FMD final.ppt
- 1. Foot and Mouth Disease Dr.Bipin Kumar
- 2. •Foot-and-mouth disease virus (FMDV; family Picornaviridae; genus Aphthovirus) causes an acute disease of cloven-hoofed animals characterized by fever, lameness and vesicular lesions of the feet, tongue, snout and teats. • other names: Aphthous fever, Aftosa, Epizootic aphthae
- 3. •The history of FMD may be traced to era of Hieronymus Fracastorius, a monk who described a disease outbreak in 1546 A.D. that occurred in cattle near Verona, Italy •Almost 400 years later, in 1897, Friedrich Loeffler and Paul Frosch demonstrated that a filterable agent is responsible for FMD •foot-and-mouth disease virus (FMDV) is classified within the genus Aphthovirus in the family Picornaviridae
- 4. •The virus exists in the form of seven serologically and genetically distinguishable types, namely, O, A, C, Asia1, SAT1, SAT2, and SAT3, but a large number of subtypes have evolved within each serotype •Type O for Oise in France and type A for Allemagne (Germany) •Later type C was recognized as an additional type in Germany •Pirbright laboratory in England demonstrated 3 novel serotypes of FMDV in sample collected from the FMD outbreak in South Africa and called SAT1, SAT2, and SAT3 •seventh serotype, that is, Asia 1 was first recognized in a sample from Pakistan
- 5. •FMDV is a single stranded (ss) positive sense RNA virus with the whole virus particles having sedimentation coefficient of 146S •The viral genome is translated as a single polyprotein, which is posttranslationally cleaved by viral proteases into four structural proteins (VP1, VP2, VP3, and VP4) and several nonstructural proteins (L, 2A, 2B, 2C, 3A, 3B, 3C, and 3D) •Among the 4 structural polypeptides, VP1 is the most immunogenic protein of FMDV having its G-H loop protruded from the surface , and is maximally exposed on the capsid surface forming large part (54%) of virus surface
- 7. •The highly contagious nature of the virus and severity of economic impacts associated with the disease determine FMD’s status as the most important disease limiting trade of animals and animal products throughout the world •FMD affects extensive areas worldwide and is included in the list of diseases notifiable to the World Organization for Animal Health (http://www.oie.int/eng/en index.htm) • The disease affects domestic cloven- hoofed animals, including cattle, swine, sheep, and goats, as well as more than 70 species of wild animals, including deer, and is characterized by fever, lameness, and vesicular lesions on the tongue, feet, snout, and teats
- 8. Resistance to physical and chemical action: Temperature: •Preserved by refrigeration and freezing •temperature of 70°C for at least 30 minutes inactivates the virus •survive for more than 60 days in bull semen frozen to -79°C (-l lO°F) pH: Quickly inactivated by pH <6.0 or >9.0 Disinfectants: •Inactivated by sodium hydroxide (2%), sodium carbonate (4%), citric acid (0.2%), acetic acid (2%), sodium hypochlorite (3%), potassium peroxymonosulfate/sodium chloride (1%), and chlorine dioxide •Resistant to iodophores, quaternary ammonium compounds, and phenol, especially in the presence of organic matter
- 9. Survival: •Survives in lymph nodes and bone marrow at neutral pH, but destroyed in muscle at pH <6.0 i.e. after rigor mortis •Survives in frozen bone marrow or lymph nodes • Residual virus survives in milk and milk products during regular pasteurisation, but is inactivated by ultra high- temperature pasteurisation • Can persist in contaminated fodder and the environment for up to 1 month
- 10. Geographic Distribution : •Foot and mouth disease is endemic in parts of Asia, Africa, the Middle East and South America •In parts of Africa, virus persistence in wild African buffalo makes eradication unfeasible •North America, New Zealand, Australia, Greenland, Iceland and most of Europe are free of this disease
- 12. •Out of the possible seven, only four serotypes, e.g., ‘O’, ‘A’, ‘C’ and Asia 1 were ever recorded in India. •Serotype ‘C’ too has not been recorded in the country since 1995. Vaccination against FMD is grossly inadequate in the country. •Similarly in the North-Eastern region of India, the frequency of FMD is highest with type “O”, which is always followed by other types including the type “Asia-1” •There are records of about 5,000 outbreaks to occur in India annually affecting nearly three lakh animals with an estimated staggering loss of Rs. 4,300 cores to the economy annually FMD in India
- 13. •In India zoning approach with properly campaigned 6 monthly vaccinations of cattle and buffalo followed by rigorous surveillance and sero-monitoring done to create disease free zones (DFZ) • A trivalent (O,A,Asia 1) vaccine is currently used in vaccination programme • FMD surveillance in India carried out by 8 regional FMD centres and 15 FMD network units and a central FMD laboratory at Muktehwar •Serotyping of the clinical materials is done by using sandwich ELISA FMD in India
- 14. • ELISA negative samples are subjected to multiplex PCR for further diagnosis •Molecular epidemiological analysis based on P1/1D gene sequence and studies of antigenic relationship of the field outbreakstrains with currently used vaccine strains FMD in India
- 15. Regional prevalence: •Maximum proportions (43%) occurred in eastern region •Southern region – 31.5% •North eastern region – 11.6% •Central region – 5% •Western region – 4.4% •Northern region – 4% (S.Subramaniam et al., 2013)
- 16. Serotype prevalence: •Serotype O widely prevalent – 80%, Asia1 and A – 12% and 8% respectively (S.Subramaniam et al., 2013)
- 17. Seasonal prevalence: •FMD outbreaks in India were recorded in all the season •Highest number of outbreaks / cases recorded in the month of September (63.4± 18.4) – end of monsoon •Lowest in June (23.6± 11.1) – start of monsoon •High relative humidity and heavy rain during rainy season inhibit aerosol transmission •Maximum incidences occurred at the end of the monsoon and post-monsoon season – dry weather and moderate RH
- 18. Emergence and re-emrgence of different genotypes: •In serotype O - six genetic groups of virus ( Branch A,B,C-I,C-II, C-III and C-IV) •The currently used vaccine strain (INDR2/1975) belongs to lineage branch B •Serotype A – 26 global genotypes ( 2, 10, 16 and 18 were recorded in India) •Serotype Asia 1 – three lineages (B,C,D)
- 20. Monthly incidence of FMD outbreaks in India
- 21. Transmission: •by inhalation •by ingestion •Carriers (cattle, sheep and goats may become carriers, but pigs do not) •Wild fauna may serve as FMD reservoir •Humans are often a vehicle for transmission •movement of infected animals, or indirectly by the transportation of virus on inanimate objects
- 22. Factors involves in aerosol transmission: •sufficient virus particles •strength of the virus source (infected pigs excrete 1000-3000 times) • the wind speed • Humidity (critical RH range of 55-60%) • diurnal variation • sea over land (60 km over land 250 km over sea)
- 24. Animated FMD virus plumes. Red squares indicate for the positions of infected farms, brown points for the more than 1000 susceptible farms in the 25 km x 25 km model domain. Yellow particles are located in the lowest 10 m above ground. Green, cyan, blue, magenta and black points mark viruses at higher vertical layers (upper panel). Risk areas for cattle (green), sheep (blue) and swine (red) based on critical FMD virus concentrations (lower panel).
- 25. Stages of FMD Pathogenesis in Cattle 1. Pre-viraemia: the period from when an animal is first infected with FMDV until virus is first detected within the intravascular (i.e. blood) compartment with a sustained and quantitatively increasing trend (determined by virus isolation (VI) or detection of viral RNA) 2. Establishment of viraemia: temporally occurs within pre- viraemia. However, because of the importance of this transition, it will be treated as a distinct event 3. Viraemia: the period during which FMDV can be detected within the intravascular compartment with kinetics suggestive of active viral replication (determined by VI or detection of viral RNA). This period typically coincides with the clinical phase of disease.
- 26. 4. Post-viraemia: the period following viraemia starting with the first negative assay on blood (determined by VI or detection of viral RNA) which includes: (i) Resolution of clinical signs (ii) Short-term persistence of infectious virus, antigen and/or RNA in specific tissues (iii) Persistent infection (carrier state): the period after 28 dpi in which infectious FMDV may be detected on at least one of multiple oesophageal–pharyngeal (OP) samples (as defined by OIE) (iv) Chronic long-term sequelae including hirsutism, heat-intolerance (panting) and thyroid dysfunction, which have been reported in recovered cattle
- 28. Pathogenesis in Swine: •Pigs are the second most commonly studied subjects for FMD pathogenesis research • Recent and historic reviews have supported the notions that compared to cattle, pigs are more refractory to aerogenous infection, more susceptible to infection via the gastrointestinal route and generate greater quantities of aerosolized virus
- 29. Clinical signs: Cattle: •there is an incubation period of 3-6 d •fall in milk yield and a high fever (40-41"C; 104- 106°F) •Acute painful stomatitis •abundant salivation, the saliva hanging in long, ropy strings, a characteristic smacking of the lips •Vesicles and bullae (1-2 cm in diameter) appear on the buccal mucosa, dental pad and tongue •vesicles appear on the feet, particularly in the clefts and on the coronet •lame, often recumbent, with a marked, painful swelling of the coronet
- 31. Sequelae to FMD: • complex of clinical signs variably referred to as ‘heat-intolerance syndrome (HIS)’, ‘hairy panters’, ‘asoleadas’ (in Argentina) and ‘peludas’ (in Brazil; M. Hugh-Jones, personal communication) • intolerance to increased environmental temperatures •Pronounced panting during hot weather is the striking feature accompanied by increased body temperature and pulse rate (Minett, 1948; Maqsood et al., 1958) •abnormal hair growth described as either hirsutism (Ghanem and Abdel-Hamid, 2010) or • hypertrichosis as a result of failure of cyclic seasonal shedding of hair (Minett, 1948; Maqsood et al., 1958) •anaemia, hyperphosphataemia, lactic acidosis, hypoproteinaemia, and hypocalcaemia (Mullick, 1949)
- 32. •serum Na, Cl, Mg, Zn, Fe, albumin, cholesterol, and cortisol were significantly reduced •Serum levels of ALT, AST, and monoaldehyde were significantly increased in affected cows •serum glucose was also significantly increased although hyperglycaemia was considered mild and below the cut-off for the diagnosis of diabetes mellitus in affected cattle •The morphology was generally consistent with thyroid hyperplasia and suggestive of hyperthyroidism •Disruption of these endocrine organs could, in part, account for the biochemical changes reported by Ghanem and Abdel- Hamid in heat intolerant cattle and the hyperglycaemia and hypoinsulinaemia detected during clinical FMD (Yeotikar et al., 2003; Ghanem and Abdel- Hamid, 2010; Nahed, 2010)
- 33. •Proper assessment of thyroid function requires evaluation of serum T3/T4 levels in the context of serum TSH and TRH •As such data have never been reported for HIS cattle, the relationship between HIS and thyroid function remains undetermined •Early investigators’ efforts to elucidate the functional derangements of HIS have included administration of thyroxin which aggravated the symptoms (Mullick, 1949) and administration of thiouracil which alleviated the symptoms (Maqsood et al., 1958).
- 34. Myotropism of FMDV • a malignant form of the disease in adults in which acute myocardial failure occur •typical course initially but a sudden relapse occurs on days 5-6 with dyspnea, a weak and irregular heart action, and death during convulsions •myocardial lesions in cattle which died shortly after infection, including inflammatory infiltrates and cardiomyocyte degeneration and necrosis (Huguenin, 1924) •In addition to young cattle, FMDV- associated myocardial necrosis and myocarditis has been documented in pigs (Potel, 1967), sheep (Salyi, 1939), goats (Bhalla and Sharma, 1965), adult cattle, gazelle (Shimshony, et al., 1986)
- 35. Diagnosis: • determination of the type of the virus involved and to differentiate the disease from vesicular stomatitis, vesicular exanthema and swine vesicular disease •Fresh vesicular fluid and surrounding epithelial tissue should be collected in glycerol-saline for laboratory tests •blood should be collected, along with esophageal-pharyngeal (OP) fluid samples from ruminants or throat swabs from pigs
- 36. Principle of FMD diagnosis:
- 37. 1. Identification of the agent in tissue or fluid (a)Virus isolation (b) Immunological methods: (ELISA, CFT, Nucleic acid recognition methods) (c) Virus morphology by electron microscopy 2. Serological tests for specific antibody response to FMD structural or nonstructural proteins: Virus neutralization, Solid- phase competitive ELISA, Liquid-phase blocking ELISA 3. Experimental transmission Principle of FMD diagnosis:
- 38. Virus Isolation: •by inoculation into cell cultures or unweaned mice •The FMD virus is cultivable on tissue culture and in hen eggs •Primary cell culture of bovine, ovine and porcine origin has exhibited susceptibility to FMDV from infected tissues •Some stable cell lines, like IBRS-2, MVPK-1 clone 7 , LFBK cell line and BHK-21 are also susceptible to FMDV and so are most desirable for diagnostic system •the cell culture system is laborious, time consuming, and relatively low sensitive • It also requires careful handling of specimens and a biosafety laboratory
- 39. Complement Fixation Test (CFT): •used extensively for distinguishing different strains of FMDV • CFT was a fast method it needed high virus load and results were sometimes affected by pro-and anti-complementary activities of the test sample •uses the activation of the classical complement pathway by antibody to antigen complexes •affected by pro- or anti-complementary factors
- 40. Enzyme-Linked Immunosorbent Assay (ELISA): •ELISA and its various modifications were applied for detection, typing, and strain differentiation of FMDV isolates with better sensitivity than CFT •At the FAO/WRL for FMD, the preferred procedure for the detection of FMDV antigen and identification of viral serotypes is ELISA •a sandwich ELISA using convalescent bovine immunoglobulin (Igs) as capture •LPBE is being used for the detection of antibody titers against the FMD vaccinated animals
- 41. Reverse Transcription-Polymerase Chain Reaction (RTPCR): •RT-PCR to amplify RNA targets - a reliable tool for FMD diagnosis •A specific RT-PCR was developed and validated for the detection of the polymerase gene (3D) of FMD with an analytical sensitivity equal to 1000 times higher than that of a single passage virus isolation
- 42. Multiplex Polymerase Chain Reaction (mPCR): •variant of the test in which more than one target sequence is amplified using more than one pair of primers •mPCR was used for the detection and differentiation of multiple pathogens/different strains of the same pathogen •the multiplex system is designed to survey multiple regions of the genome simultaneously, thereby increasing the probability of detection The test result by multiplex-RT-PCR. Line 1 and line 2 were two negative samples; line 3 was positive sample; M was DNA marker ladder
- 43. Real-Time Polymerase Chain Reaction: •Real-time PCR assays recommended by the World Organization for animal health (OIE) for detection of FMDV incorporate universal primers and fluorescent-labeled probes that recognized conserved region within the 5 UTR or conserved gene regions within the RNA-dependent RNA polymerase gene (3Dpol) • The use of a specific probe facilitates an increase in specificity compared to conventional agarose-gel-based PCR assays •the problem of carry-over is significantly reduced because of the real-time measuring principle, which is based on closed tube system
- 44. Recombinant Antigen-Based Diagnosis: •The 3AB protein of FMDV was expressed in E. coli or in P. pastoris and has been used for the diagnosis of FMD infection in cattle •Similarly, 3ABC proteins expressed in heterologous systems were used in ELISA (3ABC ELISA) for serodiagnosis of FMD
- 45. DIVA-Based Companion Diagnostic Approach: The ability to identify and selectively delete genes from a pathogen has allowed the development of “marker vaccines” that, combined with suitable diagnostic assays, allow differentiating infected from vaccinated animals (DIVA) by differentiation of antibody responses induced by the vaccine (no antibodies generated to deleted genes) from those induced during infection with the wild-type virus
- 46. Microarray-Based Diagnosis of FMDV: •DNAmicroarrays are becoming increasingly useful for the analysis of gene expression and single nucleotide polymorphisms (SNPs)
- 47. •A rapid test which gives results in 5-10 minutes of time •A laboratory based test- ELISA which gives results in 3 hours •Both the tests can be used to detect FMD- NSP antibodies in all species of animals susceptible to FMD. Recent technology by IVRI:
- 48. Necropsy finding: •vesicles and erosions in the mouth and on the feet and udder •vesicles may extend to the pharynx, esophagus, fore stomachs, and intestines as well as trachea and bronchi •Ventricular walls appear streaked with patches of yellow tissue interspersed with apparently , normal myocardium, giving the typical 'tiger heart' appearance •heart is enlarged and flabby Focal myocarditis, 'tiger striping' on the heart of a 4-week old piglet which died due to FMD.
- 49. •homeopathic preparation of Tarentula cubensis (Theranekron®) was conducted during an outbreak of FMD in cattle in Iran •Ampicillinand Cloxacillin combination @ 10 mg/kg body weightbid intramuscularly for 3 days •Enrofloxacin @ 5 mg/kgsid intramuscularly for 3 days •Oxytetracycline @ 10 mg/kgsid intramuscularly for 3 days •Gentamicin @ 2.5 mg/kg bidintramuscularly for 3 days and • Potassium permanganate(1ppm) on the lesions •local application of antiseptic solutions viz., 2% NaOH, 2% NaHCO 3 , 4%Na 2CO3 and 5% Povidon iodine respectively •Meloxicam and paracetamol combination (Melonexplus, Intas) @ 1ml/25 kg •Administration of flunixin meglumine Treatment:
- 50. Control: (a) control by eradication and (b) control by vaccination, or (c) a combination of the two •all cloven-footed animals in the exposed groups should be immediately slaughtered and burned or buried on site •proper disinfection of human clothing, motor vehicles and farm machinery • Barns and small yards must be cleaned and disinfected with 1-2 % sodium hydroxide or formalin or 4% sodium carbonate solution •the farm should be left unstocked for 6 months and restocking permitted only when 'sentinel' test animals are introduced and remain uninfected •Immediate quarantine must be imposed on all farms within a radius of 16-24 km (10-15 miles) of the outbreak
- 51. Vaccination: •Killed trivalent (containing 0, A, and C strains) vaccines are in general use •Locally isolated virus is becoming a more common practice formalin, binary ethylene immine (BEl) •oil-adjuvanted induce higher levels of antibody than aluminum hydroxide gel-saponin adjuvanted vaccines •Attenuated vaccines have been produced by passage through white mice, embryonated hen eggs, rabbits and tissue culture •genetically engineered FMD vaccine polypeptide vaccine (protein VPl)
- 52. Alternatives to general vaccination are modified programs including 'ring‘ vaccination to contain outbreaks/frontier‘ vaccination to produce a buffer area between infected and free countries and vaccination of selected herds on a voluntary basis when an outbreak is threatened
- 53. •high contagiousness, •wide geographical distribution, •broad host range, •ability to establish carrier status, •Antigenic diversity leading to poor cross-immunity, and •relatively short duration of immunity The main constraints in controlling this disease and why it is considered as the most dreaded viral disease CONCLUSION:
- 54. THANK YOU VERY MUCH