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

Veterinary Diagnostic Laboratory
Iowa State University
North Carolina Swine Health
Seminar:
Rotavirus Update
Darin Madson
madson@iastate.edu

Acknowledgments
• Dr. Paulo Arruda
• Dr. Greg Stevenson
• Dr. KJ Yoon

Outline
• Rotavirus
• “The pathogen”
• Rotavirus
• “Diagnostics”
• Rotavirus
• “Immunity and prevention”

“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

“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

“The Pathogen”
www.niaid.nih.gov

“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

“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

“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)

“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

“The Pathogen”
Normal small intestine
Note the numerous “fingers”

“The Pathogen”
Normal small intestine

“The Pathogen”
Acute infection – enterocyte swelling The virus in enterocytes

“The Pathogen”
Rotavirus infection
infects numerous cells

“The Pathogen”
End result Normal

“The Pathogen”

“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

“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)

“The Pathogen”
• Singular infection (only rotavirus)
– Generally only 2-3 days
• Co-infections
– Longer?
• Other issues
– Feed transition, feed diet changes,
environment

DIAGNOSTICS
Rotavirus

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

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

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

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

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

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

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

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

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.

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

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

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

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%

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

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

IMMUNITY AND PREVENTION
Rotavirus

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

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

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

Prevention
• Sanitation
– Crates, mats, etc
– Bleach appears to be the best disinfectant
• Vaccination
• Feedback??
• Good colostral immunity
– Assure all piglets get colostrum

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

Questions & Discussion?
Thanks for your attention

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

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