LIQUID PHASE BLOCKING ENZYME LINKED IMMUNOSORBENT ASSAY FOR SERO-MONITORING OF FOOT

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LIQUID PHASE BLOCKING ENZYME LINKED IMMUNOSORBENT ASSAY
FOR
SERO-MONITORING OF FOOT AND MOUTH DISEASE PREVACCINATED ANIMALS

BY

ASWATHY. G. M

DEPARTMENT OF BOTANY & BIOTECHNOLOGY
MAR IVANIOS COLLEGE,
NALANCHIRA.P.O.
THIRUVANANTHAPURAM.
APRIL 2011

PREFACE

Foot And Mouth Disease is not a concern for human health. However it can cause severe problems for animals with cloven hooves, such as cattle, pig, sheep, goats and deer, as once it's contracted , the disease can spread rapidly. This disease is having a major influence on the international trade of animals and their products. In India, outbreaks due to serotypes O, A, and Asia-1 are encountered throughout the year.

Primary diagnosis of FMD commonly involves recognition of typical clinical signs in affected animals. Before an outbreak is declared it is mandatory to demonstrate FMD virus or antigen using laboratory tests.

Laboratory diagnostic techniques such as the Enzyme Linked Immunosorbent Assay, more commonly known as ELISA, have become a mainstay in veterinary diagnosis over the past decade. The aforementioned and previously established FMD ELISAs provided the basis for FMD antigen and antibody detection.

LPB-ELISA was developed and adopted in different laboratories of the world with improved sensitivity and efficiency for diagnosis of FMD. The assessment of serum for the determination of FMD antibodies was achieved by the FMD Liquid Phase Blocking ELISA (FMD LPB-ELISA).

The project on LPB-ELISA for FMD virus monitoring is carried out as per the protocol followed by Project Directorate on Foot And Mouth Disease, IVRI Campus, Mukteswar. The reagents used are raised and standardized at Project Directorate on Foot And Mouth Disease for sero-monitoring of FMD virus isolates recovered from outbreaks in India.

The complete report is available here:
http://mediafirefile/befpapbmo0sst2t/LIQ...SAY%20.zip

INTRODUCTION
Foot-and-mouth disease (FMD) is a highly contagious vesicular disease affecting more than 33 species of cloven-hoofed animals, including domestic animals such as cattle buffalo, goats, swine and wild animals. The disease has been designated by the Office International des Epizooties, (World Organization of Animal Health) as a notifiable disease that spreads rapidly and requires socioeconomic consideration. FMD outbreaks exert a heavy economic toll due to loss of productivity, decline in product quality, the slaughter of millions of infected and in-contact susceptible animals, and trade embargos on animal products.

The etiological agent of FMD is FMD virus (FMDV), which belongs to the genus Aphthovirus of the family Picornaviridae. The FMDV genome is composed of a positive-sense single-stranded RNA molecule of about 8,500 nucleotides, containing a unique open reading frame. Like other RNA viruses, FMDV is antigenically variable and undergoes rapid mutation. There are seven distinguishable serological types, namely, O, A, C, Asia1, SAT1, SAT2, and SAT3, and more than 65 subtypes.

The Liquid Phase Blocking ELISA (LPBE) is the standard test proposed for use in Post-vaccination Sero-monitoring for FMD. Briefly, the assay methodology requires FMD antigen of a specified serotype to be incubated with test serum which is transferred to an ELISA plate coated with rabbit anti-FMD polyclonal IgG, specific for the serotype under assessment to trap any unbound antigen. The bound FMD antigen in the sample is detected by guinea pig anti-FMD polyclonal IgG, followed by the addition of an anti-guinea pig IgG-HRP conjugate and the substrate. The reaction is stopped by the addition of 1M H2SO4 and the optical density read at 492nm.

The FMD LPBE was employed using the methodological principles described by Hamblin et. al. (1986). The sero-monitoring approach used has provided valuable information on post vaccination outbreaks FMD and provides evidence of reduction of the disease through vaccination. Progress has been made to better map the emergence and spread of strains within the region, and to communicate between risk managers in the veterinary services.

REVIEW OF LITERATURE

FOOT AND MOUTH DISEASE
Foot and Mouth disease is an acute febrile highly contagious disease of cloven-footed animals. The earliest description of what was probably FMD was that by Hieronymi Fracastorii (1546). He described the disease, which occurred in Northern Italy in 1514, as being unusual and affecting only cattle. It is characterized by vesicular eruption in the epithelium of buccal cavity, tongue, nares, muscle, feet, teats, and udder (Fig 1). Lesions may also appear in the rumen pillars. The disease imposed considerable economic stress on the cattle readers due to reduction of milk, meat and working capabilities of dairy and draught animals respectively.

ETIOLOGY
FMD virus (Fig 2) is the first animal virus reported by Loeffler and Frosch in 1897.Valle and Carre (1922) were the first to recognize the antigenic multiplicity in animal viruses. The virus has a small single stranded, positive-sense RNA and belongs to the genus Aphthovirus within the family Picornaviridae. It is considered as the smallest known virus of animal origin. Galloway and Elford (1931) reported the size of the virus as 8-12 by using Gradocol membranes. On account of serological relationship, globally FMDV is divided into seven distinct serotypes and multiple subtypes. They were named as O, A, C, ASIA-1, SAT1, SAT2, SAT3.

In 1922 "O" strain was named for Oise valley in France and "A" strain for Alemand in France. In 1926 another strain could be demonstrated in Germany defined as "C" strain. A strain could also be demonstrated from Pakistan (South-east Asia) in 1954 and was defined as Asia-1.Strains designated as SAT1, SAT2, SAT3 were recognized from Southern African territories during 1934 to 1948.The above strains were isolated from the cattle of Rhodesia and Bechuanaland of South Africa. During recent years the capacity for and frequency of RNA virus variation and the biological significance of this variation in the rapid evolution of RNA viruses have become increasingly evident.

The virus is resistant to various external agents including common disinfectants and usual storage practice. The virus may remain viable to the extent of 1 year in infected premises, 10 to 12 weeks in clothing and feed, 4-5 months in hay and brans, 3 to 4 months in lymph node and bone marrow and fairly one month in hairs of animals. The virus may remain alive up to a month in frozen bull semen. Virus is most stable in pH ranging from 7.4 to 7.6.

HOST SPECIES
Foot and Mouth disease is primarily a disease of domestic and wild cloven hoofed animals, i.e., cattle, antelope, sheep, goats, deer, pigs and camels. However, a wide range of mammal species have been reported to have been infected with the FMDV and reports of clinical disease have been recorded for atleast 12 orders of animals.

The major host species for FMD virus are widely recognized to be domestic cattle, sheep, goats and pigs. The African buffalo is considered to be the definitive host for FMD virus serotype SAT1 SAT2, SAT3. Natural infection with FMD virus can cause serious disease in other species, including elephants and hedgehogs. A wide variety of species including many rodents have been experimentally infected by injection, but considerably fewer species have been infected by natural contact (experimentally or in the field/wild). Horses are accepted to be totally resistant to FMD virus and in general carnivores are resistant.

Cattle and domestic pigs play an important role in the epidemiology of as indicators and amplifiers. Pigs are considered to be amplifier host (pigs serve as amplifiers of the virus because they can excrete aerosols that contain up to 3,000 times more virus than produced by an equal number of cattle or sheep). The disease affects a variety of species of wild animals (Klos & Lang, 1926).

GEOGRAPHICAL DISTRIBUTION
The disease is present in many parts of the world although it has been successfully eliminated or excluded from Western Europe, North America, and Australia. In terms of the frequency of outbreaks, regions, countries, and zones with countries can be classified as endemic, sporadic or free.

TRANSMISSION
All the secretions and excretions like urine, faeces, and saliva remain infective. Virus may remain in the meat and bone. Free living birds may carry the infection from one place to another. Wild ruminants may remain as carrier. Carrier may transfer the virus from animal to animal.

Presence of virus in lesions
With a notable exception of the soft palate and pharynx, where it remains detectable for long periods after apparent recovery, virus can seldom be isolated from the tissue later than a few days after the end of the acute phase. The longest duration of viability in the lesions were: muzzle 7 days; tongue 8 days, palate 11 days; foot 11 days.

Saliva
The greatest concentration of virus occurs in the fluid of the vesicles and in the overlying epithelium, where leakage or rupture causes concentration of saliva; further contamination may result from direct involvement of the salivary glands during the period of viremia, with subsequent proliferation of virus in the secretory cells.

Urine and faeces
Blood borne viruses are soon excreted in the urine and in the faeces, though titres are often variable or low. The virus replicates and is liberated from infections of the kidneys. The faeces may become contaminated directly from blood, bile, or as a result of development of vesicles in the alimentary tract.

Milk
The epitheliotropic nature of FMD virus permits replication in the secretory cells of the mammary gland. Thus, in addition to passive secretion of virus carried out to this organ during the viremic phase, the tissue of the udder begin to produce virus at the rate which soon leads to high concentration in the milk. The hazards of milk borne infection, often commencing during the prodormal phase have been realized for many years.

Vaginal secretion
In addition to secreting virus in body fluids, infected cows may liberate virus in their vaginal secretion.

Semen
The development of extensive programs of artificial insemination (AI) in many countries has resulted in attention being focused on the possibility that large number of female stock might become infected if semen were to be collected for AI from bulls in which the disease has entered the incubative stages.

Miscellaneous
Tears, nasal discharge and membranes of aborted or full term fetuses, and their fluids are likely to contain virus during and immediately after the peak of viremia.

Meat
The virus may be distributed widely in the tissue of animals inadvertently slaughtered during the incubative stages of the disease. It is well established that virus may remain viable in the chilled or frozen carcasses of unvaccinated animals for as long as chilled meat remains marketable and for atleast three months in frozen meat. The virus has long periods of survival, atleast 8 months.

Hides and skins
Virus contaminated hair or wool of the living animal are unlikely to remain viable for periods exceeding 4 weeks .In contaminating the body surfaces of sheep and cattle it was unable to detect survival of virus for periods greater than 20 days.

Air-borne transmission
Air-borne transmission of infection (atmospheric transmission) is a specific category of long distance transmission. Transmission of respiratory and vesicular infections occur indirectly through the air over long distance. The wind may move the air borne droplets containing the virus upto 250km over water and shorter distance over land.

INCUBATION PERIOD
The incubation period of FMD is between 2 and 14 days depending on:
Infectious dose
Strain of virus
Susceptibility of host
Proximity of host between farm spread is characterized by long incubation periods within- farm spread is more rapid and can be as short as 2 days.

PATHOGENESIS
The FMD virus initially settles in the cells of the dorsal surface of the soft palate or on the lateral wall of the pharynx following the entry. But, they do not produce any lesions in the above sites. From the sites they affect the mucous membrane of the mouth cavity and invade the basal layer of the stratified epithelium of the tongue and produce primary vesicles causing pyknotic changes in the nucleus of the cells with leukocytic infiltration. Following penetration of the mucus membrane, virus multiplies in the stratum spinosum where cytolysis takes place giving rise to small cavities in the epithelial layer. These in turn develop into characteristic FMD vesicles. The process continues for about 24 hours and thus a huge quantity of the virus is produced in the vesicles of the tongue. From the primary lesions virus invade the lymphatics and enter the blood circulation and viremia is produced. Again from the blood stream virus goes to the site of predilection that is epithelium of mouth, dental pad, feet and udder where typical vesicles are formed.(Blood & Radostits, 1992). If the lesion is not complicated by the secondary bacterial infections lesions used to heal up within a short span. Secondary bacterial infection may set up extensive damage of the tissue. Damage of feet may yield to loss of horny covering and there may be sloughing of hoof. Udder lesion leading to mastititis is not due to primary vesicles but owes to secondary bacterial invasion. Heart muscles may show acute degeneration of the myocardial fibres in calf. Cardiac musculature will have striked appearance which is termed as tiger heart (Graha, 1959). There is heavy mortality in young calves due to cardiac damage with absence of appreciable vesicular eruption. Some indigenous cattle show superficial brown necrotic lesion of the epithelium with a tendency of rapid healing.

CLINICAL FINDINGS
The disease is not usually fatal. Morbidity is 100% but mortality is less in indigenous cattle and comparatively more in pure and cross bred cattle. The severity of the infection rest on the strains and sub-strains of the virus involved and immunogenic competency of the susceptible host.
Cattle
Pyrexia, anorexia, reduction in milk production.
Rupture of vesicles leaving erosion.
Vesicles can also occur on the mammary glands.
Fever (102-104 F).
Recovery generally occurs within 8-15days.
Myocarditis- Tigroid appearance (death in young one calves )
Sheep
Lesions are less pronounced. Foot lesions may go unrecognized. Lesions in dental padare noticed sheep. Agalactica in milking sheep and goats is a feature. Death of young stock occurs.
Pigs
May develop severe foot lesions particularly when housed on concrete.
High mortality in piglets is a frequent occurrence.