dr. kent schwartz - understanding rotavirus

Diarrhea in Suckling Pigs

Role of Rotavirus

26th Annual Client Appreciation Day

Kent Schwartz

Iowa State University

Veterinary Diagnostic Laboratory

Overview

• History and changes

• Bit of science

• Causes of diarrhea in farrowing

• Role of rotavirus

• Prevention of endemic diarrhea

History

Housing Nutrition Genetics

What is “normal”?

Normal is what you are used to…

History

Housing Nutrition Genetics

What is “normal”?

Normal is what you are used to…

Continuous improvement requires open minds

ask “why”?

willing to learn via analysis

willing to implement changes

that have demonstrable benefit

History: Infectious Agents of diarrhea

Housing Nutrition Genetics

• E. coli 1900

• Salmonella 1900

• TGE 1940

• Clostridium perfringens

type C – 1960’s

• Coccidiosis – 1970’s

• Rotavirus A 1970’s

• PRRSV: 1990

• Clostridium perfringens type A:

2000

• Clostridium difficile 2005

• Rotavirus C 2010

• Rotavirus A, B 2010

• PEDV 2013

• PDCV 2013

• Seneca virus A 2015

• E. coli 1900

• Salmonella 1900

• TGE 1940

• Clostridium perfringens

type C – 1960’s

• Coccidiosis 1970’s

• Rotavirus A 1970’s

History: Infectious Agents of diarrhea

Housing Nutrition Genetics

• PRRSV: 1990

• Clostridium perfringens A: 2000

• Clostridium difficile 2005

• Rotavirus C 2010

• Rotavirus A, B 2010

• PEDV 2013

• PDCV 2013

• Seneca virus A 2015

• Cryptosporidia

• Adenovirus

• Next Generation Sequencing

• Microbiome and “biotics”

• More…..

• E. coli 1900

• Salmonella 1900

• TGE 1940

• Clostridium perfringens

type C – 1960’s

• Coccidiosis 1970’s

• Rotavirus A 1970’s

Extreme and constant vigilance

EXTERNAL BIOSECURITY

The rest are nearly always present

INTERNAL BIOSECURITY

MANAGEMENT

HYGIENE

HERD IMMUNITY

Secret to prevention of endemic diseases?

Some people try to find things in this game

that don’t exist, but football is only two

things – blocking and tackling” (Vince Lombardi)

One of the things young people always ask me about is

what is the secret to success. The secret is there is no

secret. It’s the basics. Blocking and tackling. (Chris Gardner)

Wins come from flawless execution of basics - the

blocking and tackling – every day.

“Science”: Sterile gut experiences “waves” of invadersMicro-ecology changes and variations are infinite

Ongoing from birth through adult: Metagenomics & Biomes

BirthTypes and numbers of bacteria

CHANGING

Physiology

pH

Anti-trypsin

Enzymes

Microflora

Diets

Age

Immunity

High numbers (billions) of C. perfringens, C. difficile, E. coli within hours

Log b

acte

ria c

ounts

Time

“Science”: Completely reliant on Passive immunity

• Systemic: Absorbed from COLOSTRUM (absorbed first day)

– Antibody (IgG) mediated immunity

• Systemic bacteria & viruses, toxins of Clostridium

– Immunocytes (cells) that help direct immune response

• cells, cytokines, immunomodulators)

• Mucosal: Continuous via milk ingestion (every 4 hours)

– Antibody (IgA) mediated immunity

• Mucosal pathogens

– Rotaviruses, PEDV, E. coli, TGE

– IgA is gone with weaning (or low milk ingestion / agalactia)

Science: NEONATE Risk factors: Anatomy / Physiology

Small Intestine

• Has longer villi and shorter crypts (7-20x more villus height

• More mature enterocytes more receptors (virus & bacteria)

• Slower to regenerate epithelium / villi (5 days)

Colon not completely functional

• Decreased ability to resorb water & salt or maintain pH balance

Flow: rate? Peristaltic Waves Orderly Motility?

Bacteria

“ Science”: Host physiology AND bacterial physiology

Piglets: high stomach pH, high metabolic rate, limited colon function,

limited regulation of body temperature & gut motility, no trypsin, more…

Bacteria: Dose, maternal immunity, bacteria replication rate, toxin genes

“on” or “off”, more …

RISKS

Chilling and heat stress

Pathogen load in environment

Cross fostering

Compromised passive immunity

The “mathematics” of enteric disease

Severity of disease = DOSE x virulence

Animals’ RESISTANCE

Dose can determine the outcome of infection

Subclinical infection Disease Death

DOSE can be controlled – HYGIENE / SANITATION

Internal Biosecurity: Sanitation - Management - Commitment

Severity of disease = dose x VIRULENCE

animal’s resistance

Virulence is difficult to influence with management/nostrums

EXTERNAL BIOSECURITY: A barrier to outside pigs/sources

Do NOT introduce “new” pathogens to a herd

Severity of disease = dose x virulence

Animals’ RESISTANCE

“IMMUNITY”PASSIVE IMMUNITY

Colostrum contains IgG and Cells – immunomodulation

Colostrum offers systemic immunity; not much mucosal immunity

Milk (Lactogenic) immunity to provide IgA antibody for mucosal (IgA

and IgM) but not systemic immunity; GONE at Weaning

ACTIVE IMMUNITY

Vaccination or infection stimulates antibody and cellular response

What about Animal Science and RISK FACTORS?

Risk Factors influencing piglet resistance to disease

Temperature / chilling / drafts (these are BIG)

Nutritional status and condition of dams

Condition (too fat / too thin)

Ease of farrowing: no hypoxia

Ongoing supply of milk

Genetics, genotype, litter size, milking ability, consumption

Nutrition: quality and quantity of macro- and micro-nutrients

“Herd immunity” – infections consistent across population

Avoid “subpopulations” all gilts/dams exposed to all potential pathogens

Active immunity via mitigated infection or vaccinations

Good supply of colostrum – litter size – birth weight

Colostrum from immune dams

Acclimation & Vaccination of gilts and sows

Feed-back to gestating dams ???

Severity of disease = dose x virulenceAnimals’ RESISTANCE

Concept: Many enteric swine “pathogens” are ENDEMIC

“Potential

Pathogens”

“immunity, nutrition, genetics”

“facilities, management”

“infectious diseases”

Dose x Virulence

Most agents of diarrhea are ENDEMIC

We can find them with “tests”

Why is disease being expressed?

INSULTAge (days)

Mortality0-4 5-8 8-weanAgalactia x x x can be high

E. coli x x can be highClostridium perfringens A x x lowClostridium perfringens C high

Clostridium. difficile x lowRotavirus A x x lowRotavirus B x lowRotavirus C x x low

PEDV / TGEV x x x highPorcine deltacoronavirus x x variable

Isospora (coccidia) x x lowPRRSV x x x variable

Salmonella x variable

BIAS: We can find a “bug” but often it is risk factors that allow disease expression

“find something infectious”

Assign blame

Bias AND

Diagnosis: Causation or correlation?

(Confirming bias or objective analysis?)

Collect Information

Diagnostic testing

DIAGNOSTIC ACCURACY

Does it “make sense”?

Treatment, Control

Identify Opportunities

Continuous Improvement

Achieving an ACCURATE DIAGNOSIS IS A PROCESS

Think, Analyze, Research

Refine

Repeat

process

Case definition and epidemiologyInformation to gather BEFORE making a “diagnosis”

• OBJECTIVE:

– Clinical signs

– Epidemiology

– Timeline

• SUBJECTIVE

– Farm History

– Area history

“Assessing sick piglets

and diarrhea”

Sow problem?

Milk flow?

Sow condition?

Feeding & Water?

Piglet problem?

Clinical signs

Lesions

Laboratory tests

Management problem?

Hygiene

Environment

Process compliance

Cross fostering

Look – Ask – Verify – Think

Necropsy: acutely affected vs moribund pig?

Full

Stomach

White streaks

(Chyle - milkfat)

Subgross examination: Villi

Materials:

• Freshly euthanized pig

• Glass tube (red stopper blood tube)

• Water

Procedure:

• Immediately place 1 cm section of small

intestine in tube

• Add water

• Gently swirl and wait for 1 minute

• Examine directly or with magnifying glass

Interpretation:

• Length: duodenum>jejunum>ileum

• Age: villi get shorter with age

<5 days like photos

Do your own comparisons

10% formalin Refrigerated Test types Agents

Cecal-colon

contents

NO 3-10 ml PCR, ELISA,

isolation

TGE, rotavirus,

Cdiff toxin

Small intestine 6-8 one-inch

segments/lesions

6” ileum

6” jejunum

Histo, IHC,

isolation,

impression

smear, PCR

E. coli, CptA,

CptC, Salmonella,

Isospora

Colon Cross-section of

several loops

Remainder of

colon intact

Histo, isolation C.difficile, AEEC,

Salmonella

Other affected

organs

½” slice Golf ball size Depends Multiple

BAL (lung) NO 1-5 ml (or pieces) PCR PRRSV

Specimens: Select the right pigs!!3 Acutely Affected Pigs:

Colon Contents

Colon fresh and fixed

Ileum fresh and fixed

Jejunum fresh and fixed

Pool BALs or lung (R/O PRRSV)

Enterotoxigenic E coli: Diagnosis

E. coli (secretory diarrhea)

• Observations:– Watery diarrhea

– Villi intact

– Chyle in lymphatics

– Bacterial adherence

• Frequently detected

• Frequently blamed

• Is it primary or potentiated by risk factors?

E. coli Genotyping

Animal ID Gene Result

A, 107 EAST1 (toxin) Positive

LT(toxin) Positive

STa(toxin) Positive

STb(toxin) Positive

Stx1 (toxin) Negative

Stx2 (toxin) Negative

Stx2e(toxin) Negative

F18(pilus) Negative

F41(pilus) Negative

K88(pilus) Positive

K99(pilus) Negative

987P(pilus) Negative

AIDA (adhesin) Negative

EAEA (adhesin) Negative

PAA (adhesin) Positive

E. Coli will always be detected … but is it important?

Histopathology

Virulence-related disease: genotyping

Clostridium perfringens Type C

Disease fairly limited to defined geographical locations

Midwest USA: once common, now uncommon

Quite dramatic when it occurs

Coccidiosis – Isospora suis

- ~5 days through post-weaning

Dose-related disease

Prevention is preferred!

Facility – Hygiene

Internal biosecurity

0

10

20

30

40

50

60

70

80

90

Number of cases of COCCIDIOSIS in suckling pigletsISU VDL

Control of Isosporosis: Example of dose and hygieneMust eliminate “sticky” oocysts (also bacteria, viruses)

Bugs “build up” from previous litters – transmit by people

Change flooring – no concrete or wood

All-in/all-out management

SANITATION –

• Soak, soap, degreaser

• Eliminate films

• Appropriate disinfectants

MANAGEMENT

• Mats and mat management

• Cross-fostering pigs

• Hands, boots, fomites, carts, utensils Blocking and tackling of

hygiene

management

environment

Clostridium difficile

Necrotizing typhlocolitis in humans, horses, piglets

Opportunist with severe consequences

in medicated humans and horses

Common (normal) flora in swine:

• MOST (>95%) of herds are colonized

• MOST piglets are colonized by 2 days

Increased frequency of “disease diagnosis”

• Little evidence for “antibiotic induced”

Colon important for absorbing water and

determines feces consistency

Clostridium perfringens type A

• Clostridium perfringens type A is common = ubiquitous

– Environment

– In gut from D0 (high - 108 populations by 12 hr) through adult

• CptA: There are no consistent experimental models using biologically achievable inoculum to critically evaluate pathogenesis, treatment and control in enteric disease

Type Alpha Beta Epsilon Iota

A + - (beta2) - -B + + + -

C + + - -D + - + -

E + - - +

PED/TGE: segmental or entire small intestine

Isospora: Lower small intestine

E. coli: mid to lower small intestine

Clostridium: upper small intestine

Rotavirus: segmental but mid to lower small intestine

C. difficile: colon

Swine Coronaviruses associated with diarrhea

Transmissible GastroEnteritis - TGEV

Porcine Epidemic Diarrhea - PEDV

Deltacoronavirus - PDCoV

Swine Rotaviruses

Rotaviruses Group A

Rotaviruses Group B

Rotaviruses Group C

No cross-protection between groups

Villi are covered with infected

epithelial cells stained brown

Crypt epithelium is spared and will

be the source of new cell growth

Just 36 hours post-infection in a neonatal piglet: • Villi are gone damage done / gut compromised

• Only a few brown-staining cells remain

diagnosis only from ACUTELY affected)

• Millions to billions of virus particles have been shed into the environment

• Tipping point is reached cows are out of the barn!!

Extraordinary efforts required to regain control

PEDV and TGEV: Don’t get them!!

EXTERNAL Biosecurityhave and follow protocols)• Biosecurity protocols: review, strict, tighten, update

– People, supplies, feed, food, etc.

• Limit traffic: people and equipment

• Clean and disinfect incoming: anything

• Enforce downtime

• Disposal of dead stock

• Animal isolation, monitoring and surveillance: diagnostic tests

• Shower, clothing and boots: thorough / change

– Truck and trailer biosecurity

• Loading and unloading procedures

• Crew and driver protocols

• Truck wash procedures

• Manure disposal biosecurity

• Swine transportation

• Verify implementation!

http://www.aasv.org/aasv%20website/Resources/Diseases/PED/PEDVBiosecurity.pdf

Vaccination???

PEDV booster of previously positive sows

Not for “priming” – only after exposure

Rotaviruses cannot be eliminated

• Non-enveloped: very resistant in environment (it’s everywhere!)

• Can cause severe atrophic enteritis just like TGEV/PEDV• 6 structural proteins (VP)

– VP4

• 26 antigenic P genotypes with 7 P serotypes

• cleaved to VP5+VP8 for infectivity

– VP6: most abundant

– VP7: 15 G genotypes are antigenic with 10 G serotypes

• 6 non-structural proteins (NSP)

– NSP4 may be hypersecretory (like E. coli)• Structure

– Outer layers: VP7, VP4

– Inner layer: VP6

– Core: VP1, VP2, VP3

• VP6 determines antigenically distinct serogroups 1-7

• Antibody to VP6, VP7, NSP2, NSP4.

– 3 serogroups (A, B, C) and multiple serotypes

are not cross-protective

– “influenza of the gut”

0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

2007 2008 2009 2010 2011 2012 2013

Rotavirus Detection: IHC (group A only) prior to 2009 PCR started in 2009 now usually positive for rotavirus

%B & C

%C only

%B only

%A

NOTE: IHC detects rotavirus A PCR detects rotaviruses A, B and C.

Impact of PCR test sensitivity on “frequency of diagnosis”

Age Distribution versus Serotype

29%

25%

39%

7%

Group A only (N = 479)

< 1 week

1-3 weeks

3- 6 weeks

> 6 weeks

22%

16%

31%

31%

Group B only (163)

< 1 week

1-3 weeks

3- 6 weeks

> 6 weeks

56%

10%

25%

9%

Group C only (N = 521)

< 1 week

1-3 weeks

3- 6 weeks

> 6 weeks

PCR Positive Cases by Month

No seasonal effect

84%

73%

85%88%81%

89%79%81% 83%

72% 72%80%

0%10%20%30%40%50%60%70%80%90%

100%

% Positive

% Positive

Cases tested in year 2012 (N = 1696)

Diagnosis: Rotavirus• Experimentally, all serogroups cause similar disease in suckling pigs

• DX = clinical signs + detection + microscopic lesions + rule out others

– Gross and microscopic lesions are not specific

– Lesions segmental look at multiple (>6) sections

– PCR Ct value (lower is more virus) depend on stage of disease

Sample ACUTELY affected piglets

Sacrifice of 2-3 typical pigs better than simple fecal PCR

• Specific Laboratory Tests

– IHC: Serogroup A

– PCR: Serogroups A, B & C

– Histopathology: compatible lesions

• No serology test available or helpful

Risk Factors / Noninfectious contributors• Hygiene (ALL OUT with sanitation / disinfection) impacts DOSE

• Environment impacts RESISTANCE

• Chilling (wet, drafts, temperature, heat lamps, mats)

• Mat management; disinfection and timely removal

• Management impacts DOSE and RESISTANCE

– Sow factors: genetics, nutrition

– People and farm culture: teams with pride and engagement

– Farrowing processes and procedures

– Colostrum management

• Immunity impacts RESISTANCE

– Proper acclimation

– Influencing maternal immunity / dam immune status

– Acclimation

– Vaccination?

Vaccination???

PEDV booster of previously positive sows

Not for “priming” – only after exposure

Where are the data for rotaviruses?

Rotavirus: Controlling “Flare-ups”

Double down on basics

Blocking and tackling operate in PEDV mode

Farrowing room processes and procedures / McRebel

Optimize piglet acquisition of colostrum and milk

Manage chilling, drafts, hygienic practices

Can we increase dam immunity?

Acclimation: expose incoming gilts to “prime” their immune system to resident viruses and bacteria

Vaccine?

Booster maybe?

Lots of effort & Lots of “technology”:

Commercial, autogenous, subunit and otherwise

NO GOOD DATA (vs human vaccines)What about “feed-back”?

Evolution of concepts: Can feedback help?

• Protect the offspring from disease via IgA and/or IgG and/or CMI

– Passive mucosal immunity – may prevent disease

• Prototypes: TGE, PED and E. coli

• Best IgA response if boostered 10-14 days prefarrow

– Passive systemic immunity – may delay infections

• Systemic disease that are IgG / CMI mediated (Clostridium toxins)

• Best IgG response if boostered 4-5 weeks prefarrow

• Protect the unborn fetus during gestation

– Active systemic immunity prevents viremia in dam

• Protect unborn fetuses

• Prototype: porcine parvovirus

• PRRSV, perhaps other “SMEDI” or unknowns

“Assumptions

– Prototype: PRRSV … but may not be via “Feedback”

– Extrapolations start to get fuzzy

– BTY: Feedback and controlled exposure are NOT the same as vaccination

What do we really know about

feedback to gestating dams?Proceedings, AASV and SDC

Robbins and Byers: Field Study

1. No statistical differences in outcome over 1 year period

2. Feedback can contain agents that are unexpected

Arruda: Controlled Study

No differences measured in colostrum, sows

or piglet diarrhea after feedback of feces

containing Clostridium spp, E. coli, and rotaviruses

Feedback: understanding pathogenesis, mechanisms

and virulence factors

Moeser AJ, Blikslager AT: J Am Vet Med Assoc 231: 56-

67.

• Fimbria, Pili, Adhesins: Attachment is common 1st step

F18, K88, …. MANY and not just for E. coli

• Viruses and coccidia cause individual cell necrosis

• Pathogenicity islands (code for nanomachine-like proteins) for invasion

• Toxins:

Exotoxins: Clostridia, E. coli

Mediators and modulators: permeability and altered cellular machinery

• Stimulate inflammation

Helpful?

Overzealous?

What is the mechanism

for immunity for

the target agent?

Agents: 4000 in “microbiome” PLUS “Diseases of Swine”

Variation in strains, virulence, cross-protection

Bacteria:

• Actinobacillus sp / type

• Bordetella

• Brachyspira

• Brucella

• Clostridium

• Erysipelothrix

• E. coli: toxins, pili, other

• Haemophilus

• Lawsonia

• Lepto

• Mycobacterium

• Mycoplasma

• Pasteurella multocida A/D

• Salmonella

• Staphylococcus

• Streptococcus

Viruses:

• Coronaviruses (TGE, PDCV, PEDV, PRCV)

• Enteroviruses (Teschovirus)

• Influenza

• PCMV

• Porcine pestiviruses (CSF, swine pesti)

• Porcine parvoviruses

• Porcine circoviruses

• PRRSV

• PRV

• Rotavirus

• Senecavirus

• Swine poxvirus

• Transboundary: numerous– Paramyxovirus: Blue-eye, Nipah, Menangle

– Japanese B

• FEED BACK: Objectives? ENHANCE HERD IMMUNITY

– “Priming” mode First exposure to a specific strain of pathogen

• Sow Farms: assure uniform first exposure (PEDV)

• Gilt acclimation PEDV, PRRSV, parvoviruses and MORE

– “Boostering” mode rotavirus, E. coli, Clostridium sp.

• Boostering previously exposed animals requires HIGH Dose

• Can feedback spread bugs or increase persistence????

– Is that a good thing or bad thing?

• Clostridium perfringens type C or type A or C. difficile?

• Rotaviruses, PRRSV and PCV2

• Erysipelas, Brachyspira, Salmonella, Lawsonia, toxigenic E. coli

• Parasites: Isospora, nematodes

What are some obstacles to

“boostering immunity” via re-infection?• “Hypo-responsive” to feed matrix / microbiome / diet antigens

• Physiologic: Stomach pH, digestion

• Epithelium

• Innate resistance mechanisms, including

– Mucus flux

– Tight Junctions

– Antimicrobial peptides (kill zone)

– Inflammatory response

• Adaptive immunity – antibody and CMI

– Systemic

– Mucosal

Slight perturbations may not “booster” immunity

– Happens locally

– No need to call in all the “reinforcements”

WHAT and WHEN to feed back?

• For Rotavirus ( enteric viruses, bacteria, piglet scours)

– Feces from acute piglets, 10-14 days prior to farrowing

– Source?

• Feces, wipes, processing carts, anything with poop

• Macerated gut tissue from dead pigs

• Feces/content from intentionally infected pigs??

– Colostrum deprived and sow milk deprived

– Harvest feces and intestines from 1-2 day old pigs

– Easily 1000x more virus particles/unit (Ct 25 15)

• Disclaimer: no published, peer-reviewed research!!

• To protect unborn piglets (congenital tremors, PPV, SMEDI)

– Entrails (guts/viscera/mummies) given prior to breeding

– Eviscerate and macerate (garbage disposal or meat grinder)

IF doing feedback, suggestions are: 1. What is your stated objective? booster IgA to control rotavirus

2. DOSE is important

– High dose better than multiple doses for “booster”

– One dose done right probably better than multiple doses

(Antigenic mass / agent replication sufficient to induce response)

3. TIMING is probably important

– 10-14 days for IgA booster (to control scours)

– 5 weeks for IgG booster (to increase antibody for systemic diseases or toxins of Clostridium, influenza, IAV, erysipelas)

4. Define and refine the process and procedures

– What are the risks?

– What material to use? Feces/intestines?

– Dilute with cold saline and use immediately or refrigerate < 3 days

– Can retain for future use (for a while) by freezing

5. Be tidy – spend time cleaning

Controlling endemic scour flare-upsIf it’s not TGE or PED, fall back to basics Go into “PED elimination mode” in farrowing rooms

Develop a checklist / McRebel

Gilt acclimation (truly acclimated)

Sow condition, colostrum management, attended farrowings

Sow farrowing processes and procedures

Farrowing hygiene between groups and throughout suckling phase

Stop cross-fostering

Stop transmitting feces (boots, hands, fomites, carts, etc.)

Scrupulous hygiene It is an OB ward!!!

Attempt to develop good herd immunity

Gilt acclimation, controlled exposure, vaccination, feedback

Feedback to gestating dams

Assess risk factors

Establish a process

Reinforce basic concepts

One big dose more important than lots of little doses

Risk Factors potentiate endemic pathogens

Confirmation bias in making decisions?

Thank you!

ADDED COMMENTS

Does diagnosis matter?

Endemic flareups…

specific interventions?

We can control some level of disease just by management

Hygiene, sanitation, not spreading it, etc

All interventions work better if

Sows are healthy and well fed

Facilities are clean

People are not spreading infectious agents between litters/rooms

People not tranfering disease by moveing pigs or holding them back to

Infect younger pigs

Sometimes it is useful to know the specific agent, particularly if

There are specific interventions!!!