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Bovine Ephemeral Fever & Three day sickness
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  • 1. Some Studies on Bovine Ephemeral FeverSome Studies on Bovine Ephemeral Fever Mohamed Zakaria Sayed-AhmedMohamed Zakaria Sayed-Ahmed For the Degree of Master in Vet. Med. Science
  • 2. Acute infectious arthropod-born viral diseaseAcute infectious arthropod-born viral disease Acute diseaseAcute disease Transient fluctuating feverTransient fluctuating fever Muscular shiveringMuscular shivering Stiffness & lamenessStiffness & lameness Enlargement of the peripheral lymphnodesEnlargement of the peripheral lymphnodes Short course and spontaneous recoveryShort course and spontaneous recovery BackgroundBackground Bovine Ephemeral Fever -Bovine Ephemeral Fever - BEFBEF Ephemerovirus, RhabdoviridaeEphemerovirus, Rhabdoviridae Cattle & Water buffaloesCattle & Water buffaloes
  • 3. Tropical and subtropical areas of AfricaTropical and subtropical areas of Africa Middle EastMiddle East AsiaAsia AustraliaAustralia Reported for 1st time in Egypt by Piot, 1896Reported for 1st time in Egypt by Piot, 1896 Described clinically by Rabagliati, 1924Described clinically by Rabagliati, 1924 Progressively moved down the Nile valley from Aswan to CairoProgressively moved down the Nile valley from Aswan to Cairo Summer monthsSummer months Further outbreaks in 1991, 2000, 2001 and 2004Further outbreaks in 1991, 2000, 2001 and 2004 EpidemiologyEpidemiology HistoryHistory in Egyptin Egypt
  • 4. Vector bornVector born - Culicoides, Culex and Anopheles- Culicoides, Culex and Anopheles mosquitoesmosquitoes Blood transmissionBlood transmission Small amount of infected blood can be transmitted theSmall amount of infected blood can be transmitted the disease through intravenous inoculationdisease through intravenous inoculation Close contactClose contact,, body secretions, or aerosol droplets dobody secretions, or aerosol droplets do not transmit BEF virusnot transmit BEF virus The virus does notThe virus does not seem to be transmitted in semenseem to be transmitted in semen and is rapidly inactivated in meatand is rapidly inactivated in meat TransmissionTransmission
  • 5. Inoculation of the suspected buffy coatInoculation of the suspected buffy coat intracerebrally inintracerebrally in suckling baby mice, Vero, BHK21 cellssuckling baby mice, Vero, BHK21 cells Rapid diagnosis by IFT, ELISA and PCRRapid diagnosis by IFT, ELISA and PCR Short courseShort course Spontaneous recoverySpontaneous recovery High economic lossesHigh economic losses Drop in production in dairy herdsDrop in production in dairy herds Production in animal conditionProduction in animal condition Disruption of national and international tradeDisruption of national and international trade Finally a verity of complications and deathsFinally a verity of complications and deaths Clinical signsClinical signs DiagnosisDiagnosis
  • 6. Various vaccines have been produced in:Various vaccines have been produced in: -- South AfricaSouth Africa -- JapanJapan -- AustraliaAustralia -- EgyptEgypt in trials to control the diseasein trials to control the disease VaccinationVaccination
  • 7. The present study was designed toThe present study was designed to:: Investigate the presence of BEF in cattle throughInvestigate the presence of BEF in cattle through serosurvay on randomly collected serum samplesserosurvay on randomly collected serum samples Isolate BEF virus from suspected naturally infected cattleIsolate BEF virus from suspected naturally infected cattle Estimate the immune response of cattle to locally producedEstimate the immune response of cattle to locally produced live and inactivated BEF vaccineslive and inactivated BEF vaccines Follow up the maternal immunity induced in calves bornFollow up the maternal immunity induced in calves born to vaccinated dams with inactivated BEF vaccineto vaccinated dams with inactivated BEF vaccine Study some biochemical parameters in naturally infectedStudy some biochemical parameters in naturally infected &&vaccinated cattle with locally produced live andvaccinated cattle with locally produced live and inactivated BEF vaccineinactivated BEF vaccine
  • 8. AnimalsAnimals CattleCattle A- Clinical investigated animalsA- Clinical investigated animals Before outbreaks of 2004Before outbreaks of 2004 A total of 83 mixed breed cattle of differentA total of 83 mixed breed cattle of different agesages apparently healthy by clinical examapparently healthy by clinical exam.. Located in Dakahlia and Damietta governorates were investigated for neutralizing antibodies of BEF in their serum
  • 9. During outbreaks of 2004During outbreaks of 2004 AA total of 305 mixed breed cattle of differenttotal of 305 mixed breed cattle of different ages Located in Dakahlia and Damiettaages Located in Dakahlia and Damietta governorates were clinically investigated forgovernorates were clinically investigated for:: Body temperatureBody temperature Nasal and ocular dischargeNasal and ocular discharge SalivationSalivation StiffnessStiffness LamenessLameness Epidemiological pattern - age, sex and seasonEpidemiological pattern - age, sex and season
  • 10. B-B- Experimental vaccinating cattleExperimental vaccinating cattle:: Sixteen mixed breed cattleSixteen mixed breed cattle Nine cross breed bullsNine cross breed bulls Used For studies the antibodies titer against inactivated and liveUsed For studies the antibodies titer against inactivated and live attenuated BEF vaccineattenuated BEF vaccine
  • 11. Experimental vaccinating cattleExperimental vaccinating cattle:: Sixteen mixed breed cattle For studies the neutralizing BEF antibodies in their sera and maternal immunity for their offspring's Seven pregnant cattle in late stage of pregnancy
  • 12. The experiment was applied during June 2003 and ended atThe experiment was applied during June 2003 and ended at December 2003December 2003 All animals were screened using SNT and found to be freeAll animals were screened using SNT and found to be free from BEF antibodies before vaccinationfrom BEF antibodies before vaccination All animals were kept under observation for six months in silkAll animals were kept under observation for six months in silk screened house and receiving balanced diet and adequate waterscreened house and receiving balanced diet and adequate water
  • 13. 310310suckling mice of two to three days old were used for trialssuckling mice of two to three days old were used for trials of BEF virus isolation from naturally suspected cattle during theof BEF virus isolation from naturally suspected cattle during the summer of 2004summer of 2004 Suckling mice
  • 14. SamplesSamples Buffy coatBuffy coat During outbreak of 2004, 62 buffy coat samples fromDuring outbreak of 2004, 62 buffy coat samples from clinically suspected cattle were obtained at viremic stageclinically suspected cattle were obtained at viremic stage From Dakahlia 41From Dakahlia 41 From Damieta 21From Damieta 21 Collected from clinically suspected cattleCollected from clinically suspected cattle
  • 15. Serum samples from field animalsSerum samples from field animals During summer of 2003, 83 serum samples wereDuring summer of 2003, 83 serum samples were obtained randomly from clinically normal cattle to detectobtained randomly from clinically normal cattle to detect BEF antibodies in the sera of such animalsBEF antibodies in the sera of such animals Dakahlia 55Dakahlia 55 Damietta 28Damietta 28
  • 16. Serum samples for biochemicalSerum samples for biochemical analysisanalysis Inactivated BEF vaccine Live attenuated BEF vaccine Booster with inactivated 4 weeks post first dose and from naturally infected animals Serum samples were obtained from vaccinated nimal with
  • 17. Serum samples from experimentallySerum samples from experimentally vaccinated cattlevaccinated cattle Weekly post vaccination up to 4 weeks and then monthly upWeekly post vaccination up to 4 weeks and then monthly up to 6 months to follow up the level of induced BEF neutralizingto 6 months to follow up the level of induced BEF neutralizing antibodiesantibodies Also serum samples were obtained from the calves born toAlso serum samples were obtained from the calves born to the vaccinated cows weekly up to 4 weeks and then monthlythe vaccinated cows weekly up to 4 weeks and then monthly up to 6 months of ageup to 6 months of age Serum samples obtained from vaccinated animalsSerum samples obtained from vaccinated animals
  • 18. Bovine ephemeral fever vaccinesBovine ephemeral fever vaccines Both live attenuated and inactivated cell culture BEF vaccine were used in the present study were supplied kindly by the Vet . Ser. Vac. Res. Inst. Abbasia, Cairo These vaccines were used to vaccinate the experimental cattle The local strain of the BEF virus, BEF-AVS, adapted to BHK cell culture supplied kindly by the Department of Pet Animal Vaccine Research, Vet. Ser. Vac. Res. Inst. Abbasia, Cairo. It had a titer of 10-7 TICD50 per ml
  • 19. Hyper immune seraHyper immune sera Hyper immune sera was prepared locally by inoculation of rabbits with BEF virus ConjugateConjugate Anti-bovine non-conjugated and conjugated with fluorescence isothiocyanate against the local strain of BEF virus were prepared and supplied by Department of Pet Animal Vaccine Research
  • 20. Baby hamster kidney (BHK 21) cellsBaby hamster kidney (BHK 21) cells It was supplied kindly by the department of pet animal vaccine research, vet. ser. vac. res. inst. abbasia, Cairo, Cell mono layers were grown in Eagle's minimum essential media (MEM( Cell culture infectionCell culture infection Cell culture media and solutionsCell culture media and solutions Hank's balanced salt solution- HBSSHank's balanced salt solution- HBSS It was prepared according to Hank and Wallace,1949 and used in the process of cell culture passages as diluents for the stock trypsine solution and in the serum neutralization test as diluents for serum samples
  • 21. Trypsin was used as a dispersing agent for the cell culture lines Trypsine solutionTrypsine solution Salt solution of sodium bicarbonate 4.4% was prepared & sterilized by autoclaving and kept at 4◦c2 Bicarbonate solutionBicarbonate solution It was prepared as 100 x soln by dissolving 1gm of streptomycin and 1 million I.U of penicillin G.sodium in 100 ml of HBSS. 1% of such solution was added to each 100 ml of any cell culture solution to yield a final concentration of 100 mg of streptomycin and 100 i.u of penicillin-G-sodium in each ml of the cell culture solution Antibiotic solutionAntibiotic solution
  • 22. Newly born calf serum- mycoplasma and virus screened was supplied by Gibco limited, Paisley, scuttled, UK Bovine serumBovine serum Test kits- Diamond Co. were used to estimate serum total protein, albumin and globulin. Also serum calcium and phosphorus in the sera of in naturally infected cattle and vaccinated with locally produced live and inactivated BEF vaccines ChemicalsChemicals MEM was supplied by Gibco and prepared according to the manufacture directions and used for BHK21 cell cultures Minimum Essential Medium- MEMMinimum Essential Medium- MEM
  • 23. Clinical examination 305mixed breed cattle of different ages located in Dakahlia and Damietta governorates were clinically examined followed the direction of Kelly (1990(
  • 24. Virus isolation and identification The buffy coats obtained from suspected animals were inoculated intra- cerebrally at the dose of 0.01 ml / suckling mouse The inoculated baby suckling mice were kept under observation for one week post inoculation Inoculation of cell culture The suspected Buffy coats were inoculated in BHK21 cell culture to demonstrate the CPE of the BEF virus Mice inoculation test Direct Fluorescent Antibody Technique (FAT) It was carried out on the obtained buffy coats of suspected animals and impression smears of baby mice brain and inoculated BHK21 cell line . Positive results were detected by the appearance of fluorescence apple green color
  • 25. Agar gel precipitation test, AGPT It was carried out on the experimentally infected mice brain which showed positive signs of bovine ephemeral fever The test was applied to estimate the titer of BEF neutralizing antibodies during serosurvay and in vaccinated animals Serum Neutralization Test, SNT
  • 26. Vaccination Nine cross breed bulls Sixteen mixed breed cattle seven pregnant cattle at last stage of pregnancy 5pregnant cattle injected by inactivated BEF vaccine at last month of pregnancy in two doses 2ml/sc with two weeks intervals. The 2nd dose before expected parturition by 2 weeks 3animals Kept as control 3animals Injected the 1st dose by live attenuated BEF vaccine 2ml/sc and the 2nd dose by inactivated BEF vaccine with four week in between 3animals Injected by inactivated BEF vaccine in two doses 2ml/sc with two weeks intervals 2animals Kept as control
  • 27. The blood samples were collected from all animals in the 5 groups weekly after the 1st dose up to 4 weeks and then monthly up to 6 months The borned calves were subjected to blood collection after 1st week of life up to 4 wks and then monthly up to 6 months old Estimation of biochemical parameters in naturally infected cattle and vaccinated with locally produced live and inactivated BEF vaccines spectrophotometrically by using of special kits
  • 28. Symptoms No. of animals % Fever No. of clinically suspected animals = 62 62 100 Inappetance& Anorexia 62 100 Nasal discharge & salivation 42 67.74 Stiffness and lameness 11 18 Respiratory distress 4 6 Subcutaneous emphysema 2 3 Recumbancy 3 5 Clinical and epidemiological investigation of BEF suscepected cattle
  • 29. Subcutaneous emphysemaNasal discharge Recumbency
  • 30. Age group NO. of affected animals % Less than one year No. of clinically suscepected animals = 62 4 6.45 One < Two years 28 45.16 Two <Three years 25 40.32 Three < Four years 5 8.07 Incidence of BEF in relation to age of animals
  • 31. Incidence of BEF in relation to different ages 0 5 10 15 20 25 30 35 40 45 50 <1year 1-2 y 2-3 y 3-4 y % Age group 45.16% 40.32% 8.07% 6.45%
  • 32. Morbidity and mortality rates due to BEF during outbreak of 2004 No. of examined animals Morbidity rate Mortality rate Number % Number % 305 62 20.33 3 4.84
  • 33. Virus isolation on baby mice and tissue culture Locality NO. of examined samples Number of +ve Baby mice inoculation BHK21 inoculation Dakahlia Dekrness 9 5 5 Mansoura 10 5 5 Belkas 22 16 16 Damietta 21 14 14 Total number 62 40 40 Total + ve % 64.4 64.4
  • 34. Identification of the obtained isolates by VNT, FAT and AGPT NO. of Isolated virus Applied test VNT FAT AGPT 40 40 40 31 % 100 100 77.50
  • 35. Antibody titre = the reciprocal of the final serum dilution which neutralized and inhibited the CPE of 100-200 TCID50 of BEF virus Investigation of BEF neutralizing antibodies in examined cattle sera
  • 36. Antibody titre against BEF antibodies by SNT 0 2 4 6 8 10 12 14 16 18 20 NO. of examined animals 1/2 1/4 1/8 1/16 1/32 Antibody titre 19 16 14 11
  • 37. Mean BEF neutralizing antibody titre in vaccinated individual bulls (Group I( Animal No Time Control I II III Mean Prevaccination 0 0 0 0 0 1st dose 1week 0 4 8 4 5.34 2 week 0 16 16 8 13.33 2nd dose 3 week 0 32 16 16 21.33 1 month 0 64 32 32 42.67 2 month 0 32 64 32 42.67 3 month 0 32 64 32 42.67 4 month 0 16 32 64 37.33 5 month 0 32 32 64 35.5 6 month 0 32 32 32 32
  • 38. Mean BEF neutralizing antibody titre in vaccinated individual bulls (Group I( 2W 3W 1M 2M 3M 4M 5M 6M Antibody titre mean Time
  • 39. Mean BEF neutralizing antibody titre in vaccinated individual bulls (Group II( Animal NO Time Control I II III Mean Prevaccination 0 0 0 0 0 1st dose with live attenuated BEF vaccine 1week 0 4 8 8 6.67 2 week 0 8 16 16 13.34 3 week 0 32 32 32 32 4 week 0 64 64 64 64 Booster dose with inactivated BEF vaccine 5week 0 128 128 128 128 6week 0 128 128 128 128 8 month 0 128 64 64 128 12 month 0 64 64 64 64 16 month 0 64 64 64 64
  • 40. Mean BEF neutralizing antibody titre in vaccinated individual bulls (Group II( 0 10 20 30 40 50 60 70 80 90 100 110 120 130 Antibody titre mean 1W 2W 3W 4W 5W 6W 8W 12W 16W Time
  • 41. Mean BEF neutralizing antibodies titre in vaccinated individual cows with BEF inactivated vaccine Animal NO. Time Pregnant cattle number Control I II III IV V Mean Prevaccination 0 0 0 0 0 0 0 1st Dose 1week 0 4 4 8 4 4 4.8 2week 0 8 16 16 8 16 6.6 2nd Dose 3week 0 32 32 16 16 32 21.3 1month 0 64 64 32 32 46 42.6 Period post parturition 2 month 0 32 32 64 32 32 53.3 3 month 0 32 32 32 32 16 42.6 4 month 0 32 16 64 64 64 32 5 month 0 32 32 32 64 32 26.6 6 month 0 32 32 32 32 32 21.3
  • 42. Mean BEF neutralizing antibody titer in vaccinated individual pregnant cattle Abtitermea 1st dose 2nd dose Parturition 3M2 W 4M 5M 6M1W 1 M3 W 2M Time
  • 43. Mean BEF neutralizing antibody titer in vaccinated individual pregnant cattle Antibody titer= the reciprocal of the final serum dilution which neutralized and inhibited the CPE of 100-200 TCID50 of BEF virus
  • 44. Mean maternal BEF neutralizing antibody titers in calves born to vaccinated dams 0 10 20 30 40 50 60 70 1W 2W 3W 1M 2M 3M 4M 5M 6M Abtitermean Time
  • 45. Mean ±SD values of serum total protein, albumin, globulin values and A/G ratio in diseased cattle with BEF Animals Items Diseased animals with BEF Two weeks after recovery Controlled animals T.protrin (gm/dl) 7.48 ± 0.1 * 7.46 ± 0.13 * 7.45 ± 0.7 * Albumin (gm/dl) 3.33 ± 0.1 * 3.32 ± 0.3 * 3.34 ± 0.4 * Globulin (gm/dl) 4.13 ± 0.6 * 4.14 ± 0.10 * 4.11 ± 0.36 * A/G 0.74 ± 0.21 * 0.75 ± 0.12 * 0.76 ± 0.2 * Significant different at P < 0.01 using ANOVA test The mean ±SD carry the same letter in the same row have no significance difference
  • 46. Mean ±SD values of serum total protein values, albumin, globulin and A/G ratio in animals vaccinated with locally produced live attenuated and inactivated BEF vaccines Significant different at P < 0.01 using ANOVA test The mean ±SD carry the same letter in the same row have no significance difference Animals Items Live attenuated booster with inactivated Inactivated BEF vaccine Controlled animals 1st week 2nd week 1st week 2nd week T.protrin (gm/dl) 7.82 ± 0.64 * 7.97 ± 0.23 # 7.60 ± 0.44 * 7.75 ± 0.41 # 7. 54 ± 0. 20 * Albumin (gm/dl) 3.52 ± 0.22 * 3.47 ± 0.16 # 3.31 ± 0.32 * 3.41 ± 0.11 # 3.13 ± 0.12 * Globulin (gm/dl) 4.30 ± 0.10 * 4.50 ± 0.45 # 4.29 ± 0.26 * 4.34 ± 0.21 # 4.29 ± 0.13 * A/G 0.72 ± 0.17 * 0.61 ± 0.12 # 0. 69 ± 0.17 * 0. 65 ± 0.17 # 0.71 ± 0.17 *
  • 47. Mean ±SD values of serum calcium and phosphorus in diseased cattle with BEF and two weeks after recovery Significant different at P < 0.01 using ANOVA test The mean ±SD carry the same letter in the same row have no significance difference Animals Items Diseased animals with BEF Two weeks after recovery Controlled animals Calcium (mg/dl) 5.74 ± 0.5 # 8.13 ± 0.13 * 8.10 ± 0.19 * Phosphorus (mg/dl) 2.78 ± 0.14 # 4.12 ± 0.3 * 4.14 ± 0.07 *
  • 48. Mean ±SD values of serum calcium and phosphorus in animals vaccinated with locally produced live attenuated and inactivated BEF vaccines Significant different at P < 0.01 using ANOVA test The mean ±SD carry the same letter in the same row have no significance difference Animals Items Live attenuated booster with inactivated Inactivated BEF vaccine Controlled animals 1st week 2nd week 1st week 2nd week Calcium (mg/dl) 7.97 ± 0.62 * 7.45 ± 0.41 # 8.07 ± 0. 1 * 8.09 ± 0.31 * 8.10 ± 0.19 * Phosphorus (mg/dl) 3.95± 0.20 * 3.45 ± 0.12 # 4.10 ± 0.30 * 4.12 ± 0.21 * 4.14 ± 0.07 *
  • 49. From the obtained result in the present work, it was concluded that: Regular serosurvay to farm animals to estimate the neutralizing antibodies to control the BEF in Egypt and decrease the severity of outbreaks All animals above 6 months old should be vaccinated FAT more sensitive and rapid test for diagnosis of BEF Live attenuated BEF vaccine boostered with inactivated one is more potent and save the time and money ll dams should be boostered before parturition to maximizing passive immunization Hypocalcaemia is the common cause of recumbancy, so calcium therapy is indicated