Rabies

 

• Also known as hydrophobia is an acute, highly fatal viral disease of the central nervous system, caused by Rhabdovirus - transmitted by bites or licks of rabid animals, especially from the saliva..
• Rabies is characterized by acute inflammation of the brain and spinal cord (encephalomyelitis)
• It is primarily a zoonotic disease of warm-blooded animals, particularly  dogs, cats etc.
• It is the only communicable disease of man that is almost always fatal.
• About 50,000 death occur due to rabies, annually. India alone accounts for 20,000 death per year.
• The causative agent Rhabdovirus is a bullet shaped neurotropic RNA containing virus.

• It belongs to the family Rhabdoviridae - (Lyssavirus genus) [Rhabdos (G)= rod ]
• Rhabdovirus ssRNA genome
• Infect mammals, reptiles, birds, fishes etc

 

Structure

• Rhabdovirus - large rod or bullet shaped enveloped virus, measuring 7 X 180nm diameter (50-95 nm diameter, 130-139nm length)
• Bullet shaped-one end conical/rounded and the other end planar/concave

• Envelope- The lipid bilayer envelope derived from the host cell membrane contains
• Numerous knob like spikes present on the envelope, these are made up of virus encoded glycoprotein, (G-spikes).
• These glycoproteins are found to bind specifically to host cellular receptor and are responsible for neurotropism in infected cells. Thus, glycoprotein is necessary for viral attachment & also induce protective antibodies.
• Closely associated with G-protein is a second membrane known as matrix protein (M protein) which has a role in viral budding.
• Nucleocapsid- The nucleocapsid is spiral or helical composed of RNA, phosphorylated nucleoprotein and RNA dependent RNA polymerase I.
• The viral genome consists of unsegmented, linear, negative sense single stranded RNA.

 

Antigenic Components

Rabies virus particles contain two distinct, major antigens

(1)   Glycoprotein (G protein)   antigen from the virus membrane

(2)   an internal nucleoprotein antigen

(3)   Two membrane proteins, glycolipids, RNA dependent RNA polymerase are also antigenic in nature

 

1. Glycoprotein (G protein) antigen

• The glycoprotein is the only antigen capable of inducing the formation of virus-neutralizing antibodies- (the presence of virus neutralizing antibodies in the blood of man and animals is considered an index of protection against infection with rabies virus).
• Thus, G protein antigen is important in pathogenesis, virulence and immunity

(1)   Mediates binding of virus to acetyl choline receptors in neural tissues- causes neurotrophism of virus

(2)   Induces protective antibodies (hemagglutination inhibiting and neutralizing antibodies)

(3)   Stimulates cytotoxic T cell immunity

• Purified glycoprotein is a safe, effective subunit vaccine

 

2. Nucleoprotein antigen

• Induces complement fixing antibodies- Not protective in nature
• Important in diagnostic immunofluorescence testing

 

Resistance

• The rabies virus can be inactivated by lipid solvents (soap solutions, ether, chloroform, acetone), 1% sodium hypochlorite, 2% glutaraldehyde, 45-75% ethanol, iodine preparations, quaternary ammonium compounds, phenol, formalin, beta propionolactone  or a low pH.
• The virus is also susceptible to ultraviolet radiation or heat of 1 hour at 50°C.
• It is rapidly inactivated in sunlight, and it does not survive for long periods in the environment except in a cool dark area.
• Preserved at -70°C or by lyophilisation.

Host/Source of Infection

All warm blooded animals including man are susceptible to rabies.

The source of infection to man is the saliva of rabid animals.

Rabies in man is a dead-end infection.

Laboratory staff working with rabies virus, veterinarians, dog handlers, hunters face bigger risks of rabies.

Animals reported in India, include

Domestic:-Dogs & Cats, Sheep & Goats, Pigs, Donkeys, Horses, Camels

Wild:-Foxes & Jackals, Monkeys, Mongoose, Bears

 

Mode of transmission

1. Animal Bite: People are infected following a deep bite or scratch by an infected animal.

• Dogs are the main host and transmitter of rabies.
• Bats are the source of most human rabies deaths in the USA and Canada.

2. Licks-Licks on broken skin and mucosa can transmit the disease.

3. Aerosol- by inhalation of aerosol infected with virus

• In lab, personnel can become infected
• In caves, where rabid bats are present

4. Person to person (rare) - Human-to-human transmission is possible through corneal grafting because rabies antigen has been detected in the corneal cells of patients

 

Epidemiological cycles

• Rabies is maintained in two epidemiological cycles, - urban and sylvatic.
• In the urban rabies cycle, dogs are the main reservoir host. This cycle predominates in areas of Africa, Asia, and Central and South America where the proportion of unvaccinated and semi-owned or stray dogs is high. It has been virtually eliminated in North America and Europe; although sporadic cases occur in dogs infected by wild animals.

• The sylvatic (or wildlife) cycle is the predominant cycle in Europe and North America. It is also present simultaneously with the urban cycle in some parts of the world. The epidemiology of this cycle is complex; factors affecting it include the virus strain, the behavior of the host species, ecology and environmental factors.

Street Virus and Fixed Virus

The virus is excreted in the saliva of the affected animals.

The virus recovered from naturally occurring cases of rabies is called "street virus". It is pathogenic for all mammals and shows a long variable incubation period. It is capable of producing Negri bodies (intracytoplasmic eosinophilic inclusion body) in the neurons which is pathognomonic of rabies. Cause fatal encephalitis in 1-12 weeks.

pathognomonic (characteristic or indicative of a particular disease or condition)

Fixed virus- street virus when attenuated in the lab, after several serial intracerebral passages in rabbit brain is least virulent, more neurotropic and has short fixed incubation period (2-5) days. Virus isolated at this stage is called a fixed virus. It does not form Negri bodies. It no longer multiplies in extra-neural tissues. It has lost its pathogenicity on human being but retains its antigenicity. The fixed virus is used in the preparation of ant rabies vaccine. Intracerebral inoculation causes fatal encephalitis in a short period (2-5 days)

 Growth in Laboratories

• Chick embryos- virus inoculated in yolk sacs-serial propagation led to the development of attenuated vaccine strains Flury and Kelev
• Duck eggs- strains adapted to duck eggs, used in inactivated vaccine production
• Tissue culture- Primary and continuous cell cultures such as chick embryo fibroblast, porcine/hamster kidney- strains with not much cytopathic effects, but in low yield. Fixed virus strains adapted for growth in human diploid cell lines, chick embryo and Vero cell cultures- used in vaccine production

 Rabies- (Rabidus (L)=mad) 

Disease transmitted by the bite of “mad dogs”. 

Rabies virus demonstrated in the brain of infected animals by Pasteur in 1881. 

He obtained fixed virus by serial intracerebral passages in rabbits- pieces of spinal cord from rabbits dried for various periods- used as vaccine- In 1885, Joseph Meister (9-year-old)- course of 13 inoculations- first successful vaccination. 

Rabies in man is called hydrophobia-patient exhibits fear of water- incapable of drinking though subject to intolerable thirst (attempts to drink-painful spasms of pharynx and larynx- choking& gagging, dread at the sight and sound of water)

 Pathogenesis

• Rabies virus is excreted in saliva of rabid animal so, human acquire virus by the bite of rabid animals.
• Virus multiplies in muscle or connective tissue at the site of inoculation and then enter peripheral nerves at neuromuscular junction and spread up to the CNS.
• It spreads from the site of infection centripetally via the peripheral nerves towards the central nervous system.
• Following infection of the central nervous system, the virus spreads centrifugally in peripheral nerves to many tissues, including salivary glands. 

• Virus, however, can also enter directly into CNS without local multiplication.
• It multiplies in the grey matter in brain and propagates through efferent nerves to salivary gland and other tissues like kidney, heart, cornea, retina, pancreas.
• The highest titre of virus is seen in submaxillary gland (salivary gland)

The virus is neurotropic. Presence of virus in saliva and the irritability/aggression brought on by encephalitis ensure transmission and survival of virus in nature

• Rabies virus produces a specific eosinophilic cytoplasmic inclusion with basophilic granules called the Negri bodies in the brain of animals
• The Negri body is filled with viral nucleocapsid and forms the pathognomonic basis of rabies diagnosis.

Incubation period

Susceptibility to rabies infection and incubation period may depend on host age, genetic makeup, immunity, viral strain involved, inoculum size and the distance virus has to travel from the point of entry to CNS.

The incubation period in man is highly variable, commonly 1-3 months but may be as short as  weeks to as long as many years. It is usually shorter in children than in adults.

   The incubation period depends on the

• Site of the bite,
• severity of the bite,
• number of wounds,
• amount of virus injected,
• species of the biting animal,
• protection provided by the clothing and
• treatment undertaken, if any.

In general, incubation period tends to be shorter in severe

• exposures and bites on face, head, neck and upper extremities
• and bites by wild animals.

Clinical spectrum of rabies can be divided into three phases.

Prodromal phase: It lasts for 2-10days and is characterized by any of the following non-specific symptoms, malaise, anorexia, headache, photophobia, nausea and vomiting. There is pain and tingling or numbness at the site of the bite.

Acute neurological (Encephalitic) phase: It lasts for 2-7 days and is characterized by the presence of nervous system disorder like nervousness, apprehension, hallucination, lacrimation, pupillary dilation, and increased salivation, hydrophobia and painful throat muscles. 

Widespread excitation and stimulation of all parts of nervous system involving, the sensory system, the motor system, the sympathetic and mental system.

The patient is intolerant to noise, bright light (photophobia) or air (aerophobia).

Mental changes include fear of death, anger, irritability and depression. 

Swallowing liquid become unsuccessful. At later stage the mere sight or sound of water may provoke spasm of the muscles of deglutition. This characteristic symptom of hydrophobia (fear of water) is pathognomic of human rabies and is absent in animals.

The duration of illness is 2 to 3 days, but may be prolonged to 5-6 days in exceptional cases.

Coma and death:  Neurological phase is followed by coma and death. Death is due to respiratory arrest, convulsion or choking.

Paralytic rabies: In about 20% cases, muscle weakness is prominent - flaccid paralysis at the site of bite, quadriparesis (weakness in both arms and both legs) and facial weakness in 2-10days. Ultimately, multi organ failure, death. 

Lyssaphobia/Hydrophobiophobia -Persons exposed to real/imaginary risk of rabies. Psychological disorder- anxiety, irritability and exaggerated hydrophobia; afebrile- need sedation and reassurance

Rabies in Dogs

Incubation period- 3-6 weeks. 

Prodromal stage - Alert/troubled appearance, restlessness, snapping at imaginary objects, licking, gnawing at the site of bite etc.

Then, dogs may manifest either of the two types of rabies- dumb or furious rabies. Both equally infectious

Dumb rabies- paralytic form- animal lied huddled- no feeding- may not bite, but attempts to feed can be dangerous.

Furious rabies- Runs wild, bite without provocation and indiscriminately. Lower jaw drops- saliva drooling from mouth- paralysis, convulsions and death follow

Rabid dogs usually die in 3-5 days.

Hookworm - Ancylostoma duodenale

·       Widely distributed in tropical and sub-tropical countries

·       One of the small intestine nematodes

·       Cause intestinal hookworm disease in humans

·       Important cause of Iron deficiency anaemia

 Belongs to family Ancylostomatidae- 2    important human Spp:

·     Ancylostoma duodenale -named so, since anterior end of adult worm is bent -(Ankylos (Greek)=hooked, stoma=mouth) /Old world hookworm

·       Necator americanus (American Murderer)/New World hookworm

 Ancylostoma duodenale

·       Causes ancylostomiasis

·       Dubini in 1843 described the parasite;  Arthur Loss in 1898 described pathogenesis and mode of infection

Epidemiology

·       Globally 900 million people infected. 

·      Hookworm infection is prevalent in the tropics and subtropical countries – Asia, Africa, America, China and Southern Europe

·       In India – prevalent in Punjab, Uttar Pradesh and Bihar

·       Males & young adults commonly affected

·       Anaemia severe in children & pregnant women

 

Morphology

 Adult Worm:

·       small, stout, greyish white and cylindrical

·       Body is curved, dorsal surface concave and ventral surface convex,

·       Anterior end- bent dorsally - hence name hookworm

·       Oral aperture- directed towards dorsal surface

·       Mouth located dorsally-prominent Buccal capsule- has 6 teeth, 4 hook-like on the ventral surface and 2 knob-like on the dorsal surface. 

 Male worm differs from female worm

 

Adult male worm

Smaller- 8 to 11 mm long x 0.4 mm wide. Posterior end expanded into a copulatory bursa. Situated within the copulatory bursa, is the cloaca into which the rectum and genital canal open. There are two long retractile bristle-like copulatory spicules, which project from the bursa.

Copulatory bursa -Present in male, For attachment with female during copulation. Consists of 3 lobes, one dorsal and two lateral lobes. Each lobe: supported by 13 chitinous rays.

Adult female worm

Larger /longer than male 10 to 13 mm long x 0.6 mm wide. Posterior end is conical, with a subterminal anus situated ventrally. Vulva opens ventrally at the junction of the middle and posterior thirds of the body. Vagina leads to two intricately coiled ovarian tubes which occupy the hind and middle parts of the worm.

During copulation, the male attached its copulatory bursa to the vulva- copulating pair appears Y-shaped.

Life span of adult worm in human intestine: 3 to 4 years.

Differences between Adult male and female A. duodenale

Characteristic	Male	Female
Size	Smaller- 8 to 11 mm long x 0.4 mm wide	larger /longer than male 10 to 13 mm long x 0.6 mm wide
Posterior end	Expanded in an umbrella like fashion - as copulatory bursa	Tapering and no expanded bursa
Genital opening	Posterior and opens with cloacae	Present at junction of posterior and middle third of body

 

Egg

·       Oval/elliptical; 65 µm long x 40 µm wide

·       Colourless- non-bile stained

·       Surrounded by a thin transparent hyaline shell-membrane

·       Contains a segmented ovum with four (4) blastomeres

·       Clear space between egg shell and segmented ovum.

·       Egg floats in saturated salt solution

·       Single female worm- lays about 25 000 to 30 000 eggs/day

·       Eggs are excreted in faeces 4-7 weeks after infection

 

Life cycle of Hookworm

Definite host: man

Habitat: Small intestine of infected persons, mostly in jejunum- less often in duodenum and ileum

Infective Stage: Filariform larva

·      

 Eggs are passed in the stool   , and under favourable conditions (moisture, warmth, shade), larvae hatch in 1 to 2 days and become free-living in contaminated soil.

These released rhabditiform larvae (250 µm in length- feeds on bacteria and other organic matter in the soil) grow in the feces and/or the soil, and after 5 to 10 days (and two moults) they become filariform (third-stage) larvae that are infective. 

 Filariform (third-stage) larvae (500-600 µm long- non-feeding) live in the soil for 5-6 weeks. These infective larvae can survive 3 to 4 weeks in favourable environmental conditions.

On contact with the human host, typically bare feet, the larvae penetrate the skin and are carried through the blood vessels to the heart and then to the lungs. They penetrate into the pulmonary alveoli, ascend the bronchial tree to the pharynx, and are swallowed. They undergo third moulting during migration or on reaching the oesophagus. The final fourth moulting occurs in small intestine, they develop the buccal capsule, attaches to small intestine, where they reside and mature into adults.

Adult worms live in the lumen of the small intestine, typically the distal jejunum, where they attach to the intestinal wall with resultant blood loss by the host.

 Most adult worms are eliminated in 1 to 2 years, but the longevity may reach several years.

 Some A. duodenale larvae, following penetration of the host skin, can become dormant (hypobiosis in the intestine or muscle). These larvae are capable of re-activating and establishing patent, intestinal infections. 

Infection by A. duodenale may probably also occur by the oral and the trans mammary, transplacental route. 

 

    In small intestines: Adult worm -   inhabit small intestine of man-   Attachment to mucous membrane by help of mouth parts

·       Eggs contain segmented ova with 4 blastomeres passed out in faces of infected person (non-infective)

  In soil: Rhabditiform larva hatches from each egg (24- 48 hours) -moults twice on 3rd and 5th day-   develops into filariform larva-is the infective form- penetrates unbroken skin (of toes, dorsum of foot and medial aspect of sole) -Remains infective up to 6 weeks

  

N. americanus has similar morphology (though smaller in size) and life cycle. However, they do not appear to be infective via the oral or trans-mammary route.

People at risk

·       Walking barefoot on soil containing filariform larva

·       Farm workers: larva penetrates skin of hands -reaches subcutaneous tissues and enters lymphatics or small venules- heart - pulmonary capillaries- - migrates to bronchi, trachea and larynx- pharynx - gets swallowed. During migration through oesophagus, undergo 3rd moulting.

·       Settles in small intestines-undergo 4th moulting and develop into adult worms. -Attach to small intestine by their mouth parts -After 6 weeks- mature sexually -Fertilization occurs -Female begin to lay eggs in faeces -cycle repeats

      Pathogenesis 

The overall prevalence and intensity of hookworm infection are higher in males than in females, because males have greater exposure to infection. However, women and young children have the lowest iron stores and are most vulnerable to chronic blood loss as the result of hookworm infection.

The most serious effects of hookworm infection are the development of anemia and protein deficiency caused by blood loss at the site of the intestinal attachment of the adult worms. 

dyspnoea-difficult or laboured breathing

koilonychia- a nail disease that can be a sign of hypochromic anemia, especially iron-deficiency anemia

Loeffler syndrome - a rare, transient, self-limiting (less than one month), and benign pulmonary eosinophilia 

Adult hookworms cause morbidity in the host by producing intestinal hemorrhage. The adult hookworms then ingest the blood, rupture the erythrocytes, and degrade the hemoglobin. Therefore, the disease causes silent blood loss leading to iron deficiency anemia and protein malnutrition.

Diagnosis

The standard method for diagnosing the presence of hookworm is by identifying hookworm eggs in a stool sample using a microscope. Because eggs may be difficult to find in light infections, a concentration procedure is recommended.

The diagnostic approaches include:

Occult blood- not easily seen with the naked eye-due to  bleeding in the digestive tract

Treatment

Anthelminthic medications (drugs that rid the body of parasitic worms), such as albendazole and mebendazole, are the drugs of choice for treatment of hookworm infections. Infections are generally treated for 1-3 days. Morbidity can be reduced through anthelminthic drug treatment (also known as “deworming”).

Iron supplements may also be prescribed if the infected person has anemia.

Prophylaxis

The best way to avoid hookworm infection is not to walk barefoot in areas where hookworm is common and where there may be human faecal contamination of the soil. Also, avoid other skin contact with such soil and avoid ingesting it. Use footwear and gloves.

Infection can also be prevented by not defecating outdoors and by effective sewage disposal systems.

Treatment of patients and carriers, limit the source of infection.

The global disease burden from hookworm exceeds all other major tropical infectious diseases with the exception of malaria, leishmaniasis, and lymphatic filariasis. 

Hookworm infection is a major health threat to adolescent girls and women of reproductive age, with adverse effects on the outcome of pregnancy. Severe iron-deficiency anemia during pregnancy has been linked to increased maternal mortality, impaired lactation, and prematurity and low birth weight. Infants and preschool children are particularly vulnerable to the developmental and behavioural deficits caused by iron-deficiency anaemia. In addition, hookworm has been associated with impaired learning, increased absences from school, and decreased future economic productivity.