesh, India. World Journal of Vaccines, 8, 8-20. https://doi.org/10.4236/wjv.2017.81002 Received: December 5, 2017 Accepted: February 2, 2018 Published: February 5, 2018 Copyright © 2018 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access


Introduction
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 20 th day of exposure and another after 35 th 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 [1] but still PEP failures in cattle have been reported from all parts of the Globe [2]. Even vaccine antigen titers when raised to 4.0 IU or even higher resulted in failure [3] underlining the inappropriateness of rabies vaccine alone in animal post exposure prophylaxis. mited studies in cattle [4] [5]. 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 [6]. Since the rabies immunoglobulins given based on body weight in animals have proven their efficacy in saving the life of animals like Sheep [7] (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 [8] 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.

Methods
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

Procedure/Technique of Intra-Dermal Vaccination
This involves injection of 0.

Rapid Fluorescent Focus Inhibition Test (RFFIT)
RFFIT for estimation of rabies virus neutralizing antibodies (RVNA) was per-

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).

Animal Subject Protection
Ethical approval was obtained from the Institutional Ethical Committee for  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

Three Phases of the Study Protocol
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.

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 [10]. Test of Sig-    [12] 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 [13].
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 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    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 [14] [15].

Conclusion
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.

Recommendations
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. [15].