Transcript Understanding Rotavirus – Dr. Kent Schwartz

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
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
•
•
•
•
• PRRSV:
1990
• Clostridium perfringens A: 2000
• Clostridium difficile
2005
• Rotavirus C
2010
• Rotavirus A, B
2010
• PEDV
2013
• PDCV
2013
• Seneca
virus A
2015
Extreme and constant vigilance

• Cryptosporidia
EXTERNAL BIOSECURITY
• Adenovirus
The rest are nearly always •present
Next Generation Sequencing
INTERNAL BIOSECURITY
• Microbiome and “biotics”
MANAGEMENT
• More…..
HYGIENE
E. coli
1900
Salmonella 1900
TGE
1940
Clostridium perfringens
type C –
1960’s
• Coccidiosis 1970’s
• Rotavirus A 1970’s
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 invaders
Micro-ecology changes and variations are infinite
Ongoing from birth through adult: Metagenomics & Biomes
Log bacteria counts
High numbers (billions) of C. perfringens, C. difficile, E. coli within hours
CHANGING
Physiology
pH
Anti-trypsin
Enzymes
Microflora
Diets
Age
Immunity
Time
Types and numbers of bacteria
Birth
“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
“ 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 …
Flow: rate?
Peristaltic Waves
Bacteria
RISKS
Chilling and heat stress
Pathogen load in environment
Cross fostering
Compromised passive immunity
Orderly Motility?
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?
Severity of disease = dose x virulence
Animals’ RESISTANCE
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 ???
Most agents of diarrhea are ENDEMIC 
We can find them with “tests”
 Why is disease being expressed?
Age (days)
INSULT
Agalactia
E. coli
Clostridium perfringens A
Clostridium perfringens C
Clostridium. difficile
Rotavirus A
Rotavirus B
Rotavirus C
PEDV / TGEV
Porcine deltacoronavirus
Isospora (coccidia)
PRRSV
Salmonella
0-4
x
x
x
5-8
x
8-wean
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
Mortality
can be high
can be high
low
high
low
low
low
low
high
variable
low
variable
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?)
Achieving an ACCURATE DIAGNOSIS IS A PROCESS
Collect Information
Diagnostic testing
Think, Analyze, Research
DIAGNOSTIC ACCURACY
Does it “make sense”?
Refine
Repeat
process
Treatment, Control
Identify Opportunities
Continuous Improvement
Case definition and epidemiology
Information to gather BEFORE making a “diagnosis”
• OBJECTIVE:
– Clinical signs
– Epidemiology
– Timeline
• SUBJECTIVE
– Farm History
– Area history
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
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)
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
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 will always be detected … but is it important?
Histopathology
Virulence-related disease: genotyping
E. coli Genotyping
Animal ID
Gene
A, 107 EAST1 (toxin)
LT(toxin)
STa(toxin)
STb(toxin)
Stx1 (toxin)
Stx2 (toxin)
Stx2e(toxin)
F18(pilus)
F41(pilus)
K88(pilus)
K99(pilus)
987P(pilus)
AIDA (adhesin)
EAEA (adhesin)
PAA (adhesin)
Result
Positive
Positive
Positive
Positive
Negative
Negative
Negative
Negative
Negative
Positive
Negative
Negative
Negative
Negative
Positive
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
Number of cases of COCCIDIOSIS in suckling piglets
ISU VDL
90
80
70
60
50
40
30
20
10
0
Control of Isosporosis: Example of dose and hygiene
Must 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
Type
Alpha
Beta
Epsilon
Iota
A
+
- (beta2)
-
-
B
+
+
+
-
C
+
+
-
-
D
+
-
+
-
E
+
-
-
+
• 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
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 Biosecurityhave 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
Vaccination???
• Loading and unloading procedures
PEDV booster of previously positive sows
• Crew and driver protocols
Not for “priming” – only after exposure
• Truck wash procedures
• Manure disposal biosecurity
• Swine transportation
Verify implementation!
http://www.aasv.org/aasv%20website/Resources/Diseases/PED/PEDVBiosecurity.pdf
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”
Impact of PCR test sensitivity on “frequency of diagnosis”
Rotavirus Detection: IHC (group A only) prior to 2009
PCR started in 2009  now usually positive for rotavirus
60.00%
50.00%
40.00%
%B & C
30.00%
%C only
%B only
%A
20.00%
10.00%
0.00%
2007
2008
2009
2010
2011
2012
2013
NOTE: IHC detects rotavirus A  PCR detects rotaviruses A, B and C.
Age Distribution versus Serotype
Group A only (N = 479)
Group B only (163)
7%
< 1 week
1-3 weeks
3- 6 weeks
> 6 weeks
29%
39%
25%
31%
22%
16%
31%
Group C only (N = 521)
9%
25%
56%
10%
< 1 week
1-3 weeks
3- 6 weeks
> 6 weeks
< 1 week
1-3 weeks
3- 6 weeks
> 6 weeks
PCR Positive Cases by Month
No seasonal effect
Cases tested in year 2012 (N = 1696)
% Positive
100%
85% 88% 81% 89%
90% 84%
80%
79% 81% 83%
73%
80%
72% 72%
70%
60%
50%
40%
30%
20%
10%
0%
% Positive
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
Vaccination???
– Colostrum
management
PEDV booster of previously positive sows
NotRESISTANCE
for “priming” – only after exposure
• Immunity impacts
– Proper acclimation
Where are
the data
for rotaviruses?
– Influencing
maternal
immunity
/ dam immune status
– Acclimation
– Vaccination?
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
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-ups
If 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!!!