Veterinary Diagnostic Laboratory
Iowa State University
North Carolina Swine Health
Seminar:
Rotavirus Update
Darin Madson
...
Veterinary Diagnostic Laboratory
Iowa State University
Acknowledgments
• Dr. Paulo Arruda
• Dr. Greg Stevenson
• Dr. KJ Yo...
Veterinary Diagnostic Laboratory
Iowa State University
Outline
• Rotavirus
• “The pathogen”
• Rotavirus
• “Diagnostics”
• ...
Veterinary Diagnostic Laboratory
Iowa State University
“The Pathogen”
• Rotavirus
– Major cause of diarrhea
• Humans, calv...
Veterinary Diagnostic Laboratory
Iowa State University
“The Pathogen”
• Non-enveloped virus
• = More resistance to environ...
Veterinary Diagnostic Laboratory
Iowa State University
“The Pathogen”
www.niaid.nih.gov
Veterinary Diagnostic Laboratory
Iowa State University
“The Pathogen”
• Groups
– Designated “A, B, or C” based on the VP 6...
Veterinary Diagnostic Laboratory
Iowa State University
“The Pathogen”
Group A rotavirus
• 1st identified (1970‟s)
• Furthe...
Veterinary Diagnostic Laboratory
Iowa State University
“The Pathogen”
• Group A
– Highly prevalent (near 100% of adult swi...
Veterinary Diagnostic Laboratory
Iowa State University
“The Pathogen”
• Infection
– Fecal-oral transmission
– Virus infect...
Veterinary Diagnostic Laboratory
Iowa State University
“The Pathogen”
Normal small intestine
Note the numerous “fingers”
Veterinary Diagnostic Laboratory
Iowa State University
“The Pathogen”
Normal small intestine
Veterinary Diagnostic Laboratory
Iowa State University
“The Pathogen”
Acute infection – enterocyte swelling The virus in e...
Veterinary Diagnostic Laboratory
Iowa State University
“The Pathogen”
Rotavirus infection
infects numerous cells
Veterinary Diagnostic Laboratory
Iowa State University
“The Pathogen”
End result Normal
Veterinary Diagnostic Laboratory
Iowa State University
“The Pathogen”
Veterinary Diagnostic Laboratory
Iowa State University
“The Pathogen”
• Infects mature enterocytes
– Baby piglets – born w...
Veterinary Diagnostic Laboratory
Iowa State University
“The Pathogen”
• Infection
– Most prevalent = 3-5 weeks of age
– Ca...
Veterinary Diagnostic Laboratory
Iowa State University
“The Pathogen”
• Singular infection (only rotavirus)
– Generally on...
Veterinary Diagnostic Laboratory
Iowa State University
DIAGNOSTICS
Rotavirus
Veterinary Diagnostic Laboratory
Iowa State University
Swine disease diagnosis:
ISU-VDL
25.2
22.4
1.1
1.7
12.3
0.6
0.7
27....
Veterinary Diagnostic Laboratory
Iowa State University
Enteric diseases
33.9
18.3
17.8
9.2
8.3
7.0
3.1
1.9
0.2 0.2
0.1 0.0...
Veterinary Diagnostic Laboratory
Iowa State University
0
100
200
300
400
500
600
700
2002 2003 2004 2005 2006 2007 2008 20...
Veterinary Diagnostic Laboratory
Iowa State University
Numbers of rotavirus-associated
enteritis cases at ISUVDL
0
200
400...
Veterinary Diagnostic Laboratory
Iowa State University
Incidence of rotavirus in feces or
intestinal content
35%
42%
18%
5...
Veterinary Diagnostic Laboratory
Iowa State University
Age distribution of pigs positive
for rotavirus shedding
33%
10%
51...
Veterinary Diagnostic Laboratory
Iowa State University
Age distribution (cont‟d)
12%
7%
71%
10%
A+B
≤ 7
8 - 20
21 - 42
> 4...
Veterinary Diagnostic Laboratory
Iowa State University
Summary
• Diagnostic data
– Group A
• More common post-wean
– Group...
Veterinary Diagnostic Laboratory
Iowa State University
ISU-VDL rotavirus study
Main objective
Compare viral titers and dur...
Veterinary Diagnostic Laboratory
Iowa State University
Material and Methods
• PRRSv negative pregnant sows
– Derivation of...
Veterinary Diagnostic Laboratory
Iowa State University
Material and Methods
Inoculation and housing
• Groups separated by ...
Veterinary Diagnostic Laboratory
Iowa State University
Results – clinical disease
• No diarrhea or clinical signs in negat...
Veterinary Diagnostic Laboratory
Iowa State University
Results – fecal shedding
• No detection in negative controls
• No c...
Veterinary Diagnostic Laboratory
Iowa State University
Results – histopathology cont‟d
24 hpi Mean villous height (µm) by ...
Veterinary Diagnostic Laboratory
Iowa State University
Summary
• Rotavirus study
– All groups cause diarrhea in neonatal C...
Veterinary Diagnostic Laboratory
Iowa State University
IMMUNITY AND PREVENTION
Rotavirus
Veterinary Diagnostic Laboratory
Iowa State University
Immunity
• Suckling piglet
– Colostrum/milk antibodies; neutralizin...
Veterinary Diagnostic Laboratory
Iowa State University
Immunity
• Systemic and local response
• Predominate response is to...
Veterinary Diagnostic Laboratory
Iowa State University
Immunity
• No cross protection across
– Different groups (A, B, and...
Veterinary Diagnostic Laboratory
Iowa State University
Prevention
• Sanitation
– Crates, mats, etc
– Bleach appears to be ...
Veterinary Diagnostic Laboratory
Iowa State University
Prevention
• Vaccine
– Multiple commercial products
• Type A
– New ...
Veterinary Diagnostic Laboratory
Iowa State University
Questions & Discussion?
Thanks for your attention
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Dr. Darin Madson - Rotavirus Update: Pathogen, Diagnostics, Immunity and Prevention

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Rotavirus Update: Pathogen, Diagnostics, Immunity and Prevention - Dr. Darin Madson, Iowa State University, Veterinary Diagnostic Lab, from the 2013 Boehringer Ingelheim Swine Health Seminar, August 16-18, 2013, Wrightsville Beach, NC, USA.

More presentations at http://www.swinecast.com/2013-boehringer-ingelheim-carolina-swine-health-seminar

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Dr. Darin Madson - Rotavirus Update: Pathogen, Diagnostics, Immunity and Prevention

  1. 1. Veterinary Diagnostic Laboratory Iowa State University North Carolina Swine Health Seminar: Rotavirus Update Darin Madson madson@iastate.edu
  2. 2. Veterinary Diagnostic Laboratory Iowa State University Acknowledgments • Dr. Paulo Arruda • Dr. Greg Stevenson • Dr. KJ Yoon
  3. 3. Veterinary Diagnostic Laboratory Iowa State University Outline • Rotavirus • “The pathogen” • Rotavirus • “Diagnostics” • Rotavirus • “Immunity and prevention”
  4. 4. Veterinary Diagnostic Laboratory Iowa State University “The Pathogen” • Rotavirus – Major cause of diarrhea • Humans, calves, pigs, and other species – Rotavirus are species specific » Swine rotavirus only infects swine • Generally confined to the gastrointestinal tract – Small intestine – Intensely raised/larger production sites • Positive for rotavirus • High infection rate, low mortality
  5. 5. Veterinary Diagnostic Laboratory Iowa State University “The Pathogen” • Non-enveloped virus • = More resistance to environmental degradation • RNA virus with 11 double-stranded segments • Recombination is gene segments is possible with coinfection • Similar to swine influenza – Three-layered viral capsid • Outer = VP 4 and VP 7 • Middle = VP 6 • Inner = VP 2 – Subdivided into groups based on VP 6 • 7 total groups • 5 infect swine: A, B, C, E, & H
  6. 6. Veterinary Diagnostic Laboratory Iowa State University “The Pathogen” www.niaid.nih.gov
  7. 7. Veterinary Diagnostic Laboratory Iowa State University “The Pathogen” • Groups – Designated “A, B, or C” based on the VP 6 gene – Groups E and H have not be identified in the US. • VP 7 and VP4 (outer capsid) are responsible for protective immunity – The major antigenic sites – VP 7; highest immune response
  8. 8. Veterinary Diagnostic Laboratory Iowa State University “The Pathogen” Group A rotavirus • 1st identified (1970‟s) • Further subtyped – G type; based on VP 7 – P type; based on VP 4 • Cultivable by virus isolation techniques • Detection – VI, Antigen ELISA, IHC, PAGE, and PCR Group B and C rotavirus • 1st identified in the early „80s • No subtypes at this point – Diversity is known • Virus isolation is extremely difficult • Detection – PAGE, PCR
  9. 9. Veterinary Diagnostic Laboratory Iowa State University “The Pathogen” • Group A – Highly prevalent (near 100% of adult swine) – Diarrheic samples = around 66% in young pigs • Group B and C – Relatively unknown • Likely common as PCR detection methods are being used • Coinfections – Pigs can be infected or re-infected with multiple group A viruses • Antigenic difference in VP 7 and VP 4 • Likely the same for groups B and C – U of MN data – Group combinations • Also common (more later)
  10. 10. Veterinary Diagnostic Laboratory Iowa State University “The Pathogen” • Infection – Fecal-oral transmission – Virus infects mature villous enterocytes • Destroys enterocytes – Villous shorten and fusion • Reduced ability to absorb feed – “malabsorptive diarrhea” – Non-absorb sugars (disaccharides) also pull fluid into the lumen causing further dehydration
  11. 11. Veterinary Diagnostic Laboratory Iowa State University “The Pathogen” Normal small intestine Note the numerous “fingers”
  12. 12. Veterinary Diagnostic Laboratory Iowa State University “The Pathogen” Normal small intestine
  13. 13. Veterinary Diagnostic Laboratory Iowa State University “The Pathogen” Acute infection – enterocyte swelling The virus in enterocytes
  14. 14. Veterinary Diagnostic Laboratory Iowa State University “The Pathogen” Rotavirus infection infects numerous cells
  15. 15. Veterinary Diagnostic Laboratory Iowa State University “The Pathogen” End result Normal
  16. 16. Veterinary Diagnostic Laboratory Iowa State University “The Pathogen”
  17. 17. Veterinary Diagnostic Laboratory Iowa State University “The Pathogen” • Infects mature enterocytes – Baby piglets – born with mature enterocytes along the entire length • Potential more severe disease – >7 days • Only villous tip enterocytes are mature – Just physiology
  18. 18. Veterinary Diagnostic Laboratory Iowa State University “The Pathogen” • Infection – Most prevalent = 3-5 weeks of age – Can range from 1 day to adults – Can be infected with multiple groups at the same time. – A, B, C combinations – Can be infected with different viruses in the same group at the same time – Multiple A‟s, B‟s, or C‟s » May serotypes within each group (VP7 and VP4)
  19. 19. Veterinary Diagnostic Laboratory Iowa State University “The Pathogen” • Singular infection (only rotavirus) – Generally only 2-3 days • Co-infections – Longer? • Other issues – Feed transition, feed diet changes, environment
  20. 20. Veterinary Diagnostic Laboratory Iowa State University DIAGNOSTICS Rotavirus
  21. 21. Veterinary Diagnostic Laboratory Iowa State University Swine disease diagnosis: ISU-VDL 25.2 22.4 1.1 1.7 12.3 0.6 0.7 27.2 8.8 Respiratory Gastroenteric Neurologic Reproductive Systemic Arthritis Toxicosis Others No Dx Pneumonia Diarrhea Septicemia Just PCR testing
  22. 22. Veterinary Diagnostic Laboratory Iowa State University Enteric diseases 33.9 18.3 17.8 9.2 8.3 7.0 3.1 1.9 0.2 0.2 0.1 0.0 Rotavirus E. coli Salmonella spp Lawsonia Clostridium spp TGEV Coccidia Brachyspira spp Cryptosporidia PCV2 Parasites PRRSV Rotavirus
  23. 23. Veterinary Diagnostic Laboratory Iowa State University 0 100 200 300 400 500 600 700 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 #ofcases Year Frequency of diagnosis, 2003-2011 (ISU Diagnostic laboratory) C. diff Rotavirus TGE C. perfringens type C C. perfringens type A Coccidia E. coli Swine cases
  24. 24. Veterinary Diagnostic Laboratory Iowa State University Numbers of rotavirus-associated enteritis cases at ISUVDL 0 200 400 600 800 2003 2004 2005 2006 2007 2008 2009 2010 2011 NOTE: Data includes only confirmed tissue cases. 2012 863
  25. 25. Veterinary Diagnostic Laboratory Iowa State University Incidence of rotavirus in feces or intestinal content 35% 42% 18% 5% PCR detection of rotaviruses 0 1 group 2 groups 3 groups 24% 9% 32% 8% 11% 8% 8% Breakdown of positives for group A B C A+B A+C B+C A+B+C Singular infection N=1186 N=777 KJ Yoon Confidential
  26. 26. Veterinary Diagnostic Laboratory Iowa State University Age distribution of pigs positive for rotavirus shedding 33% 10% 51% 6% Group A only ≤ 7 8 - 20 21 - 42 > 42 28% 7% 35% 30% Group B only ≤ 7 8 - 20 21 - 42 > 42 56% 5% 29% 10% Group C only ≤ 7 8 - 20 21 - 42 > 42 KJ Yoon Confidential
  27. 27. Veterinary Diagnostic Laboratory Iowa State University Age distribution (cont‟d) 12% 7% 71% 10% A+B ≤ 7 8 - 20 21 - 42 > 42 29% 5% 60% 6% A+C ≤ 7 8 - 20 21 - 42 > 42 14% 10% 70% 6% B+C ≤ 7 8 - 20 21 - 42 > 42 7% 11% 75% 7% A+B+C ≤ 7 8 - 20 21 - 42 > 42 KJ Yoon Confidential
  28. 28. Veterinary Diagnostic Laboratory Iowa State University Summary • Diagnostic data – Group A • More common post-wean – Group B • Seen equally pre- and post wean – Group C • More common < 1 wk of age – Co-infections are common
  29. 29. Veterinary Diagnostic Laboratory Iowa State University ISU-VDL rotavirus study Main objective Compare viral titers and duration fecal shedding, and location and extent of microscopic lesions across mono-infected and co-infected challenge groups.
  30. 30. Veterinary Diagnostic Laboratory Iowa State University Material and Methods • PRRSv negative pregnant sows – Derivation of CDCD pigs on day 113 of gestation – Randomized into groups and inoculated Experiment Groups n Age Inoculation Comparative Study 1 6 1 day None (negative control) 2 6 1 day Rotavirus group A 3 6 1 day Rotavirus group B 4 6 1 day Rotavirus group C 5 6 1 day Rotavirus group A & B 6 6 1 day Rotavirus group A & C 7 6 1 day Rotavirus group B & C 8 6 1 day Rotavirus group A, B, & C
  31. 31. Veterinary Diagnostic Laboratory Iowa State University Material and Methods Inoculation and housing • Groups separated by room • Pigs individually housed in plastic totes – No contact between pigs • Oro-gastric inoculation – 5 hrs post delivery – Titered to a standard dose • Ie. all the same • Tube fed milk replacer 3x daily • Strict biosecurity Sample collection • Fecal swabs – Prior to inoculation – Every 12 hrs thereafter • Necropsy – ½ pigs at 24 hrs post infection (hpi) – Remaining pigs at 72 hpi • Necropsy samples – Colonic contents – 5 sections of small intestine • Duodenum, proximal, mid, and distal jejunum and ileum
  32. 32. Veterinary Diagnostic Laboratory Iowa State University Results – clinical disease • No diarrhea or clinical signs in negative controls • Singular infected groups (A, B, & C) • No diarrhea at 12 hrs • ~ 50% diarrhea at 24 hrs (all groups) • 100% diarrhea at 48 hrs (all groups) • Coinfected groups; rotaviral combinations • No diarrhea at 12 hrs • ~ 50% diarrhea at 24 hrs (all groups) • 100% diarrhea at 48 hrs (all groups) • Diarrhea and emaciation; severe at 72 hrs
  33. 33. Veterinary Diagnostic Laboratory Iowa State University Results – fecal shedding • No detection in negative controls • No cross contamination • Only inoculated virus was recovered; by group Serogrou p Fecal Shedding 12 hpi 24 hpi 36 hpi 48 hpi 60 hpi 72 hpi A 0% 100% 100% 66% 66% 66% B 50% 50% 0% 0% 30% 0% C 83% 100% 100% 100% 100% 100%
  34. 34. Veterinary Diagnostic Laboratory Iowa State University Results – histopathology cont‟d 24 hpi Mean villous height (µm) by location Group Duodenum Prox. Jejunum Mid jejunum distal jejunum ileum Negativ e 897 1028 1029 819 786 A 931 677* 537 443 791 B 346* 209 226 232 192 C 331* 234 241 233 348 A/B 492 279 300 362 343 A/C 256* 196 187 167 251 B/C 425 237 231 249 249 A/B/C 654 192 174 174 212 72 hpi Mean villous height (µm) by location Group Duodenum Prox. Jejunum Mid jejunum distal jejunum ileum Negativ e 973 917 909 840 863 A 797* 214 180 154 146 B 221 288 318 272 234 C 299 275 302 303 287 A/B 258 244 237 252 377 A/C 338 417 198 194 201 B/C 345 336 304 334 403 A/B/C 292 284 280 334 375
  35. 35. Veterinary Diagnostic Laboratory Iowa State University Summary • Rotavirus study – All groups cause diarrhea in neonatal CDCD pigs – Viral shedding • May be dependent on specific combinations – Group C was more consistent – Group B less consistent – All groups cause atrophic enteritis • Group A; more mid to distal SI • Groups B and C; diffuse atrophic change
  36. 36. Veterinary Diagnostic Laboratory Iowa State University IMMUNITY AND PREVENTION Rotavirus
  37. 37. Veterinary Diagnostic Laboratory Iowa State University Immunity • Suckling piglet – Colostrum/milk antibodies; neutralizing • Binds rotavirus within the gut lumen to prevent enterocyte infection • Frequent suckling helps “bath” the gut with these antibodies – Colostrum  IgG • These are initially absorbed and generally not re- secreted into the lumen • However, will help decrease the severity
  38. 38. Veterinary Diagnostic Laboratory Iowa State University Immunity • Systemic and local response • Predominate response is to VP 6 – The “group” antigen – But are not sufficient for protection • VP 7 and VP 4 (outer capsid) are next in line • G and P types – G and P type confers homologous protection, but not to heterologous • Similar to Influenza A virus
  39. 39. Veterinary Diagnostic Laboratory Iowa State University Immunity • No cross protection across – Different groups (A, B, and C) – Different serogroups • “different G and P types of a A, B, or C virus Basically, immunity is only protective against a single rotavirus isolate There is not broad immunity Immunity is VERY specific
  40. 40. Veterinary Diagnostic Laboratory Iowa State University Prevention • Sanitation – Crates, mats, etc – Bleach appears to be the best disinfectant • Vaccination • Feedback?? • Good colostral immunity – Assure all piglets get colostrum
  41. 41. Veterinary Diagnostic Laboratory Iowa State University Prevention • Vaccine – Multiple commercial products • Type A – New vaccines? • Harrisvaccines » VP 7 sequence » Can do rotavirus B and C • Newport Labs – Type C vaccine • Others – Currently looking into
  42. 42. Veterinary Diagnostic Laboratory Iowa State University Questions & Discussion? Thanks for your attention

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