2. Bovine Neosporosis Benjamin (BJ) Newcomer, DVM bwn0001@auburn.edu February 6, 2009

3. Outline Background Background Life cycle Life cycle Transmission Transmission Clinical presentation Clinical presentation Diagnostics Diagnostics Prevention/control Prevention/control

4. History First recognized in dogs in 1984 First recognized in dogs in 1984 –encephalomyelitis and myositis in 6 boxer puppies Onset: 2-6 mo old Onset: 2-6 mo old Extensive inflamm. Extensive inflamm. lesions in CNS and skeletal muscle Neg. toxo titers Neg. toxo titers

5. History First associated with cattle in 1987 abortion storm First associated with cattle in 1987 abortion storm –29 abortions over 5 months 1987 - weak calf born 1987 - weak calf born assoc with protozoan dis. assoc with protozoan dis. –negative for T. gondii –negative for Sarcocystis sp.

6. History In 1988, determined to be a new genus and species In 1988, determined to be a new genus and species –Neospora caninum Often misdiagnosed as Toxoplasma gondii Often misdiagnosed as Toxoplasma gondii Stillborn calf in 1974 Stillborn calf in 1974 Toxoplasma

7. Affected species Definitive host Multiple abortions N. caninum T. gondii

8. Distribution Found in 6 continents Found in 6 continents Endemic in CA since 1984 Endemic in CA since 1984 Primary abortifacient Primary abortifacient –California –New Zealand –The Netherlands

9. Life cycle Typified by 3 infectious stages Typified by 3 infectious stages –oocysts –tachyzoites –tissue cysts (bradyzoites) Definitive hosts: Definitive hosts: –dogs –coyotes –other canids also?

10. Life cycle Definitive hosts shed unsporulated oocysts >4 days after ingestion Definitive hosts shed unsporulated oocysts >4 days after ingestion Variable shedding Variable shedding –Up to 4.5x10 6 oocysts/gram of feces –1 day to 4 months

11. Life Cycle Puppies shed higher # oocysts Puppies shed higher # oocysts Shedding: bovine cysts > murine cysts Shedding: bovine cysts > murine cysts Corticosteroids increase shedding Corticosteroids increase shedding –immunocompromised dogs?

12. Life Cycle After 1-3 days, oocysts sporulate After 1-3 days, oocysts sporulate –form two sporocysts –each contain 4 sporozoites

13. Life cycle Sporulated oocysts ingested by intermediate hosts (cattle) Sporulated oocysts ingested by intermediate hosts (cattle) Sporozoites released in GIT and penetrate GIT lining Sporozoites released in GIT and penetrate GIT lining

14. Life cycle Tachyzoites spread throughout body Tachyzoites spread throughout body –neural tissue –muscle tissue, myocardium –placenta, fetus Once infected, always infected Once infected, always infected

15. Life cycle When faced with a strong immune response, tachyzoites encyst as bradyzoites When faced with a strong immune response, tachyzoites encyst as bradyzoites Cysts are minimally inflammatory Cysts are minimally inflammatory Remain latent until host is immunosuppressed Remain latent until host is immunosuppressed Cysts reinfect definitive host if consumed Cysts reinfect definitive host if consumed

16. Encysted bradyzoites Thick-walled cysts differentiate N. caninum from T. gondii www.cmgm.stanford.edu www.k-state.edu www.liv.ac.uk

17. Dubey JP, Lindsay DS. Vet Parasitol. 2;67(1-2):1-59.

18. Transmission in cattle Horizontal infection Horizontal infection –1-2% of infections in cattle Cow-to-cow transmission not observed Cow-to-cow transmission not observed –no excretion of live organisms Prevalence doesn’t Prevalence doesn’t increase with age increase with age

19. Transmission High efficacy of transplacental infection High efficacy of transplacental infection –lower efficacy in low prevalence herds “Silent infections” “Silent infections” Monitor by precolostral sampling Monitor by precolostral sampling –false negatives (?)

20. Transmission Experimental transmission in colostrum Experimental transmission in colostrum –N. caninum tachyzoites or DNA –not successful in dogs Not believed to be a natural route of transmission Not believed to be a natural route of transmission

21. Transmission N. caninum DNA has been identified intermittently in semen N. caninum DNA has been identified intermittently in semen Sexual transmission produced experimentally with 10 4 tachyzoites Sexual transmission produced experimentally with 10 4 tachyzoites –dose-dependant response Not believed to be Not believed to be transmitted naturally transmitted naturally

22. www.neosporosis.com

23. Wildlife www.vetmed.illinois.edu

24. Potential definitive hosts

25. Potential intermediate hosts

26. So what?

27. Prevalence Neosporosis historically a dairy disease Neosporosis historically a dairy disease –sampling bias (?) Evidence of infection common in both beef and dairy herds Evidence of infection common in both beef and dairy herds

28. Prevalence - Dairy 15-40% of dairy cattle tested (+) 15-40% of dairy cattle tested (+) –in multi-herd studies Up to 100% (+) within herds Up to 100% (+) within herds Up to 100% of herd in endemic areas Up to 100% of herd in endemic areas

29. Prevalence - Dairy Prevalence study on 3 GA dairies Prevalence study on 3 GA dairies Milk samples assayed by Western blot Milk samples assayed by Western blot Results: Results: FarmPositive/Total% A28/11524.4 B30/13921.6 C47/7364.4 TOTAL105/32731.1 Ortega, Y.R. et al., Vet Parasitol. 2007(144):353-355.

30. Prevalence - Beef 5-23% of beef cattle tested (+) 5-23% of beef cattle tested (+) Up to 67% (+) within herds Up to 67% (+) within herds –avg 19% 36-100% herds positive 36-100% herds positive

31. Dairy vs. Beef Serology data difficult to compare Serology data difficult to compare –different methods and cut-off points Multi-national study Multi-national study –Bartels, et. al, Vet Parasitol. 2006 137:17-27 –>1000 herds, ~25,000 head Random herds: all cattle >2 yrs tested Random herds: all cattle >2 yrs tested –standardized ELISA

32. Herd type # tested# positive Prevalence (%) SwedenDairy1123430 GermanyBeef1064341 Dairy10050 The Netherlands Beef825871 Dairy1088680 SpainBeef37217146 Dairy29118463

33. Herd type # tested# positive Prevalence (%) SwedenDairy1123430 GermanyBeef1064341 Dairy10050 The Netherlands Beef825871 Dairy1088680 SpainBeef37217146 Dairy29118463

34. Prevalence - Dogs Cheadle MA, Lindsay DS, Blagburn BL. Cheadle MA, Lindsay DS, Blagburn BL. Vet Parasitol. 1999; 85(4):325-30. 1077 serum samples from US/Canada 1077 serum samples from US/Canada IFAT testing at single lab IFAT testing at single lab

35. 9/158 (6%) 14/190 (7%) 30/313 (10%) 3/145 (2%)

36. Financial losses $35 million in CA annually $35 million in CA annually $25 million in TX beef industry $25 million in TX beef industry 30,000-60,000 pregs/year in Canada 30,000-60,000 pregs/year in Canada $25-35 per head in $25-35 per head in herds with 20% herds with 20% prevalence prevalence

37. Clinical Picture Reproductive losses Reproductive losses Decreased milk production Decreased milk production Decreased weight gain Decreased weight gain Premature culling Premature culling

38. Reproductive losses 4 manifestations of disease 4 manifestations of disease 1.Early embryonic death 2.Abortion 3.Stillbirths or weak calves born alive 4.Apparently healthy calf Depends on pregnancy status and stage of gestation Depends on pregnancy status and stage of gestation

39. Reproductive losses When a naïve, open cow is infected, development of CMI leads to CNS cysts When a naïve, open cow is infected, development of CMI leads to CNS cysts –seroconversion This cow poses a low risk of infection to her herdmates This cow poses a low risk of infection to her herdmates

40. Reproductive losses If a cow is <3 months pregnant, early embryonic death may occur If a cow is <3 months pregnant, early embryonic death may occur –through pro-inflammatory cytokines active at the placental connection www.drostproject.vetmed.ufl.edu

41. Reproductive losses Cows in mid-gestation most susceptible Cows in mid-gestation most susceptible Cow’s immune system down regulated Cow’s immune system down regulated –placental type 2 cytokine on T-cells Calves’ immune system incompetent Calves’ immune system incompetent Tachyzoites infect CNS Tachyzoites infect CNS –abortion –stillbirth, CNS deficits

42. Abortion Risk of abortion is 4-7x higher in seropositive cows Risk of abortion is 4-7x higher in seropositive cows Cows that abort once Cows that abort once –6 times more likely to abort again than seropositive cows w/out abortion

43. Reproductive losses Cows that abort are not clinically ill Cows that abort are not clinically ill 4-6 months of gestation 4-6 months of gestation Aborted fetus usually autolyzed Aborted fetus usually autolyzed Placentas not retained Placentas not retained Two patterns Two patterns –endemic –epidemic

44. Endemic abortion Persistent elevation greater than 5% in annual abortion rate Persistent elevation greater than 5% in annual abortion rate Abortions occur throughout the year Abortions occur throughout the year Abortions in both cows and heifers Abortions in both cows and heifers Abortion rate due to N. caninum Abortion rate due to N. caninum –10-17% on two CA dairies

45. Epidemic abortion Abortion outbreaks Abortion outbreaks 30% of pregnant cattle over several months 30% of pregnant cattle over several months Likely due to a horizontal transmission event Likely due to a horizontal transmission event

46. Abortion Some farms may have both Some farms may have both –prolonged history of sporadic abortions –occasional abortion outbreaks

47. Reproductive losses Cows in late gestation Cows in late gestation Clinically affected live calves uncommon Clinically affected live calves uncommon Variable deficits Variable deficits –limb dysfunction mild proprioceptive deficits mild proprioceptive deficits complete paralysis complete paralysis Multifocal encephalomyelitis Multifocal encephalomyelitis –grey matter of spinal cord

48. Reproductive losses Cows in late gestation Cows in late gestation Calves’ immune’s system fully developed Calves’ immune’s system fully developed –calf able to fight off infection infection –most common result of transplacental infection transplacental infection

49. Reproductive losses Majority of calves infected in utero are clinically normal Majority of calves infected in utero are clinically normal High precolostral antibody titer High precolostral antibody titer –80-100% calves born to seropositive dams are seropositive Important for maintenance of disease within herds Important for maintenance of disease within herds

50. Reduced milk production Thurmond MC, Hietala SK. JAVMA. 1997; 210(5):672-4. Thurmond MC, Hietala SK. JAVMA. 1997; 210(5):672-4. 118 (+) and 254 (-) heifers 118 (+) and 254 (-) heifers Seropositive heifers: Seropositive heifers: –2.5 lb/hd/day less milk –0.14 lb/hd/day less fat Other studies show Other studies show variable results variable results

51. Decreased weight gain >1000 weaned steers from 92 TX herds >1000 weaned steers from 92 TX herds Seropositive steers: Seropositive steers: –Lower postweaning ADG (0.05 kg/d) –Lower slaughter weight (7.5 kg) –No difference in feed conversion

52. Premature culling Study of 8 Canadian beef herds: Study of 8 Canadian beef herds: –Waldner CL, Janzen ED, Ribble CS. JAVMA 1998; 213(5):685-90. –20-50% herd prevalence Seropositive cows 1.9x risk of culling Seropositive cows 1.9x risk of culling 2.5x risk of reproductive failure 2.5x risk of reproductive failure –abortion and stillbirth Seropositive cows Seropositive cows younger at culling younger at culling

53. Clinical presentation Any system can be affected –neuro and muscular deficits predominate No reports of abortion No reports of abortion –mummies, resorption, weak pups exp. Purebreds overrepresented Purebreds overrepresented –Boxers –Greyhounds –Labradors –Golden Retrievers –Basset Hounds

54. Puppies Puppies affected most frequently Puppies affected most frequently –onset: 3-9 weeks of age Ascending paralysis Ascending paralysis –pelvic limbs more affected Arthrogryposis Arthrogryposis –scar formation/myositis Intracranial manifestations Intracranial manifestations absent absent

55. Older dogs LMN flaccid paralysis LMN flaccid paralysis Multifocal CNS involvement Multifocal CNS involvement –+/- polymyositis Likely reactivation of prior infection Likely reactivation of prior infection Cutaneous neosporosis Cutaneous neosporosis seen with seen with immunosuppression immunosuppression

56. Clinical Presentation Sporadic disease has been seen in: Sporadic disease has been seen in: –goats and sheep –horses Cats susceptible experimentally Cats susceptible experimentally Never isolated from humans Never isolated from humans –low antibody titers –2 rhesus monkeys infected

57. Diagnostics Abortions Abortions –one or more aborted fetuses –placenta –sera from aborting dam

58. Diagnostics Pescador, et al. Vet Parasit 2007; 150:159- 163 Pescador, et al. Vet Parasit 2007; 150:159- 163 258 aborted fetuses submitted to d-lab 258 aborted fetuses submitted to d-lab –21% due to N. caninum Aborted fetus usually autolyzed Aborted fetus usually autolyzed Serosanguinous fluid in body cavities Serosanguinous fluid in body cavities 2 cases – pale, white foci in lungs 2 cases – pale, white foci in lungs

59. Neospora abortus

60. Histology FOCAL ENCEPHALITIS FOCAL ENCEPHALITIS –necrosis & nonsuppurative inflammation Scattered foci of nonsuppurative cellular infiltrates in brain sections Scattered foci of nonsuppurative cellular infiltrates in brain sections nonsuppurative inflammation also in: nonsuppurative inflammation also in: –heart –skeletal muscle –liver –lungs

61. Immunohistochemistry Effective at identifying parasites Effective at identifying parasites –tissue cysts (bradyzoites) –tachyzoites Most commonly in brain Most commonly in brain Can also be used in Can also be used in mummified fetuses mummified fetuses –diminished accuracy

62. Serology Indirect Fluorescent Antibody Test (IFAT) Indirect Fluorescent Antibody Test (IFAT) –Can be used in fetuses > 5-6 mo old Enzyme-linked Immunosorbent Assay (ELISA) Enzyme-linked Immunosorbent Assay (ELISA) Each lab will establish cut-off points Each lab will establish cut-off points –often >1:50 –CSF titers of lesser magnitude

63. Serology Used as a herd screening test Used as a herd screening test Titers fluctuate throughout gestation Titers fluctuate throughout gestation –do not revert to seronegative status Most cows seropositive at time of abortion Most cows seropositive at time of abortion –individual cows may be NEGATIVE

64. Diagnosis Compatible gestational age Compatible gestational age Autolyzed fetus Autolyzed fetus Disseminated inflammatory lesions Disseminated inflammatory lesions Presence of N. caninum parasites (IHC) Presence of N. caninum parasites (IHC) –or serological evidence Absence of other abortifacients Absence of other abortifacients

65. Prevention – negative herds Close herd Close herd Open herd - only buy seronegative imports from herds with excellent repro or negative herd tests Open herd - only buy seronegative imports from herds with excellent repro or negative herd tests Keep dogs away from feed areas Keep dogs away from feed areas –feral canids

66. Prevention – positive herds Serological testing of young stock Serological testing of young stock –pre-colostral samples –after 6 mo of age Dispose of aborted fetuses and dead animals Dispose of aborted fetuses and dead animals Embryo transfer Embryo transfer Minimize contact with canids Minimize contact with canids

67. Control strategies Cull females with mid-term abortion or stillborn calves Cull females with mid-term abortion or stillborn calves Whole-herd test, cull seropositives, replace with seronegatives Whole-herd test, cull seropositives, replace with seronegatives Whole-herd test, exclude heifer replacements from seropositive dams Whole-herd test, exclude heifer replacements from seropositive dams –best cost-benefit ratio

68. Vaccination One licensed vaccine in U.S. One licensed vaccine in U.S. –Bovilis ® NeoGuard Inactivated tachyzoites Inactivated tachyzoites –$3-4/dose 2 doses initially 2 doses initially –5 ml subQ in 1 st trimester –2 nd dose 3-4 weeks later Questionable efficacy Questionable efficacy –herd variability Vaccinates are seropositive Vaccinates are seropositive

69. Questions?