Rabies: Incurable Biological Threat

Abstract

Rabies is a lethal zoonotic disease that affects all the homeotherms, including humans, and is caused by the Rabies virus of Rhabdoviridae family. Every year, this disease kills about 55,000 individuals globally. The stray dog is a key player in the spread of rabies. The disease is usually transmitted through the bite of a rabid animal. After being exposed to the virus, the virus must travel to the brain before generating symptoms. Delirium, unusual behaviour, hallucinations, hydrophobia and insomnia may occur as the condition advances. Diagnostic tests such as direct fluorescent antibody test (dFAT), direct rapid immunohistochemical test (dRIT), lateral flow assay (LFA), reverse transcriptase polymerase chain reaction (RT-PCR), nuclear sequencing, etc. are used in diagnosis of this dreadful disease. The genotype and lineage of the rabies virus can be determined via N gene sequencing and phylogenetic analysis. There is no effective treatment for rabies. Even though a tiny number of people have survived rabies, the disease is usually fatal. Rabies can be completely avoided in people if they receive timely and adequate medical treatment. Vaccinating and sterilising the dogs in our neighbourhoods effectively and humanely limit their population and eliminate rabies in both dogs and humans.

1. Introduction

Rabies is a highly fatal viral infection of the central nervous system caused by the Rabies virus, which belongs to the genus Lyssavirus of the Rhabdoviridae family that affects all warm-blooded animals and is a major occupational anthropozoonosis. It is one of the oldest recognised diseases. The word rabies comes from the Latin word rabere, which meant to be insane, enraged or ravenous. It is one of numerous zoonotic diseases that go unnoticed, resulting in more than 55,000 deaths per year. According to the Association for Prevention and Control of Rabies in India (APCRI) in 2003, a person dies of rabies every 9 minutes. It is one of the most important zoonotic diseases in history, with a fatality rate of around 100% and a global distribution [1, 2]. While rabies is thought to affect all mammalian species, including nonhuman primates and humans, it is not predominantly a human disease. Human infection is an unintended consequence of the disease’s reservoir in wild and domestic animals. In the natural world, rabies is a disease that affects wild carnivores, with reservoirs and vectors including dogs, cats, wolves, foxes, coyotes, jackals, raccoons, skunks and bats. Bats are the main tanks for 10 known lyssavirus serotypes [3]. In 400 BC, Aristotle stated that ‘dogs suffer from the madness. This causes them to become very irritable and all animals they bite become diseased’.

Rabies is sustained in two epidemiological cycles, one urban and the other sylvatic. Dogs are the principal reservoir host in the urban rabies cycle. This cycle is most prevalent in areas of Africa, Asia, Central and South America where there are a large number of unvaccinated, semi-owned or stray dogs. In Europe and North America, the sylvatic (or wildlife) cycle is the most common. In animals, disease patterns might be relatively stable or evolve into a slow-moving epidemic.

The skin or mucous membrane is the most common site of rabies virus entrance in humans and animals, where the virus enters the muscle and subcutaneous tissue through biting, licking or scratching by a rabies-virus-infected animal. Acute encephalomyelitis is the pathogenic manifestation in the CNS. In animals, disease can manifest itself in two ways. The classical or encephalitic (furious) form of rabies accounts for 80–85% of rabies cases. Hydrophobia, pharyngeal spasms and hyperactivity are all symptoms of the furious type of rabies, which can lead to paralysis, coma and death. The dumb type, also known as the paralytic form, is characterised by the development of pronounced and flaccid muscular weakness and is less prevalent. In humans, symptoms of cerebral dysfunction, agitation, anxiety and confusion develop. Later the person experiences abnormal behaviour, delirium, hallucinations, insomnia and respiratory failure. Once the symptoms develop, the disease is often fatal.

Even though Louis Pasteur achieved his first breakthrough against rabies with post-exposure vaccination in 1885, the disease continues to haunt the mankind, particularly in impoverished countries, more than 125 years later [4]. Despite recent advances in diagnosis, post-exposure treatment, the production of human and veterinary vaccines and the control of rabies in dogs and wild animals, rabies remains a major health hazard in many countries in Africa, South America and Asia and an economic burden for both developed and developing countries. Rabies is currently found on all continents except Antarctica, although Asia and Africa account for more than 95% of human mortality. Domestic/wild animals, as well as humans, are the primary transmitters. Many countries, including Japan, the United Kingdom, Denmark, Sweden, Greece, Scandinavia, Iceland, Portugal, New Zealand and Australia, are rabies-free, according to the World Health Organisation (WHO). Vaccination, public awareness, responsible participation, continued cooperation among stakeholders and the removal of the stray dog population are some of the measures to avoid rabies [5].

2. History

The deity of death was accompanied by a dog as the ambassador of death in India about 3000 BC. Rabid canines continue to kill 20,000 people each year in modern-day India. The Mosaic Esmuna Code of Babylon, written around 2300 BC, is the first documented record of rabies causing death in dogs and humans. Babylonians had to pay a fine if their dog communicated rabies to another person. Democritus, in the fifth century BC, accurately described the disease in dogs, as did Aristotle in the third century BC [6].

The medical literature of the ancient world was littered with ineffective folk cures. Scribonius Largus, a physician, proposed a poultice of cloth and hyena skin, while Antaeus suggested a concoction prepared from a hung man’s skull. The Roman scholar Celsus correctly predicted that rabies was transmitted through the saliva of the bitten animal in the first century A.D. He wrongly suggested that placing the victim under water would cure rabies. Those who did not drown succumbed to rabies. In eighteenth-century America, the most intriguing rabies therapy was the usage of madstones. Madstones are calcified hairballs found in ruminant stomachs including cows, goats and deer. They were supposed to have healing properties since they drew the craziness from the bite wound.

In the 1880s, the first effective rabies treatment was developed. When Louis Pasteur, a French chemistry instructor, was experimenting with chicken cholera, he discovered that virulent cultures exposed to the elements no longer caused sickness. He also discovered that chickens inoculated with this weaker or ‘attenuated strain’ were immune to fresh, virulent cultures. Pasteur then attempted an attenuated anthrax vaccination in cattle. It was successful! He next turned his attention to the world’s scourge, rabies. Pasteur wanted more time to purify his attenuated vaccine before trying it on himself, despite the positive results of his initial animal experiments. In the year 1885, a rabid dog mauled a 9-year-old kid named Joseph Meister. The wounds were treated by a local doctor, who informed Joseph’s family that Louis Pasteur was the only person who could rescue him. Pasteur consented only after speaking with a few of genuine doctors, who stated Joseph was a ‘dead lad walking’. Joseph recovered completely after receiving 13 inoculations in just 11 days. The nerve tissue vaccine developed by Louis Pasteur in 1885 was a success, and it was modified over time to decrease the typical severe side effects [7].

3. Mode of transmission

Although dogs are the predominant reservoirs, other domesticated animals and wildlife also play a role in rabies transmission [8]. The virus can easily be passed from one mammal to another, whether they are of the same species or not. Humans are most infected with rabies after being bitten or scratched by an infected dog or cat. Bats, foxes, coyotes, skunks, raccoons, wolves, opossums and other animals are among the commonly infected wild or feral animals. Rabid dogs infect most people in poor countries. These dogs are frequently aggressive and drool frequently, although they act very withdrawn. Humans and domestic animals contract the disease after coming into contact with infected saliva.

Bites, non-bite exposure and human-to-human transmission are all possible routes for rabies transmission. The most common way to contract rabies is through a bite from a rabid animal, although infection can also be spread through skin wounds contaminated by infected saliva. The incubation period is the time between the bite and the onset of symptoms, and it can span anywhere from weeks to months. Because the virus has not yet made it to the saliva, a bite by the animal during the incubation stage carries no danger of rabies. Other inoculation routes are uncommon. The rabies virus can enter the body through wounds or direct contact with mucous membranes. The virus cannot pass through intact skin. The chance of contracting rabies from a bite (5–80%) is at least 50 times higher than the risk of contracting it from a scratch (0.1–1%). Virus particles are present in all the body secretions 2 days after it first enters the CNS, and the victim is fully contagious. At or shortly after this point, clinical symptoms develop.

Non-bite exposures are uncommon sources of transmission. Non-bite exposure includes scratches, abrasions, open wounds or mucous membranes infected with saliva or materials such as rabid animal brain tissue. Inhalation of aerosolised rabies virus is another non-bite route of infection, although most people, except for laboratory personnel, are unlikely to encounter an aerosolised rabies virus. Rare cases of rabies in humans have been reported because of breathing air in a cave home to thousands of bats. As rabies virus can be found in the milk of infected animals, milk can be a vehicle for virus transmission. During the consumption of infected milk, an ulcer, abscess or other lesion in the mouth may trigger rabies. Transmission between humans is extremely rare, although it can happen through transplant surgery or even more rarely through bites, kisses or sexual relations. There were outlined a number of cases of rabies transmission from human to human through cornea transplant. Some dogs slaughtered for human consumption may be infected with the rabies virus, exposing handlers of dog meat to the disease because the virus may be present in the meat’s nerves. Rabies transmission to butchers is increased during handling, catching, loading, transportation and holding prior to slaughter.

4. Pathogenesis

The virus enters the body via transdermal inoculation (wounds) or direct contact with infectious materials (saliva, cerebrospinal fluid, nerve tissue) on mucous membranes or skin lesions. The virus is incapable of penetrating intact skin. After its entry in the skin, it can undergo eclipse phase, which is not easily detected. Virus replication begins in non-nervous tissue such as striated muscle cells at the site during this phase [9]. The virus can survive for a long time here, influencing the incubation period (the time between exposure and the development of sickness) in different individuals. The virus uses nicotinic acetylcholine receptors to connect to the cells at the inoculation site. The amount of virus acquired through the bite, the amount of tissue innervated and the tissue’s proximity to the brain all influence how long it takes for clinical indications to appear. The faster the signals appear, the higher the dose and the closer it is to the central nervous system. It can last anywhere from 4 days to several years, but it usually lasts between 20 and 90 days. Muscle cell replication occurs without causing any noticeable signs. It normally does not elicit an immunological response at this time, but if antibodies are present, it can be neutralised. Because the virus is neurotropic, its absorption into peripheral neurons is critical for infection progression. The neuromuscular spindles are a critical entrance point for viruses into the neurological system. Motor end plates can also be used to gain access to the nervous system by the virus [9].

The rabies virus can infect a variety of cell types, although it is most seen in neurons. Virus infection and replication include several processes, including:

1. Adsorption: It is the process of fusing the rabies virus envelope to the host cell membrane, which may entail contact with the G protein and certain cell surface receptors.

2. Penetration: Infection of the host cell by the virus by pinocytosis (via clathrin-coated pits).

3. Uncoating: Virions clump together in large endosomes (cytoplasmic vesicles), and viral membranes merge with endosomal membranes, which results in uncoating which exposes the virus’s genetic content.

4. Transcription: Rhabdoviridae are negative-stranded RNA viruses that need the utilisation of an RNA polymerase (L gene) enzyme to convert the negative-stranded mRNA segment to a positive-stranded segment before translation.

5. mRNA: RNA that acts as a template for the synthesis of proteins.

6. Translation: The process of converting the mRNA code into N, P, M, G and L proteins.

7. Replication: In the host cell, the virus genetic material is amplified.

8. Assembly: Virus components are assembled. The ribonucleoprotein (RNP) core is formed by the N–P–L complex encasing negative-stranded genomic RNA, while the M protein forms a capsule, or matrix, surrounding the RNP.

9. Budding: The completed virus buds from the M–RNP complex’s interaction with the glycoprotein in the plasma membrane.

After replication in the originating neuron’s cell body, infection spreads through multiple neurons by retrograde axonal transport and transsynaptic dissemination. The ability of a virus to proliferate within the CNS via synaptic connections is known as transsynaptic spread. The rabies virus infects neurons, causing changes in neurotransmitter function that impact serotonin, GABA and muscarinic acetylcholine transmission. After that, acinar cells are infected, and the virus is discharged into the oral cavity. This explains why the virus can be found in saliva.

5. Pathology

In rabies, there are no visible lesions. Rabies lesions are microscopic, restricted to the CNS and have a wide range of severity. Except for early necrosis of neurons with cytoplasmic inclusion bodies in the afflicted nerve cells, they may be difficult to detect. Pathologic evidence of rabies encephalomyelitis (inflammation) in brain tissue and meninges includes the following:

1. Mononuclear infiltration

2. Perivascular cuffing of lymphocytes or polymorphonuclear cells

3. Lymphocytic foci

4. Babes nodules consisting of glial cells

5. Negri bodies

There are diffuse perivascular cuffing, neuronophagic nodules and other alterations for neuron destruction throughout the brain in some cases. The hippocampus in the brain stem and the gasserian ganglia are notably affected. Lesions in the gasserian ganglia are more particular, occur earlier and are more consistent than lesions in other parts of the body. Babes nodules, which are clumps of growing glial cells, are the major lesion. Most of the histopathologic markers of rabies were identified by 1903, but rabies inclusions had yet to be discovered. Dr. Adelchi Negri reported the discovery of the Negri body, which he believed to be the etiologic agent of rabies. Negri bodies are round or oval inclusions within the cytoplasm of nerve cells of rabies-infected animals. The size of Negri bodies can range from 0.25 to 0.27 metres. The pyramidal cells of Ammon’s horn and the Purkinje cells of the cerebellum are the most potential sites for them. They’re also found in medulla cells and a variety of other ganglia. Negri bodies can be detected in the salivary glands, tongue and other organs’ neurons. They’re generally found in the hippocampus in dogs, but they are more common in the Purkinje cell of the cerebellum in cattle. In preparations stained with Mann’s or Seller’s stain, a granular, somewhat basophilic interior structure can be detected. When the virus infects the salivary glands centrifugally, the acinar epithelium undergoes degenerative alterations that lead to necrosis, primarily affecting the mucogenic cells of the mandibular salivary glands. Fluorescent antibody methods and electron microscopy can easily show virus within these cells. The degenerative alterations are accompanied by a moderate infiltration of lymphocytes and plasma cells.

6. Clinical signs

Rabies clinical indications are rarely conclusive. Rabid animals of all species show similar symptoms of CNS abnormalities, with slight differences across species. It’s likely that the animal is seeking solitude. Rabid wild animals may lose their fear of humans, and traditionally nocturnal species may be found walking around during the day. The clinical course can be split into three stages: prodromal, furious and dumb.

Prodromal form—The term prodromal is initial period of rabies with non-specific period.

Furious form—It refers to animals in which the aggression is pronounced.

Dumb form—It refers to animals in which the behavioural changes are minimal, and the disease is manifest principally by paralysis.

6.1 Prodromal form

It is initiated when rabies virus travels up the peripheral nerve axons to the spinal ganglia which form the junction between the peripheral and central nervous systems.

6.2 Furious form

During this stage, there is very little evidence of paralysis. The animal becomes restless and may lash out with its fangs, claws, horns or hooves at the slightest provocation. These animals lose their fear of other animals and lose their caution. Carnivores infected with this strain of rabies are known to roam freely, attacking other animals, including humans and moving objects. Rabid dogs may shatter their teeth by chewing the wire and frame of their cages. Saliva either flows out of the mouth or is churned into a foam that can stick to the lip and face. Progressive paralysis leads to death.

6.3 Paralytic form

The paralysis of the throat and masseter muscles is the initial symptom, which is typically accompanied by excessive salivation and the inability to swallow. Dogs tend to drop their lower jaw. These animals aren’t violent and only bite occasionally. The paralysis spreads quickly to all regions of the body and may lead to coma, and many die within a few hours.

7. Variations in signs and symptoms in different species

7.1 Cattle

Incubation takes about 3 weeks on average, although it can take anywhere from a few weeks to several months. Early indications of paralysis include knuckling of the hind fetlocks, sagging and swaying of the hindquarters while walking and typical deviation or flaccidity of the tail to one side. In this species, yawning is a common phenomenon. Soon after yawning, the animal begins to bellow, which continues until it reaches paralysis. One of the most common symptoms is saliva drooling. The penis of bulls in this stage is paralysed. Animals may attack other animals or inanimate objects with ferocity. Lactation in dairy cows ends abruptly. Sexual arousal is common. These symptoms linger during 24–48 hours, after which the animal collapses in a paralysed state and dies within a few weeks.

7.2 Sheep

The signs are comparable to those of cattle. Sexual arousal, attacks on humans or each other and intense wool pulling have all been observed. There are twitches in the muscles, and salivation is observed. There is no excessive bleating. Most sheep are quiet and anorectic.

7.3 Goat

Aggressive and continuous bleating seen.

7.4 Pigs

Excitement, a tendency to strike, dullness and incoordination are some of the indications that have been identified. There is nasal twitching, quick chewing movements, profuse salivation and clonic convulsions.

7.5 Horses

Show signals of anguish and agitation on a regular basis. These indications are frequently accompanied by rolling. They can bite or strike with ferocity. Abnormal postures, frequent whining, kicking, biting, colic and abrupt onset of lameness in limbs followed by recumbency are all the symptoms. Paddling convulsions and ultimate paralysis are followed by sternal and lateral recumbency.

7.6 Cats

The symptoms are identical to those seen in dogs. Two to four days after the initial symptoms start, the posterior part of the body is paralysed.

8. Clinical signs in humans

Incubation, prodromal stage, acute neurological phase, coma and death are the five stages of clinical manifestations.

8.1 Incubation phase

It takes 30–90 days for rabies to develop, although it can take anything from 5 days to more than 2 years after initial exposure. It may be slightly shorter in children and vary depending on the bite place.

8.2 Prodromal phase

During this period, the first signs and symptoms appear. Some of the symptoms include fever, fatigue, sore throat, cough, dyspnea, anorexia, dysphagia, nausea, vomiting, abdominal pain, diarrhoea, headache, vertigo, anxiety, irritability and anxiousness. Agitation, photophobia, priapism, increased libido, sleeplessness and depression are all symptoms that could indicate encephalitis, psychiatric problems or brain conditions.

8.3 Acute neurologic period

This stage starts with symptoms of central nervous system dysfunction, such as anxiety, insomnia, disorientation, agitation, strange behaviour, paranoia, terror and hallucinations and progresses to delirium. During the later stages, significant amount of saliva is produced together with an inability to swallow, resulting in hydrophobia due to paralysed throat and jaw. If hyperactivity is present, the condition is classed as furious, and if paralysis is present, the disease is categorised as dumb. Periods of rapid, uneven breathing may begin near the end of this phase, followed by coma and death.

9. The virus and its genome

Rabies virus is a single-stranded, negative-sense, unsegmented, enveloped RNA virus with a rod or bullet shape. Five proteins are encoded by the viral genome. In the cytoplasm of infected cells, viral RNA uncoils. A virion-associated RNA-dependent RNA polymerase transcribes the genome. Individual viral proteins are subsequently translated from viral RNA. The creation of progeny negative-stranded RNA begins with the synthesis of positive-stranded RNA templates [10]. The RNA is responsible for coding five genes: N, Nucleoprotein, 1424 bp; P, Transcriptase associated, 991 bp;M, Matrix, 805 bp; G, Glycoprotein, 1675 bp and L, Transcriptase, 6475 bp while one pseudogene intron of approximately 700 bp makes total genome size of ≈ 12Kb. N gene is responsible for the nucleic acid, so it holds more importance for the diagnostic and evolutionary tracking of the disease.

9.1 Important strains of the virus

Pasteur—Pasteur passaged the virulent ‘street’ virus of rabies in the rabbit by intracranial inoculation. After several passages in the rabbit, virus has modified several characteristics and started giving similar kind of only paralytic forms in rabies and similar incubation period and without producing intracytoplasmic Negri bodies. The virus is also identified as the ‘fixed’ virus. The virus is used to produce vaccine.

CVS—International bodies have approved and recommended a variety of vaccine strains for rabies vaccine production around the world. The rabies challenge virus standard (CVS-11) strain is one of these strains that has been licenced for vaccine manufacturing, is recommended for use in (Rapid Fluorescent Foci Inhibition Test) RFFIT and is utilised as a challenge virus around the world. It is a well-characterised strain that was adapted in mice from the original Pasteur rabies virus discovered in 1882.

9.2 Difference in the street and fixed viruses

Street virus	Fixed virus
Isolated from infected animal or human	Isolated from several intracranial passages from rabbits
Causes several encephalopathies after varying incubation period	Less infective but cause disease after a fixed incubation of 7–10 days
Negri bodies can be demonstrated	Negri bodies are not produced
Not utilised for vaccine production	Suitable and utilised for the vaccine production

10. Need of the genetic study of the virus

A case of human rabies is described in Siberia’s polar region by Kuzmin [11] In the year 1999. The victim had been bitten by a wolf. Monoclonal antibodies revealed that the isolate was from arctic fox virus strain. This finding reaffirmed the importance of strain typing rabies virus isolates in areas where it has not yet been done: such characterisation is important for identifying the reservoir host, learning about the virus’s natural history in the reservoir and planning future surveillance, post-exposure treatment and public education in the area.

11. Epidemiology of rabies

Epidemiology is the study of distribution and the determinants of the disease. For the better understanding of the topic, epidemiology is given in two separate sections.

11.1 Distribution of the disease

Rabies is found everywhere across the planet, except for islands. Except for Australia and Antarctica, rabies is endemic in many of the countries. Bahrain, Cyprus, Hong Kong, Japan, Malaysia, Maldives, Qatar, Singapore, Lakshdweep, India’s Andaman and Nicobar Islands and Timor-Leste are among the Asian countries free of rabies. Antigua and Barbuda, the Bahamas, Barbados, Belize, Falkland Islands, Jamaica, Saint Kitts and Nevis, Trinidad and Tobago, Uruguay of the Americas subcontinent, and Albania, E.Y.R. of Macedonia, Finland, Gibraltar, Greece, Iceland, Isle of Man, Malta, Portugal, Norway (except Svalbard), United Kingdom and Spain (except Melilla and Ceuta) have all been declared rabies-free. Cape Verde, Congo, Libya, Mauritius, Reunion and Seychelles are the only African countries free of rabies. Fiji, Cook Islands, Vanuatu, Guam, French Polynesia, New Zealand, New Caledonia, Solomon Islands and Papua New Guinea are among the Oceana group of islands that are rabies-free [12, 13]. Bangladesh and India are the most affected, followed by Nepal, Myanmar, Bhutan, Thailand and Indonesia. Nepal is one of the countries in the world with the highest number of human rabies deaths [12, 13].

11.2 Determinants of the disease

Rabies is a unique disease as it can contract a wide range of all the warm-blooded hosts.

11.2.1 Age

Children lack fundamental ethology understanding about dogs, children are more vulnerable to the sickness and animal bites. When children disturb dogs while they are feeding, resting, mating or terrified, dogs get violent and bite them. According to research, the disease claimed the lives of 37% of children aged 5–14. [14]. Although the people from all the age groups are susceptible to the disease. The common site of bite in case of children is the face, and often children tend to hide the animal bite marks due to fear of scolds from the parents [15].

11.2.2 Gender

Males are more prone to contract to the disease as they are usually accessed to go out of the homes for earning or playing. In a study it was found that the ratio of men to women suffering from the disease was 4:1 [16].

11.2.3 Area

Awareness plays a major role in the succession of the disease. In urbanised area, availability of the medical facilities and awareness is a factor which may show reduced cases of the disease contrary; in rural areas, many socio-economic and religious factors are responsible for the disease spread as in many part of Gujarat, India, there are temples of ‘Hadkai mata’, mythologically protecting the people bitten by dogs from the rabies. The bite of a rabies-infected dog causes over 95% of human cases, which disproportionately afflict rural people, particularly children, in economically challenged countries of Africa and Asia [17].

11.2.4 Dog population

Majority of the rabies cases are occurring in the Southeast Asia and that too from the dogs. More the dogs are the factors significantly contributing to the spread of the disease. Stray dogs are generally naive towards the disease while only scanty dogs are vaccinated against the disease. The unvaccinated stray dog population is the biggest factor for the spread of the disease. At least 70% vaccination in the canine population will be taking care of spread of the disease in the animal population [18].

11.2.5 Type of the exposure

11.2.5.1 Animal bite exposure

The disease occurs in two phases. First phase of the disease occurs once rabid dog bites any animal or human. The live viruses travel from the site of the bite to the brain in centripetal manner. The second phase starts when the virus after reaching the brain starts travelling from the brain to the peripheral nerves and induces the clinical signs. The course of the occurrence is directly related to the site of bite. If the site is nearer to the head, disease progression is rapid.

11.2.5.2 Non-bite exposure

The rabies virus is spread by direct contact with saliva or brain/nervous system tissue from an infected animal (such as through broken skin or mucous membranes in the eyes, nose or mouth). Aside from bites and scrapes, there aren’t many injuries. One non-bite form of exposure is inhalation of aerosolised rabies virus, although most people, except for laboratory personnel, will not meet an aerosol of rabies virus. Rabies has been transmitted through corneal and solid organ transplants, but these cases are extremely rare [19].

11.2.6 Host susceptibility range

Although all warm-blooded animals are susceptible to rabies and can transmit the RABV, there is significant interspecies heterogeneity in the ability of mammals to act as reservoirs. Rabies is mostly spread by carnivores all over the world [12]. Main cause for the transmission of the virus is wildlife or stray animals, lesser than 10% cases are reported from the domesticated animals such as dogs or cats [20]. Equine and bovine are generally susceptible to the disease, but they are considered as the dead end hosts as they generally do not transmit the disease [21].

There are two types of epidemiological cycle for the occurrence of the disease. Urban and Sylvatic cycles, both the cycles are overlapping to each other and interrelated. Dog, cat, fox, raccoon, jackal, wolf, badger, etc. are the reservoir of the disease while bats are the vector of the disease. In Asian subcontinent, majority of the cases are dog-mediated rabies while in American and European countries, bat-mediated rabies is seen [12]. In India, there are some factors which promote the growth of the stray dogs.

1. Mythologically dogs are related to ‘Kal Bhairav’, a god of Hinduism, and so from almost all the homes, last feed is offered to the dogs, which helps in the maintenance of the dog population.

2. Vulture population is getting declined day by day which is competitive exclusion parameter for dog food.

3. Open slaughter policies are providing food for sustainability to the dogs outdoor.

4. Open garbage disposal attracts dogs, and many a times they can be utilised as a source to the feed.

12. Diagnosis of rabies

Accurate and timely diagnosis is very important for proper management of the post-exposure prophylaxis and application of the public health control efforts. The disease is diagnosed using a variety of techniques. However, adequate proper collection and submission of post-mortem materials, particularly brain tissues from animals suspected of having rabies, can provide basis for rabies confirmatory diagnosis [22]. Rabies can be difficult to diagnose because, in the early stages, it is easily confused with other diseases or even with a simple aggressive temperament. The reference method for diagnosing rabies is the fluorescent antibody test (FAT), an immunohistochemistry procedure, which is recommended by the World Health Organisation (WHO).

Primary diagnostic methods, such as the direct fluorescent antibody (DFA) test, the direct rapid immunohistochemistry test (dRIT) or pan-lyssavirus polymerase chain reaction (PCR) assays, are used to identify agents. If a proper conjugate or primer/probe is employed, the DFA test, dRIT and PCR offer an accurate diagnosis in 98–100% of cases for all lyssavirus strains. In highly equipped facilities, conventional and real-time PCR may produce speedy results for a large number of samples. Histological procedures such as Seller staining (Negri bodies) are no longer suggested for diagnosis. In the incidence that main diagnostic tests (DFA test, dRIT or pan-Lyssavirus PCR) give unsatisfactory findings, further confirmatory testing (molecular tests, cell culture or mice inoculation tests) on the same sample or repeat primary diagnostic tests on different samples are recommended. Virus isolation in cell culture should be used instead of mice inoculation testing whenever possible. In specialised laboratories, the agent can be characterised utilising monoclonal antibodies, partial and whole genome sequencing and phylogenetic analyses. These approaches can tell the difference between field and vaccine strains, as well as the geographical origin of the field strains. These extremely sensitive tests should only be performed and evaluated by highly experienced experts.

Pre-exposure vaccination and boosters are necessary for all persons and laboratory workers engaged in the management of rabies suspected cases. These individuals are at risk of contracting rabies in a variety of ways. As a result, personal protection equipment (PPE) must be always worn, beginning with the necropsy procedure.

12.1 Identification of the agent

Because the rabies virus inactivates quickly, the specimens should be delivered on ice to the laboratory as soon as possible. Various approaches are used to diagnose rabies, with a focus on brain tissue, although other organs such as salivary glands are also used. Both the cerebellum and the brain stem are recommended for laboratory testing since the virus will be abundant in them and will help in laboratory detection. These portions of the brain can be acquired when the complete brain is removed during necropsy using the skull open approach.

12.2 Collection of samples

The virus can be found in the brain, spinal cord, saliva and salivary glands of a rabies-infected animal. B rain tissue is the preferred specimen for rabies diagnosis, the animal suspected of having rabies should be euthanised in such a way that the brain is not damaged. Only vaccinated and well-trained veterinarians or animal control personnel should remove the animal heads.

Brain sample collection for the accurate diagnosis of rabies is very difficult work and that can be dangerous in the field or if the person is not properly trained. The occipital foramen route of brain sampling is an alternative way of collecting brain samples that does not need open the skull.

12.3 Brain sample collection through the occipital foramen

The brain sample is collected through the occipital foramen by inserting a 5 mm drinking straw or a disposable plastic pipette with a capacity of about 2 mL or by inserting an artificial insemination sheath about 10 cm long into the foramen in the direction of the eye. Brain stem and cerebellum samples can be obtained through the juice straw or artificial insemination sheath (Figure 1). This technique of collection should be user-friendly, quick and risk-free for reliable rabies diagnosis [22]. This technique speeds up the more number of brain samples collection simultaneously.

Figure 1.

Brainstem collection through the foramen magnum technique in a dog (captured by the authors).

12.4 Animal rabies diagnosis

Laboratory procedures for diagnosing rabies were developed as early as 1800 BC. Adelchi Negri discovered the Negri bodies in 1903, and their diagnostic significance was proved by his wife Lina Negri-Luzzani in 1913 [22]. This cleared the way for the development of a multiplicity of laboratory procedures for rabies confirmation, which are described in the WHO book ‘Laboratory Techniques in Rabies’ [23] as well as the ‘OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals’ [24].

12.5 Sellar’s staining under direct microscopy

Seller’s staining method is a quick and easy test. It is a histological test used on brain impressions to show the unique cell lesions known as ‘Negri bodies’. These are viral particle aggregates visible as intracytoplasmic inclusion bodies ranging in size from 3 to 30 m in infected neural cells. The Negri bodies are round or oval structures that include basophilic granules in an eosinophilic matrix. This technique has relatively poor sensitivity for the diagnosis of rabies, that’s why nowadays this test is no longer recommended [23].

12.6 Direct fluorescent antibody assay (DFA)

The World Health Organisation (WHO) and the World Organisation for Animal Health (OIE) both endorse the direct fluorescent antibody assay as the most extensively used test for post-mortem confirming diagnosis of rabies. Goldwasser and Kissling created this gold standard test in 1958. The ‘Nucleoprotein antigen’ (N) of the rabies virus is shown here to be present in fresh brain impressions of rabies suspect animals (Figure 2). Such rabies viral inclusions do not exist in the brain impressions of non-rabid animals (Figure 3). Furthermore, in a normal laboratory, the DFA has a specificity and sensitivity of about 99% [22].

Figure 2.

Rabid animal brain impression, counterstained with Evan’s blue and stained with rabies virus antinucleocapsid IgG-FITC conjugate (rabies DFA III, light diagnostics, cat # 6500, captured by authors).

Figure 3.

Non-rabid animal brain impression stained with rabies virus anti-nucleocapsid IgG-FITC conjugate (rabies DFA III, light diagnostics, cat # 6500, captured by authors).

The DFA is accurate and sensitive. The sensitivity of this test is determined by the quality of the sample (how carefully the brain is sampled as well as the degree of autolysis), the type of lyssavirus and the diagnostic staff’s expertise. Impressions are obtained from a composite sample of brain tissue that includes the brainstem. It is air-dried before being immersed in 100% high-grade cold acetone for 1 hour to set the impressions. The impression is withdrawn from the acetone, air-dried and stained with a drop of the appropriate conjugate.

The impression is then incubated for 60 minutes at 37°C. Anti-rabies fluorescent conjugates are commercially available as polyclonal or monoclonal antibodies (MAbs) that are specific to whole virus or the N protein of the rabies virus and have been conjugated to the fluorescing dye, fluorescein isothiocyanate (FITC). The DFA slides should be inspected under a fluorescent microscope with a filter that corresponds to the wavelength of the fluorescent conjugate employed. FITC, which is stimulated at 490 nm and re-emits at 510 nm, is the most often used fluorescent dye. The presence of nucleocapsid protein aggregates can be seen by the fluorescence of associated conjugate in an apple green colour. When fresh brain tissue is used, this test is reliable. Bacterial contamination of partially decomposed brains causes nonspecific fluorescence that is difficult to differentiate from specific fluorescence owing to N antigen, making it inappropriate for this test.

12.7 Direct rapid immunohistochemistry test (dRIT)

The Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia, developed dRIT, which is one of the most important breakthroughs in the diagnosis of Rabies. This test also detects the N protein of the rabies virus in rabies brain impressions. A suspected animal brain smear is fixed with buffered formalin before being processed further. Following appropriate viral fixation, the antigen was treated with a biotinylated monoclonal antibody cocktail that was highly concentrated and purified (to N protein). After that, an indicator and streptavidin peroxidase are added. The aggregation of viral clusters is seen as brick red clusters within the cell, along the axons and throughout the brain impression (Figure 4). Negative brain impressions show no such brick red inclusions.

Figure 4.

Rabid animal brain impression tested by dRIT, captured by authors.

This 1-hour test method is helpful in field conditions since the results may be examined with an ordinary light microscope. It has been tested in several nations and confirmed to be 100% as sensitive and specific as DFA. This simple, low-cost test will be extremely useful in enhancing rabies epidemiology monitoring, particularly in underdeveloped countries where expensive fluorescence microscopes and cold storage facilities may not be accessible [22].

12.8 Lateral flow assay (immunochromatography)

The lateral flow assay is a simple and quick immunochromatographic technique. The rabies virus nucleoprotein is recognised by this test kit, which was produced utilising monoclonal antibodies. It has been tested as a quick rabies screening test. This assay is an immunodiagnostic test that provides quick findings in the field by detecting RABV antigen in post-mortem samples without the need of laboratory equipment. In summary, the detector antibodies are coupled to a membrane at two separate zones, and when the processed material is added to the device at the appropriate slot, coloured lines appear, indicating the presence of viral antigen [25].

In the case of rabies-virus-positive brain samples, coloured lines may be observed in both the ‘C’ (Control) and ‘T’ (test) zones; however, in the case of negative samples, only the ‘C’ zone displays colour development (Figure 5). Furthermore, this assay might be used to successfully identify rabies virus in cell culture [26].

Figure 5.

Lateral flow test of rabies-positive brain sample suspensions (captured by authors).

12.9 Other assays for antigen detection

Other less common antigen detection techniques are as follows:

A quick sandwich ELISA is used to identify lyssaviruses belonging to all seven genotypes that circulate in Europe, Africa, Asia and Oceania [27]. Dot-blot immunoassay for brain tissues and enzyme immunoassay (EIA) for quick diagnosis in humans and animals [28].

Various PCR-based tests are now being developed for ante-mortem and post-mortem rabies diagnosis. Because the nucleoprotein (N) gene is extremely conserved, most of these PCR variants amplify it. This method has shown to be quite efficient in detecting rabies ante-mortem.

12.10 Reverse transcriptase PCR (RT-PCR)

RT-PCR tests based on gels are also used to identify rabies virus RNA in clinical samples [29, 30, 31, 32]. The amplicons produced in these tests, notably those targeting N, G and G-L intergenic regions, have been sequenced in order to characterise the virus and analyse its phylogeny [33]. However, these assays are vulnerable to cross-contamination, which is a major problem that prevents them from being used routinely for rabies diagnosis [27].

12.11 Real-time PCR

Real-time PCR is used to identify and quantify genome copies while reducing the probability of cross-contamination. The SYBR Green real-time PCR technique is applied for rabies ante-mortem diagnosis [34] as well as finding lyssaviruses [35]. TaqMan real-time PCR tests have been shown to have high specificity [27, 36]. This was shown to be 100 times more sensitive than typical nested RT-PCR [36].

12.12 Demonstration of antibodies

Although they have certain drawbacks, the rabies virus neutralisation test, notably FAVN or RFFIT, is the test of choice for determining neutralising antibodies [37]. Various varieties of ELISA (enzyme-linked immunosorbent assay) are also utilised as an alternative since they are safe, simple and quick. Furthermore, because these tests do not involve the handling of live virus, they do not require use of high-containment facilities. The ELISA findings are shown to correspond well with the RFFIT results. ELISA based on N and G protein Mab was developed to the specially trap the rabies antigen during ante-mortem diagnosis [38].

A second-generation ELISA kit, the Platelia Rabies II, was designed to detect antibodies against the glycoprotein in blood and CSF samples. This ELISA was tested and shown to correlate well with RFFIT, making it suitable for use in laboratories without cell culture facilities [39]. However, when compared with neutralisation tests, ELISA is less sensitive [40].

13. Management of rabies

13.1 Management in human

13.1.1 Pre-exposure prophylaxis (PrEP)

PrEP (vaccination) is the most efficient way of rabies control. It not only saves the budget of the management, but it assures the prevention of the disease. There are several protocols for the prophylaxis of the disease. There are two routes for the vaccination: intra-muscular (IM) and intra-dermal (ID). Intra-dermal vaccine saves the quantity of the vaccine by 80%. The detailed protocol is given below. PrEP is recommended to the people who are associated to specific group vulnerable to rabies such as veterinarians, para-vets, animal welfare activists or people residing in endemic area. The dosage for ID is 0.1 ml at two sides while the dose of IM vaccine is whole vial. The vaccine should not be given at gluteal muscle. The protocol suggests two shots of vaccine on 0 and 7 days.

13.1.2 Post-exposure prophylaxis (PEP)

PEP is suggested after the exposure of rabies. There are three categories of the exposure listed in Table 1. There are three dosage regimes given by different institutions, Institut Pasteur du Cambodge regimen, Essen regimen and Zagreb regimen.

Category	Immunologically naive	Previously immunised
1 Touching or feeding animals, animal licks on intact skin (no exposure)	Wash exposed skin surfaces. No PEP required.
2 Nibbling of uncovered skin, minor scratches, or abrasions without bleeding (exposure)	Wound washing and immediate vaccination: - 2-sites ID on days 0, 3 and 7; OR; - 1-site IM on days 0, 3, 7 and between days 14 and 28; OR; - 2-sites IM on days 0 and 1-site IM on days 7, 21; RIG is not indicated.	Wound washing and immediate vaccination*: - 1-site ID on days 0 and 3; OR; - at 4-sites ID on day 0; OR; - at 1-site IM on days 0 and 3); RIG is not indicated.
3 Single or multiple transdermal bites or scratches, contamination of mucous membrane or broken skin with saliva from animal licks, exposures due to direct contact with bats (severe exposure)	Wound washing and immediate vaccination; - 2-sites ID on days 0, 3 and 76; OR; - 1-site IM on days 0, 3, 7 and between days 14 and 287; OR; - 2-sites IM on days 0 and 1-site IM on days 7, 218; RIG administration is recommended.	Wound washing and immediate vaccination*: - 1-site ID on days 0 and 3; OR; - at 4-sites ID on day 0; OR; - at 1-site IM on days 0 and 3; RIG is not indicated.

Table 1.

Suggested PEP according to exposure.

The major between immunologically naïve and previously immunised person with the PEP is no requirement of RIG in previously immunised person. The maximum dose of RIG is 20 (hRIG) or 40 (eRIG). If RIG is not available, thorough, prompt wound washing, together with immediate administration of the first vaccine dose, followed by a complete course of rabies vaccine, is highly effective in preventing rabies. Vaccines should never be withheld, regardless of the availability of RIG. Rabies virus is enveloped virus and so through washing of bite wound with soap solution under running tap water is advised. (Adopted from [41])

13.2 Management in animals

13.2.1 Pre-exposure prophylaxis

13.2.1.1 Parenteral prophylaxis

The rabies vaccine with the potency of RIVM >2 I.U. may be used for the vaccination. The vaccine is approved for the use for the prophylaxis of apparently healthy mammals. The vaccine may be given by the IM or SC route. Generally, a temporary palpable nodule at the site of the SC injection may be noticed, which will disappear by the time. Rarely anaphylactic reaction can be seen which can be managed by SC injection of adrenalin. It is always recommended to give the vaccine a bit earlier than the due date to ensure the protection. Many a times it is possible that the whole dose of vaccine may be failed to be administered to the animals due to faulty administration. Vaccination can begin as early as 3 months of age in dogs, ferrets and livestock. Vaccines for cats can be administered as early as 2 months of age [42].

The schedule is given below in Table 2.

Species	Age at Primary Vaccination	Revaccination
Dog & Cat	After 3 months of age*	3 years**
Cattle, Horse, Sheep & Goat	After 6 months of age*	2 years**
Ferret	After 3 months of age*	1 year**

Table 2.

Vaccination schedule for animals.

*

Primary vaccination can be administered at an earlier age, but then a repeat vaccination must be given at the age of 3 or 6 months depending on the species.

**

Annual revaccination is recommended in endemic areas.

Source: [43].

13.2.1.2 Oral prophylaxis for dogs and wildlife

The rabies vaccine bait RABORAL V-RG® contains an attenuated (‘modified-live’) recombinant vaccinia virus vector vaccine that expresses the rabies virus glycoprotein gene (V-RG). Since 1987, when the first licenced recombinant oral rabies vaccine (ORV) was released into the environment to immunise wildlife populations against rabies, over 250 million doses have been distributed globally with no reports of adverse responses in wildlife or domestic animals. V-RG is genetically stable, does not remain in the oral cavity for more than 48 hours after ingestion, is not shed into the environment by vaccinated animals and has been tested for thermostability in a variety of laboratory and field settings. V-RG has been tested in over 50 vertebrate species, including nonhuman primates, and no adverse effects have been reported regardless of method or dose. Immunogenicity and efficacy in a variety of target species have been established in the lab and in the field (including fox, raccoon, coyote, skunk, raccoon dog and jackal). The liquid vaccine is placed within edible baits (such as RABORAL V-RG, the vaccine-bait product) that are released into animal areas for target species to consume. The use of RABORAL V-RG in the field has helped to eradicate wildlife rabies in three European nations (Belgium, France and Luxembourg), as well as the dog/coyote rabies virus form in the United States (USA). With the final case reported in a cow in 2009, an oral rabies vaccination programme in west-central Texas has effectively removed the grey fox rabies virus strain from Texas. In the United States, a long-term ORV barrier effort using RABORAL V-RG is preventing significant geographic spread of the raccoon rabies virus strain. For more than a decade, RABORAL V-RG has been used in Israel to control wildlife rabies [44].

13.2.2 Post-exposure prophylaxis

PEP of the rabies should include five administrations of the vaccine on the days 0, 3, 7, 14, 28/90 days. If the animal is not immunised previously, eRIG is advised to be administered at the site of bite.

14. Control of rabies

The disease is a classical example of neglected zoonosis. The disease can be well managed by the multi-disciplinary approach. Control of the rabies in the dogs is very important. World Health Organisation (WHO) has strong measures in place to prevent rabies in dogs. These guidelines include:

1. Notification of suspected cases, with euthanasia of dogs with clinical signs and those bitten by suspected rabid animals

2. Leash laws and quarantine to limit contact between susceptible dogs

3. A mass immunisation programme with ongoing boosters

4. Stray dog control and euthanasia of unvaccinated dogs roaming freely

5. Dog registration programmes [45].