Original Research

Rabies- Still a Challenge in Developing World

Hari Mohan1*, Subhash Kharb2

1Centre for Medical Biotechnology, Maharishi Dayanand University, Rohtak-124001, India
2Departments of Animal Husbandry & Dairying, Sonepat (Haryana)-131001, India

J Innov Biol (2014) Volume 2, Issue 3: Pages: 239-244

Abstract: Rabies, a viral zoonosis transmissible to all mammals including human beings, is a highly infectious disease which is almost fatal with no cure. The disease is prevalent worldwide with largest contribution from Asia and Africa. Dogs are the major reservoir hosts in developing countries for transmission of rabies to human beings as well as domestic animals. The annual estimated human deaths due to rabies are around 50000-60000 with 99% occurring in developing countries. However, public health hazards and economic losses to the livestock sectors can be minimized by strictly following the pre-exposure (in reservoir hosts) and post-exposure (in exposed human beings as well as domestic animals) prophylactic vaccination regimens recommended by World Health Organisation. Early detection of the disease can pave the way for applying rapid infection control measures. This will prevent unnecessary medical test and will prompt timely vaccination of family members and medical staff. Conventional tests used for rabies diagnosis have several shortcomings. Use of modern rabies detection tests based on rabies antigen-antibody reaction, virus genomic RNA detection assay and biomarkers assay have overcome almost all limitation of conventional methods. These diagnostic tests have potential to complement conventional assays and revolutionize the detection of rabies in upcoming scenario.

Received: 11 July 2015
Accepted: 02 September 2015
Published Online: --- September 2015

Corresponding Author:
Mohan H.,
Centre for Medical Biotechnology, Maharishi Dayanand University, Rohtak-124001, India
email: harimohansaini@gmail.com

Keywords: Dogs, Human beings, Rabies, Vaccination, Zoonosis


Rabies (Latin word that means madness) or hydrophobia is a contagious viral disease of dogs and other warm blooded animals which is almost invariably fatal. The disease causes serious public health problem because of its zoonotic (spreading from animals to humans) nature. It ranks 10th among the causes of human death due to infectious diseases worldwide. The disease is still a challenge in developing world as 99% of total human deaths are occurring in tropical developing countries (Haupt, 1999). India alone contributes approximately 36% of these total rabies related deaths (Chatterjee, 2009). Over 55,000 people die from rabies worldwide annually, 95% of them in Asia and Africa (WHO, 2010). Most human deaths occur after a dog bite, between 30% and 60% of them are children aged less than 15 years. In India, 95-97% of human deaths due to rabies are because of dog bite and annual incidence of bite is 1.7% (Sudarshan et al. 2006). In the majority of cases, these bites go unreported and are unrecognized by their parents and health officials, resulting in a much higher risk of complications and deaths among children, compared to adults. It is likely the total number of deaths in many parts of the world is much higher than official estimates.
The etiological agent of rabies is a type species RNA virus named Lyssavirus belonging to family Rhabdoviridae in the order Mononegavirale. OIE (2014) enlisted twelve distinct species in Lyssavirus namely, classical rabies virus (RABV; genotype 1), Lagos bat virus (LBV; genotype 2), Mokola virus (MKV; genotype 3), Duvenhage virus (DV; genotype 4), European bat lyssavirus 1 (EBLV-1; genotype 5), European bat lyssavirus 2 (EBLV-2; genotype 6) and Australian bat lyssavirus (ABLV; genotype 7), Aravan virus (ARAV), Khujand virus (KHUV), Irkut virus (IRKV), West Caucasian bat virus (WCBV) and Shimoni bat virus (SHIBV). Rabies virus is a single stranded, non-segmented, negative sense, bullet shaped, enveloped RNA virus of approximately 75 nm x 200 nm size. The genetic information is packed as a ribonucleoprotein complex in which RNA is tightly bound by the viral nucleoprotein. The genome of the virus encodes five genes viz. nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and the viral RNA polymerase (L) whose order is highly conserved (Yousaf et al. 2012). The virus is highly resistant to cold, dryness and decay but susceptible to physical and chemical agents as 60 ºC for 35 sec, pH less than 3 or more than 11, 45-75% ethanol, formaldehyde, iodine, phenol, sodium hypochlorite, quaternary ammonium compounds and some other detergents (OIE, 2014).
Host range and Epidemiology
The disease has a very wide host range including all warm blooded animals, however, dogs (principal reservoir hosts in Asia and Africa) and some wild mammals as skunks and racoons (principal reservoir hosts in North America), foxes (principal reservoir hosts in Europe and North America) and bats (principal reservoir hosts in USA) are major reservoir hosts of rabies (Yale et al. 2014).
The disease is distributed all over the world except some countries and islands. De Serres et al. (2008) divided the geographical areas of rabies into three major categories namely- i) Countries with enzootic canine rabies (Asia, Latin America and Africa), ii) countries in which canine rabies has been brought under control and wildlife rabies predominates (Western Europe, Canada and United States) and iii) rabies free countries (England, Ireland, Sweden, Norway, Finland, Denmark, Iceland, Japan, Singapore, Australia, Greece, Portugal, Israel, Newzealand, Maldives, Andaman & Nicobar and Lakshadeep islands of India).
India- Rabies is prevalent throughout the country except Andaman & Nicobar and Lakshadeep islands (free from rabies). Since 1985, surveys report 25000-30000 human deaths from rabies annually and the actual incidence may be much higher because the disease is not truly reported as it is still not a notifiable disease in India (Kole et al. 2014). Among the total human deaths occurring worldwide, 36% are occurring only in India. The disease has mainly been reported in people of lower socio-economic status and children of 5-15 years age. The animals mainly responsible for causing rabies in India are dogs (96.2%) and among these most are stray dogs (Sudarshan et al. 2007).
Incubation Period
Incubation period is highly variable and may range from minimum 5 days (in cases where virus directly enters the peripheral nerves) to maximum up to 7 years (in cases where virus get localized in muscle or other cells). Generally the incubation time is considered 20-90 days (Plotkin, 2000; Jackson, 2010).
Rabies virus is a neurotropic virus that attacks on nervous system and later on excreted in saliva. Entry of virus in the host usually occurs through saliva of infected animal via bites or scratches (Fekadu et al. 1982), however, non bite exposure in humans may also occur via contamination of skin abrasion, open wound, conjunctiva, oral mucus membranes or genitalia with saliva of rabid animal, inhalation of aerosolized virus in caves inhabiting rabid bats and accidental exposure in laboratories (Dutta et al. 2005). Human to human transmission other than corneal transplantation from infected person (Gode and Bhide, 1988) has not been well known, however, there is always potential risk of getting infection as secretions from infected persons contain viable virus. Efficient transmission of virus into host depends mainly on the severity of bite, distance of bite from head and amount of virus in saliva. In deep bites and areas of muscles having high density of receptors for virus attachment, efficiency of transmission will be higher.
After entry, virus replicates in non-nervous tissues or directly enters the peripheral nerves in deep bites. The virus binds to nicotinic acetylcholine receptors on the muscles in canine rabies virus infection and to unknown receptors in bat Lyssa virus infection (Lentz et al. 1982). Virus may get localized in the muscle cells or other cells at neuromuscular junction for a long time indicating long incubation period of the disease (Charlton et al. 1997). Virus is endocytosed from the membrane of muscle cells into unmyelinated nerve endings at neuromuscular junction via the process of budding and again multiplies in the dorsal root ganglia and anterior horn cells (Tsiang, 1993). Transportation of virus from peripheral nerves to spinal cord and central nervous system (CNS) occurs by retrograde axonal transport along the neuro-anatomical connections at a rate of 50-100 mm/day (Mazarakis et al. 2001). After reaching the CNS, virus multiplies and localizes mainly in brainstem, thalamus, basal ganglia and spinal cord. Neurological dysfunctions occur due to virus induced cell death either by apoptosis or by necrosis. Centrifugal spread of virus from CNS for completing the infectious cycle occurs mainly through the parasympathetic nervous system infecting various organs as adrenal medulla, cornea, salivary glands, skin, heart etc. Virus is secreted from saliva, milk and urine of infected animals (Dutta, 2014).
Clinical Features
The clinical signs and symptoms of rabies are usually similar among all animals with high individual variations which could be because of prolonged and invariable incubation period. The disease is always fatal, once the clinical signs appear. The disease causes typical symptoms usually divided into two clinical stages of furious and dumb forms occurring after a prodormal phase of behavioural changes in animal (Cahn and Line, 2005).
Prodormal Phase: the phase lasts for 2-3 days and often is overlooked in animals. Clinical signs include change in tone of bark, chewing at the site of bite due to itching and pain, fever, loss of appetite and subtle changes in behaviour of the animal.
Furious form (Encephalitic form): This stage is also known as mad dog syndrome. Generally lasts for 2-4 days and not all rabid animals show this phase of disease. The animals exhibit erratic behavior, restlessness, irritability, episodes of aggression, craving to eat all type of objects, constant growling and barking, dilatation of pupils, unable to swallow water giving the disease’s name- hydrophobia, excessicive salivation, hyper responsive to light and sound, roaming, seizures, trembling and incoordination of muscles. As the encephalitis progresses, furious form goes away and the animal shows symptoms of paralytic form.
Dumb form (Paralytic form): There is appearance of choking, dropping of lower jaw, inability to swallow causing drooling and foaming of saliva due to flaccid paralysis of jaw, throat and chewing muscles. The paralysis then spreads to other parts of body also.
In terminal stages of both forms, convulsive seizures, coma and respiratory arrest occur, leading to death of animal within 2 to 14 days after onset of clinical signs. Furious form of the disease is more pronounced in dogs, cats and horses as compared to ruminants and laboratory animal species (Murphy et al. 2006).
Clinical signs in humans are also somewhat similar starting from prodormal phase to encephalitic type showing classical presentation of hydrophobia with or without aerophobia or photophobia and paralytic type showing flaccid paralysis of jaw and chewing muscles ultimately leading to death (Consales and Bolzan, 2007; Dutta, 2014).
As no clinical features and post-mortem lesions can be considered pathognomonic in animals, rabies can be diagnosed by laboratory tests. For accurate laboratory diagnosis, preferred samples must be transported to the laboratory using suitable preservatives. Preferred samples for diagnosis of rabies are saliva, cerebrospinal fluid, nasal and throat swabs, nuchal or facial skin biopsies in living individuals and CNS tissues specifically brain stem, Ammon’s horn, thalamus, cerebral cortex and medulla oblongata in dead individuals (Warrell and Warrell, 2004).
Laboratory diagnosis by detecting Antigen
Histological examination: Viral inclusions (Negri bodies) can be demonstrated in fresh brain sections by using different types of acidophilic stains as haematoxylin-eosin stain (Negri bodies stain pink), Mann’s stain (Negri bodies stain red), Seller’s stain (Negri bodies stain cherry red) etc. followed by examination under light or electron microscope. However, these tests have not been recommended presently because these tests are less sensitive, time consuming and more expansive (CDC, 2011).
Fluorescent antibody test (FAT): FAT is gold standard test for detection of rabies virus developed by Goldwasser and Kissling (1958) recommended by WHO and OIE. Thin touch impressions of medulla, cerebellum and hippocampus parts of brain are preferred samples for diagnosis of rabies in FAT. Smears fixed in 100% high grade cold acetone for 20 minutes are air dried and stained with anti-rabies antibodies labelled with specific conjugates as fluorescein isothiocyanate (FITC). The slides are then examined under fluorescent microscope for detection of aggregates of nucleocapsid protein. FAT is sensitive, specific and cheap and results can be obtained within 2 hrs (OIE, 2013).
Various other tests viz. immunoperoxidase test (IPT), enzyme linked immunosorbant assays (ELISA), rapid rabies enzyme immune diagnosis (RREID) can be used as alternative tests for FAT. Dip stick/Dot ELISA can also be used for rabies antigen detection in brain samples in geographically remote areas where FAT facilities are not available. This test is rapid, reagent conservative and requires less sophisticated equipments (Jayakumar et al. 1997).
Rapid latex agglutination test: This test is quick and can be performed in 15 minutes. Latex beads sensitized with rabies immunoglobulins agglutinate quickly in presence rabies virus antigens (Kasempimolporn et al. 2000).
Isolation of virus: Isolation of virus can be done in laboratory animals as Mouse inoculation test (MIT) as well as in tissue culture as Rapid tissue culture infection test (RTCIT). MIT has been replaced with tissue culture test as it avoids the use of live animals, less expensive, gives more rapid results (reducing the time of test from 30 to 4 days) and as sensitive as MIT (Webster and Casey, 1996). Highly susceptible neuroblastoma cells as N2a, CCL-131, NA-C 1300 etc are used for detection of rabies virus replication in saliva, cerebrospinal fluid or homogenised brain samples. After 24 hr incubation (37 ºC in 5% CO2) of cells mixed with test sample, the cells are fixed with acetone, stained with fluorescent antibody conjugate and observed under fluorescent microscope.
In recent years, newer diagnostic tests as direct rapid immunohistochemical test (dRIT): for detection of N proteins in brain smears using biotinylated monoclonal antibody, indirect rapid immunohistochemistry test (IRIT): for the detection and differentiation of rabies virus in prevalence, distribution and transmission studies, rapid immunodiagnostic test (RIDT): single step immunochromatographic lateral flow strip test for detection of rabies virus antigen and rapid sandwich ELISA (WELYSSA) have been developed (Mani and Madhusudana, 2013).
Serological tests
These tests are mainly used to determine antibody responses to vaccination in animals as well as high risk humans. Antibody titre of 0.5 IU/ml is considered as minimum safe level of immunity in humans as well as dogs and cats that provides protection against rabies virus infection (WHO, 1985).
Fluorescent antibody virus neutralization test (FAVN): As the neutralizing antibodies are major components of protective immunity, virus neutralization tests are considered as gold standard tests. FAVN is a prescribed test for international trade based on the principle of in vitro neutralisation of constant amount of virus before inoculating cells susceptible to rabies virus (Ondrejkova et al. 2015).
Mouse Neutralization Test (MNT): Webster and Dawson (1935) developed first serological test for detection of antibodies against rabies based on the principle that mixing various dilutions of test serum and virus, inoculating the mixture in mice and observing whether the mice dies from rabies or not. Presently, MNT is not recommended by OIE and WHO.
Rapid Fluorescent Focus Inhibition Test (RFFIT): This test is also based on the principle of MNT except the use of susceptible cell culture system as indicator system instead of mice. RFFIT is more rapid and sensitive than MNT and is prescribed for international trade (Smith et al. 1996).
Indirect enzyme linked immunosorbant assay (I-ELISA): It is a rapid test used for detection of antibodies against whole virus or envelope glycoprotein (G) of rabies virus. I-ELISA has advantages over other diagnostic tests as it avoids the requirement of handling the live rabies virus, cell culture techniques and live mice (Servat et al. 2007). Commercial indirect ELISA tests have been recommended for international movements of dogs and cats provided the kits used for evaluating vaccine responses have been validated and adopted on the OIE Register.
Molecular tests
Reverse Transcriptase PCR (RT-PCR): The DNA fragment amplified by RT-PCR assay can be used for nucleotide sequencing for virus genetic characterization and establishing phylogenetic relationship with other strains (Coertse et al. 2010). However, chances of cross contamination is a major drawback of RT-PCR assays and this preclude routine use of reverse transcription PCR for diagnosis of rabies in animals and human being
Quantitative Real-Time PCR: Due to closed tube nature of qRT-PCR assay, there is considerable reduction in cross contamination during qualitative and quantitative assaying of the virus samples. SYBR Green chemistry based Real Time PCR has been standardized for antemortem rabies diagnosis from human saliva samples and a universal assay for Lyssaviruses detection (Nagaraj et al. 2006). These assays are highly sensitive and specific but it requires upmost care to ensure specificity and also require sophisticated and expensive equipment and consumables.
Once the clinical signs appear in animal or man, there is no treatment of rabies. Only supportive treatment is given. However, only five human rabies survivor cases including a six year old girl from India have been reported. All the cases had been received some pre-exposure rabies vaccine before bite and all cases had paralytic form of rabies. Severe neurological problems had been reported in all cases showing limited survival (Warrell and Warrell, 2004). Various types of drugs viz. ribavirin, interferon-α, ketamine have been used, but no one has been found effective (Dutta and Dutta, 1994; Jackson et al. 2003).
Until 1885, when Louis Pasteur and Emile Roux developed a vaccine against rabies, most cases of rabies were fatal. The first rabies vaccine was nervous tissue vaccine (NTV) produced by growing virus in rabbits and then weakening it by drying the affected nerve tissue. The vaccine had been tested in 50 dogs before its first successful human trial on 9 year old Joseph Meister on July 6, 1885 (Geison, 1978). Because of less potency, high number of doses required and the reactogenic nature, use of NTVs has not been recommended by WHO and cell culture vaccines (CCVs) are used presently. Human diploid cell culture (HDCC) vaccine was introduced in 1967 and their after various cell culture vaccines as purified chick embryo cell vaccine (PCECV), purified vero cell rabies vaccine (PVRV), etc. are used for pre and post- exposure prophylaxis of rabies (WHO, 2007; Wu et al. 2011).
Domestic animals- Rabies in animals can be controlled by pre-exposure vaccination of reservoir hosts. Dogs are vaccinated via parentral routes (sub-cutaneous or intra-muscular) of immunization with CCVs at the age of 3 months and then annual revaccination. Post-exposure prophylaxis is done in all domestic animals following the schedule of day 0, 3, 7, 14 and 28.
Wild animals- Oral vaccination is recommended for feral and wild animals because of difficult accessibility for parenteral vaccines’ administration. The currently used oral vaccines are either modified live vaccines (MLV) or biotechnology derived vaccines (BDV) using vaccinia and adenoviruses as viral vectors (OIE, 2013).
Pre-exposure prophylaxis
Pre-exposure prophylaxis is recommended for high risk personals as veterinarians, animal attendants, laboratory workers, visitors in endemic areas of rabies etc. Schedule for pre-exposure prophylaxis is given in table 1.
Post-exposure prophylaxis
Wound management- Wash the wound gently and thoroughly with soap or detergent solution and flush the wound with running water for at least 10 minutes to reduce the risk of virus entry in the body. After thorough washing and drying, apply suitable antiseptic solution as cetrimide, chlorhexidine gluconate, chloroxylenol or povidone iodine etc. on the wound. Wound should not be curetted or sutured as it may further exacerbate the virus entry into the body by exposing the nerves. If required, suturing of wound should be done 24-48 hrs after bite and done only after RIG infiltration. Do not touch the wound with bare hands and do not use home remedies (chillies, soil, chalk etc.) at the bite site.
Passive immunization- Inject rabies immunoglobulins (RIG) at the site of bite immediately (@ 20 IU/kg body weight for human RIG and 40 IU/kg body weight for equine RIG) as RIGs have the property of binding with the virus and slow down or stop the viral progression through the nerves (Warrell, 2012; Gozdas, 2015).
Active immunization- Administer safe and potent CCVs following proper schedule recommended by WHO. Post-exposure prophylaxis is also safe for administration in children and pregnant women. Schedule for post-exposure prophylaxis is given in table 1.
Table 1. Regimen for pre- and post-exposure prophylaxis of rabies in humans recommended by WHO, 2010.
Pre-exposure prophylaxis
Intra-muscular route Intra-dermal route
Regimen Day 0, 7 and 28 Day 0, 7 and 28
Site of injection Deltoid area of arm in adults and antero-lateral area of thigh in children Deltoid area of arm in adults and antero-lateral area of thigh in children
Dose 1 or 0.5 ml
(depending on the vaccine type) 0.1 ml
Post-exposure prophylaxis*
Intra-muscular route Intra-dermal route
Regimen 5 Dose Regimen: Day 0, 3, 7, 14 and 28 Two site ID method (2-2-2-0-2):
One dose of vaccine at two sites on day 0, 3, 7 and 28
4 Dose Regimen (2-1-1): Two doses on day 0, third dose on day 7 and fourth dose on day 21 8 site ID Regimen
* Site of injection and dose of vaccine are same as in pre-exposure prophylaxis
Future perspectives
Rabies is a fatal viral zoonosis and major public health problem. The disease is endemic in most of developing countries and still a threat for developed countries due to presence of infectious agent in wildlife. As the disease is incurable, control of the disease in reservoir hosts following pre-exposure vaccination and post-exposure management of bite cases as wound management, injecting RIG and parentral vaccination by strictly following WHO recommended schedule can reduce the incidence of rabies. Control strategies should also include, making of rabies a notifiable disease for estimation of actual incidence, educating people about the disease and first aid of wound to be given to bite case and avoiding the application of harmful traditional remedies. The disease is far away from the eradication level in most of developing countries but can be controlled to a large extent by effectively vaccinating the dogs and other reservoir hosts either through parentral or oral route.

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