dr. darin madson - rotavirus update: pathogen, diagnostics, immunity and prevention

Veterinary Diagnostic LaboratoryIowa State University

North Carolina Swine Health Seminar:

Rotavirus Update

Darin [email protected]

mailto:[email protected]


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

http://www.niaid.nih.gov/topics/rotavirus/Pages/rotavirusIllustration.aspx


“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


DIAGNOSTICSRotavirus


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


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


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


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


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


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



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



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

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


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


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

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%


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


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 PREVENTIONRotavirus


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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