Dinko Novosel, PhD Croatian Veterinary Institute, Pathology department

Savska cesta 143, 10000 Zagreb, Croatia email: dinko.novosel@gmail.com

Prevention and control of transboundary and emerging viral infections of pigs in

Central Europe – Croatian situation

• 1. ASF – in a background of CSF control measures

• 2. PEDV, PDCoV

• 3. PPV 1-4

• 4 PRRSV

• 5. PCV2

• 6. HEV and TTSuV 1and 2

Instruction:

-all phylogeny is analyzed using MJ Phylogenetic networks

-immunohistochemistry – positive signal brown, background blue

-in situ hybridization, in situ PCR – postive signal blue, background green

Topics

African swine fewer -last outbreak of CSF in

2006, 1.5 year after

vaccination is

suspended

-last confirmed case in

2008.

-control measures

CSF

African swine fewer -two level biosecurity

measure, counties close

to Bosnia and Serbia

and others

-suspicious case-

pathology, serology and

PCR + obligatory

monitoring of all farms

serology and PCR for

CSFV and ASF ASF

PED and PRCoV

• PED – not present

• -in situ RT PCR tested, but not jet optimized

• PRCoV- never confirmed, antibodies in 2 wild boars in 2006.

• -lazy to purchase Mab-s for IHC

PED

Swine influenza

• -in 2012 health problem in one farm

• -PCV2 is identify as a „bad guy”

• -SIV is identify in background od PCV2 infection

• -actually this was the first confirmation of SIV in Croatia – ever

• -research is ongoing and we have several isolates in MDCK cells

• -2000. antibodies in wild boars, in 2006-2010. serorevalance in wild boars was 9.7%

PPV1-4

• PPV1 – very old and known disease

• -vaccination against reproductive failures

• -weak implication in reproductive disorders

• PPV3 – confirmed in fatteners

• - can be possibly attributed in patogenesis of pneumonia

• -Croatia ancestory region

• PPV2 – confirmed in fatteners and fetuses

• -can be possibly attributed in patogenesis of pneumonia

• PPV4 confirmed in fatteners and fetuses

• - can be possibly attributed in patogenesis of reproductive failures and lymphnode lesions in animals affacted with PMWS

PPV1 • -easy to recognize, 1985. reported in Croatia, seroprevalance was 38%

• -routine vaccination was introduced, seroprevalnce decline to 1%

• -involved in 2.4% of reproductive failure cases

Farm

ID

Total

no. of

fetuses

Number of

reproductive

animals

RF1

PRRSV

Status2

PMWS

status3

No. (%) of fetuses positive for indicated virus by PCR

Infections

found in

fetuses

PRRSV PCV2 TTSuV1 TTSuV2 PPV1 PPV2 PPV3 PPV4

A 14 4000 18% Category

1 Affected 3 (21.4)

14

(100) 1 (7.1) 2 (14.3) 0 (0)

PRRSV,

PCV2,

TTSuV1,

TTSuV2

B 10 2500 5% Category

1 Affected

10

(100) 0 (0)

2

(20.0) PCV2,

PPV4

C 8 12000 2% Category

2 Affected

8

(100) 0 (0)

1

(12.5) PCV2,

PPV4

D 11 800 5% Category

1 Affected

11

(100)

1

(9.1) 0 (0)

2

(18.2)

PCV2,

PPV2,

PPV4

E 2 3000 2% Category

3 -

2

(100) 0 (0) PCV2

F 33 3000 8% Category

1 Affected 3 (9.1)

33

(100) 1 (3.0) 0 (0)

1

(3.1)

PRRSV,

PCV2,

TTSuV1,

PPV4

G 2 200 2% Category

3 -

2

(100) 0 (0) PCV2

H 3 200 1,5% Category

3 -

3

(100)

2

(100) 0 (0)

PCV2,

PPV1

Total 83 6 (7.2) 83

(100) 2 (2.4) 2 (2.4)

2

(2.4)

1

(1.2) 0 (0)

6

(7.2)

Abreviations: 1 RF - Reproductive failures;

2 according to Holtkamp’s criteria;

3 according to PMWS 3 diagnostic key criteria.

PPV2 • -virus is in low viral load, in lungs and lymph

nodes, poorly express SLAIIDQ, almost not CD3

• -no other causative agent observed in proliferative and interstital pneumonia

• -relathionship with strains in neighbour countries

• -low prevalance in fetuses

PPV3 • -present in lungs (in bronchus) and lymph

nodes, virus is in low prevalence, in lymphocytes like cells, poorly express SLAIIDQ, CD3 even less

• -involved in pneumonia primary cause by PRRSV

• -not detected in fetuses

• -Croatia seems to be ancestor region

PPV4 • -present in lymph nodes, in lymphocyte like

cells mostly not express CD3 or SLAIIDQ antigen

• -in relationship with some lesions in lymph node in PMWS cases

• -prevalent in fetuses, some indication that it might be involved in reproductive failures and cause systemic infection in fetus

PRRSV • -introduced in 1994., two

doses of Duroc semen was smuggled from Netherland

• -devastate Croatian pig production, estimations were 17mil Euros

• -significant reproductive failures

• -one outbreak of „hot PRRSV” in 2007

• -several large pig producers close farms

• -no national control measures

• -still unknown exact prevalence

• -the first cases were confirmed by IHC

• -developed ISH, in sit RT PCR needs further optimisation

Imunohistochemistry

In situ hybridization

In situ RT PCR

PCV2

• -first outbreak in 2001.

• -devastated effect in Croatian pig production, after vaccine is introduced situation cooled down, but still unvaccinated herds suffer economical loses

• -in 2006 is reported PMWS in wild boars

Identified viruses in fetuses

Farm ID Total

samples

No. (%) positive for indicated virus

PRRSV PCV2 TTSuV1 TTSuV2 PPV1 PPV2 PPV3 PPV4

M 14 3 (21,4) 14 (100) 1 (7,1) 2 (14,3)

A 10

10 (100)

2 (20)

B 8

8 (100)

1 (12,5)

D 11

11 (100)

1 (9,1) 2 (18,2)

F 2

2 (100)

S 33 3 (9,1) 33 (100) 1 (3)

1 (3,1)

T 2

2 (100)

K 3

3 (100)

2 (100)

Total 83 6 (7,2) 83 (100) 2 (2,4) 2 (2,4) 2 (2,4) 1 (1,2) 0 (0) 6 (7,2)

PCV2-RD Diagnosis -histopathology + IHC or ISH

-common finding in almost all fetuses, but there si no specific lesion in heart and ISH or IHC are always negative -both in situ PCR methods revealed presence of PCV2 in Kupffer cells (not know!) -role in reproductive failures during subclinic infection – unknown -ongoing research...

Direct in situ PCR

Indirect in situ PCR

in situ hybridization

Imunohistochemistry

+

-

Origin of samples TTSuV1 positive

TTSuV2 positive

PCV2 RD ( Σ 63) 1 (1,58%) 2 (3,17%) Interstitial nefritis(Σ 14)

1 (7,14%) 8 (57,14%)

PCV2 SD wild boars (Σ 3)

3 (100%) 3 (100%)

Proliferative and necrotizing pneumonia (Σ 5)

1 (20%) 4 (80%)

Torque teno sus virus

-indication that TTSuV2 is involved -other researchers already made connection with TTSuV infection and glomerulonephropathy

Proliferative and necrotizing pneumonia

• High mortality, pigs per acute dies!

• Still is unresolved etiology PCV2 and PRRSV play significant role

• sever form of proliferative interstitial pneumonia

• High replication level of PCV2

• TTSuV2 identify in lymphocytes

PNP lymph node PMWS lymph node

ISH ssDNA PCV2 ISH ssDNA PCV2

ISH dsDNA PCV2 ISH dsDNA PCV2

In situ PCR TTSuV2

PCV2/PRRSV PCV2/PRRSV

no air

necrosis

TTSuV1 TTSuV2

HEV in HR

• Seroprevalence in pigs 89%

• Prevalence of virus in sera 13.3% , in liver 8.1%

Hepatitis -in PMWS affected animals -in farms with high HEV prevalence we observed outbreaks of edema gut disease without reason.

Imunohistochemistry In situ RT PCR

Take home massage:

• Many known and unknown virus circulates, real impact in health status or production effectiveness is unknown

• If you search you will find many things!

• with PCV2, everything is possible...

Acknowledgment

• PCV2 sequencing, PPVs research to Tamas Tuboly, Daniel Cadar and Attila Csagola , University in Budapest

• PCV2 phylogeny – Joaquim Segales, Marti Cortey from CReSA

• PRRSV - Tomasz Stadejek, University in Warsaw

• SIV and PED – Helena Grgic, Josepha Del Lay University in Guelph

• ISH for PRRSV – Lars Eric Larsen DTU Copenhagen

• In situ PCR – Tahar Ait Ali, Roslin Institute and Rodolfo Ocadiz-Delgado –CINVESTAV , Mexico

Thank you for your attention!