Veterinary Diagnostic Laboratory Iowa State University North Carolina Swine Health Seminar: Rotavirus Update Darin Madson [email protected]
Jun 16, 2015
Veterinary Diagnostic LaboratoryIowa State University
North Carolina Swine Health Seminar:
Rotavirus Update
Darin [email protected]
Veterinary Diagnostic LaboratoryIowa State University
Acknowledgments
• Dr. Paulo Arruda
• Dr. Greg Stevenson
• Dr. KJ Yoon
Veterinary Diagnostic LaboratoryIowa State University
Outline
• Rotavirus• “The pathogen”
• Rotavirus• “Diagnostics”
• Rotavirus• “Immunity and prevention”
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“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
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“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
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“The Pathogen”
www.niaid.nih.gov
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“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
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“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
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“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)
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“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
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“The Pathogen”
Normal small intestine
Note the numerous “fingers”
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“The Pathogen”
Normal small intestine
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“The Pathogen”
Acute infection – enterocyte swelling The virus in enterocytes
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“The Pathogen”
Rotavirus infection infects numerous cells
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“The Pathogen”End result Normal
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“The Pathogen”
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“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
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“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)
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“The Pathogen”
• Singular infection (only rotavirus)– Generally only 2-3 days
• Co-infections– Longer?
• Other issues– Feed transition, feed diet changes,
environment
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DIAGNOSTICSRotavirus
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Swine disease diagnosis: ISU-VDL
25.2
22.4
1.11.7
12.30.6
0.7
27.2
8.8
Respiratory
Gastroenteric
Neurologic
Reproductive
Systemic
Arthritis
Toxicosis
Others
No Dx
Pneumonia
Diarrhea
Septicemia
Just PCR testing
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Enteric diseases
33.9
18.317.8
9.2
8.3 7.03.1 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
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2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 20120
100
200
300
400
500
600
700
Frequency of diagnosis, 2003-2011 (ISU Diagnostic laboratory)
C. diff
Rotavirus
TGE
C. perfringens type C
C. perfringens type A
Coccidia
E. coli
Year
# o
f ca
ses
Swine cases
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Numbers of rotavirus-associated enteritis cases at ISUVDL
2003 2004 2005 2006 2007 2008 2009 2010 20110
100
200
300
400
500
600
700
NOTE: Data includes only confirmed tissue cases.
2012
863
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Incidence of rotavirus in feces or intestinal content
34%
42%
18%5%
PCR detection of rotaviruses
0
1 group
2 groups
3 groups
24%
9%
32%
8%
12%
8%8%
Breakdown of positives for group
A
B
C
A+B
A+C
B+C
A+B+C
Singular infection
Concurrent infection
N=1186 N=777
KJ Yoon Confidential
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Age distribution of pigs positive for rotavirus shedding
32%
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
Veterinary Diagnostic LaboratoryIowa 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%
69%
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
Veterinary Diagnostic LaboratoryIowa 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
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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.
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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
ComparativeStudy
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
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Material and MethodsInoculation 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
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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
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Results – fecal shedding • No detection in negative controls• No cross contamination
• Only inoculated virus was recovered; by group
Serogroup
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%
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Results – histopathology cont’d24 hpi Mean villous height (µm) by location
GroupDuodenum
Prox. Jejunum Mid jejunum distal jejunum ileum
Negative 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
GroupDuodenum
Prox. Jejunum Mid jejunum distal jejunum ileum
Negative 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
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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
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IMMUNITY AND PREVENTIONRotavirus
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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
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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
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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
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Prevention
• Sanitation– Crates, mats, etc– Bleach appears to be the best disinfectant
• Vaccination• Feedback??• Good colostral immunity
– Assure all piglets get colostrum
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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
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Questions & Discussion?
Thanks for your attention