豬圓環病毒3型相關疾病及母豬場動態流動規律 楊振 南京農業大學.pdf

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1、楊振，副教授Zhen Yang,DVM,MS,Associate Professor南京農業大學 動物醫學院College of Veterinary Medicine,Nanjing Agricultural University中國中國長沙長沙2023中國李中國李曼養豬大會曼養豬大會Porcine circovirusuPorcine circovirus(PCV),Circovirus,Circoviridae family,circular,covalently closed,single-stranded DNA(ssDNA)genomeuPCV1:Non pathogenic uP

2、CV2:PMWS,PCVADuPCV3:UnclearuPCV4:Unknown clinical relevancePCV11974PCV21990sPCV32015PCV42020(Opriessnig,T.,and et.al,2020)PCV3 genomeuA circular,covalently closed single-stranded DNA genome(2,000 nt)uOpen reading frames(ORFs):ORF1:nonstructural replicase protein Rep(296-297 aa),48%similar to the rep

3、licase protein of PCV2 ORF2：capsid protein,the structural protein(214 aa),26-36%similar to the capsid protein of PCV2 ORF3：231 aa non-structural protein of unknown functionuGenotyping:PCV3a,3b;genotypes?(Palinski et al.,2016;Phan et al.,2016;Opriessnig et al.,2020)Key features of PCV3 epidemiologyuP

4、CV3 was first reported in U.S.swine herds in 2015uPCV3 has worldwide distribution uPCV3 was detected in 1993 in Sweden,1996 in Spain and China,and 2002 in the United Kingdom(Palinski et al.,2016;Phan et al.,2016;Klaumann et al.,2018;Ye et al.,2018;Sun et al.,2018;Collins et al,2017)Key features of P

5、CV3 epidemiology uTransmission and duration of PCV3 shedding PCV3 DNA has been detected in a variety of samples,including mummified and stillborn fetal tissues,serum,heart,lung,brain,lymph node,liver,kidney,et.al.Direct contact and vertical transmission PCV3 can be detected in blood for at least 42

6、days and in nasal secretions for 28 days post infection PCV3 can be detected for more than 5 months in wild boars uPotential to cause cross-species infections Detected in domestic pigs,wild boars,cattle,chamois,dog,donkey,fallow deer,laboratory mice,mouflon,roe deer and red deer(Czyewska et al.,2020

7、;Wang et al.,2019;Franzo et al.,2019;Zhang et al.,2018;Wang et al.,2021;Jiang et al.,2019;Temeeyasen et al.,2020)DiagnosisuPCR uConventional PCR,qPCR,multiplex PCR,loop-mediated isothermal amplification,droplet digital PCR uImmunohistochemistry(IHC)and in situ hybridization(ISH)uSequencing uNext-gen

8、eration sequencing(NGS)uSanger sequencing uELISAuIndirect ELISAuCap proteins,virus-like particles(Palinski et al.,2016;Phan et al.,2016;Ha et al.,2018;Chen et al.,2019；Han et al.,2019；Wang et al.,2019；Yang et al.,2019；Hou et al.,2021；Zhang et al.,2019；Deng et al.,2012；Mora-Daz et al.,2020)Virus isol

9、ationuPCV3 isolation in cell culture is challenging Swine testicle(ST)cells and porcine kidney-15(PK-15)cells were not working initially Infectious clone reported in 2019 Isolation of PCV3 in PK-15 cells and primary porcine kidney cells in 2020 Tissue homogenates,with Ct values ranging between 7.5 a

10、nd 19.3 No cytopathic effects on PK-15 and primary porcine kidney cells(Palinski et al.,2016;Jiang et al.,2019;Mora-Daz J et al.,2020)Clinical signsuMultiple clinical signs have been reported in pigs infected with PCV3uExperimental infections were mild and inconsistentuSubclinical infectionsClinical

11、 signsField observationExperimental reproductionRespiratory EntericReproductiveIntegumentaryNeurologicWeight lossLamenessSystemicPalinski et al.,2016;Phan et al.,2016;Kedkovid et al.,2018;Arruda et al.,2020;Kim et al.,2018;Saporiti et al.,2020;Zhai et al.,2017;Jiang et al.,2019;Mora-Daz et al.,2020)

12、;Temeeyasen et al.,2021;Wang et al.,2021;Alomar et al.,2021)Histologic lesionsHistologic lesionsField observationExperimental reproductionArteriolitis/periarteritis MyocarditisSystemic inflammationEncephalitis and meningitisInterstitial pneumoniaCutaneous and renal Lymph node and spleenHepatitis(Pal

13、inski et al.,2016;Phan et al.,2016;Arruda et al.,2019;Temeeyasen et al.,2021;Mora-Daz et al.,2020;Jiang et al.,2019)Histologic lesions are not clear and more work need to be doneHistologic lesions(Phan(Phan etet al.,2016;al.,2016;Arruda B etet al.,al.,2019)Prevention and control uLimited information

14、 on PCV3 control uVaccine Cross protection between PCV2 and PCV3 is unlikely PCV2 vaccines had no viremia reduction of PCV3,and did not have an impact on PCV3 circulation RNA-particle platform Sequivity PCV3 vaccine Reduce PCV3 prevalence in processing fluids;improve reproductive outcomes?Recombinan

15、t vaccine:pseudorabies virus with TK/gE gene deletion combined with PCV3 Cap protein(Palinski et al.,2016;Taylor et al.,2020;Woniak et al.,2019;Stika et al.,2020;Yao et al.,2022)ContentsuCharacterize clinical cases associated with PCV3 in the US shortly after its discovery Frequency of porcine circo

16、virus type 3 detection and histologic lesions in clinical samples from swine in the United StatesuCharacterize PCV3 dynamic in commercial sow farms Preferred sampling methods in sow farms to detect PCV3 PCV3 dynamic in farrowing housesIntroductionuPCV3 has been detected in multiple farms in the US s

17、ince 2015;however,the overall detection frequency in clinical samples was unclearuPCV3 associated clinical signs and lesions in clinical cases were rarely reported in early yearsuLater on,diverse clinical signs and lesions have been associated with PCV3;however,a limited number of pigs and cases wer

18、e examineduDescribe PCV3 detection frequencies in veterinary diagnostic samplesuInvestigate the presence of PCV3 and its association with clinical signs and histologic lesions in a large database of clinical samples from pigs in the United StatesPCV3 frequency in diagnostic submissionsu730 diagnosti

19、c swine submissions between February 2016 and January 2018 contained 2,177 samples submitted from 474 sites located in 21 states in the United StatesuPCR assay results were positive for PCV3 for 577 of 2,177(27%)samples,255 of 730(35%)submissions,181 of 474(38%)sites,and 17 of 21(81%)states uPCV3 wa

20、s a very frequent finding in diagnostic samplesYang Z,Marthaler DG,Rovira A.Frequency of porcine circovirus 3 detection and histologic lesions in clinical samples from swine in the United States.J Vet Diagn Invest.2022 Jul;34(4):602-611.doi:10.1177/10406387221099538.Epub 2022 Jun 8.PMID:35674058;PMC

21、ID:PMC9266519.PCV3 frequency by specimen typeSpecimen type*Sample PCV3+/no.samples(%)Tissue homogenate226/1,295(17)Serum112/483(23)Oral fluid90/101(89)Processing fluid 79/98(81)Lung15/76(20)Blood11/16(69)Fluid8/12(67)Heart2/11(18)Testes5/10(50)Bulbourethral gland1/7(14)Epididymis 5/8(63)Prostate gla

22、nd4/8(50)Semen0/8(0)Vesicular gland2/8(25)Intestine 1/6(17)Brain0/3(0)Feces3/3(100)Lymph node1/3(33)Skin0/3(0)Spinal cord3/3(100)Bone marrow1/2(50)Kidney0/2(0)Placenta0/2(0)Spleen0/2(0)Feedback0/2(0)Skeletal muscle1/1(100)Of 28 different specimen types tested for PCV3,19 specimen types had 1 sample

23、positive for PCV3Tissue homogenate(a mixture of fresh tissues including lung,heart,kidney,spleen,and lymph nodes)was the most common specimen followed by serum,processing fluid,and oral fluidOral fluids and processing fluids were among the specimens with the highest positive ratesPCV3 frequency in p

24、igs with various clinical signsuClinical sign information was available for analysis from 1,323 pigs from 730 submissionsuThe positivity rate of PCV3 in pigs with various clinical signs ranged from 13%in pigs with CNS signs to 28%in pigs with lameness or weight lossuSignificant associations were lac

25、king between clinical signs and the presence of PCV3Clinical signFrequency(%)Odds ratio(95%CI)Fisher exact test,p valueRespiratory163/746(22)0.93(0.67,1.28)0.679GI78/383(20)0.84(0.62,1.14)0.280Weight loss24/87(28)1.37(0.84,2.25)0.225Lameness20/72(28)1.38(0.80,2.36)0.242Sudden death17/71(24)1.11(0.63

26、,1.95)0.767CNS7/56(13)0.48(0.22,1.08)0.097Other21/83(25)1.20(0.72,2.02)0.492Fetal death57/284(20)NANAPCV3 age distribution and PCV2+PRRSV co-detectionuPCV3 was detected in pigs of all agesuThe PCV3 positivity rates among fetuses(210),piglets(158),nursery pigs(476),and finishing pigs(289)was 1521%，an

27、d 58%in adults(77)uThe total PCV3+PRRSV,PCV3+PCV2,and PCV2+PRRSV co-detection rates were 9%,5%,and 9%,respectivelyuPCV3 detection was significantly associated with PCV2 detection(p When does it matter or what are the associated histologic lesions?uIn 58 different histologic lesions,PCV3 frequency in

28、 pigs with various lesions ranged from 0%in pigs with ear necrosis or epidermitis to 63%in pigs with cardiac vasculitis or perivasculitisuPCV3 detection significantly increased the odds of pigs having myocarditis,cardiac vasculitis or perivasculitis,or interstitial pneumoniauCt values were significa

29、ntly lower in pigs with myocarditis,cardiac vasculitis or perivasculitis,interstitial pneumonia,or hepatitisLesionPCV3 positive rateOdds ratio(95%CI)Fisher exact test,p valueCt value,median(min.,max.)Estimated Ct difference(95%CI)Wilcoxon p valueInterstitial pneumonia26/81(32)2.07(1.20,3.55)0.01026.

30、8(16.4,39.6)3.8(0.4,7.9)0.025Myocarditis11/24(46)3.51(1.50,8.21)0.00723.0(16.4,39.4)8.1(2.8,13.5)0.004Cardiac vasculitis and perivasculitis8/10(80)9.44(2.38,37.51)0.00121.5(18.5,32.9)10.6(3.8,15.2)0.001Liver,hepatitis9/31(29)1.72(0.76,3.91)0.23826.4(20.7,35.3)4.1(0.4,9.9)0.038Description of fetal de

31、ath casesPCV3(%)PRRSV(%)PCV2(%)PPV1(%)PPV2(%)Positive submissions(rate)14(22)4(6)1(2)5(8)1(2)Positive samples(rate)62(22)12(4)6(2)8(3)1(0.4)64 fetal death submissions,including 283 fetuses(184 had histologic descriptions)Most fetuses(199/283;70%)were negative for all of the tested viruses172 fetuses

32、 and was 1.533 cm with a median of 20 cm.PCV3-positive fetuses,the CR length was 1033 cm with a median of 20 cm(p 0.05)The most common lesion in fetuses was epicarditis(51/184).Significant association was lacking between epicarditis and PCV3 detection or with the PCV3 Ct valueDiscussionuDetecting PC

33、V3 is common in diagnostic submissions and a positive result alone does not indicate that PCV3 plays a role in the development of diseaseuAssociation with myocarditis,cardiac vasculitis,and interstitial pneumonia were found;however,association with encephalitis,lymphadenitis and kidney lesions sugge

34、sted in previous studies were not founduAn unexpectedly high PCV3 detection rate in fetuses,suggesting that PCV3 can cross the placental barrier easily and replicate in fetal tissues,and supports a potential role of PCV3 as a cause of fetal deathuThe results can be readily applied to the interpretat

35、ion of diagnostic investigations of clinical disease in swine,providing important information for the definition and diagnosis of PCV3 cases ConclusionsuPCV3 is widespread in the US,and it can be detected in multiple specimen types and in pigs of all ages uNo association between PCV3 detection and c

36、linical signs was founduPCV3 detection was associated with myocarditis,cardiac vasculitis,and interstitial pneumonia in pigsuA high PCV3 detection rate was observed in aborted fetuses,which suggests PCV3 could cause fetal deathLimitationsuAll tissue cases received at MN-VDL were still biased to some

37、 extent,as most of the samples were from sick populations,were not a perfect representation of the US swine industry uPCV3 positive status could not be determined at the tissue level,but at the pig level because the PCR test was performed on a tissue homogenate containing multiple organsContentsuCha

38、racterize clinical cases associated with PCV3 in the US shortly after its discovery Frequency of porcine circovirus type 3 detection and histologic lesions in clinical samples from swine in the United StatesuCharacterize PCV3 dynamic in commercial sow farms Pilot studies Preferred sampling methods i

39、n sow farms to detect PCV3 PCV3 dynamic in farrowing housesComparison of different sample types for PCV3 detectionuSurveillance uSensitivity uAgreement uProcessing fluids,under wipe and umbilical cord bloodPCV3 detection by sample typeuOut of 134 litters from a 2500 sow farm in China,the PCV3 detect

40、ion rate in udder wipe was the highest(77%),followed by processing fluid(72%)and umbilical cord blood(49%)uSignificant differences were found between piglet umbilical cord blood and udder swabsuThere was no significant difference in the distribution of PCV3 Ct values of different types of positive s

41、amples(P0.05,Kruskal-Wallis test)uThe analysis of Lins Concordance Correlation Coefficient(LCC)showed that the PCV3 Ct values of positive udder wipe and processing fluids had the best concordance VariablesOR95%CIPSample typesUdder wipeReferenceUmbilical cord blood0.2730.1580.4690.01Processing fluids

42、0.7510.4281.3180.318Yi Wang,Zhen Yang,unpublished dataParity and production parametersuSignificant difference of PCV3 detection rate between P1 and P2 was observeduPCV3 had a higher detection rate in P2 than P1 by under wipeuTotal born,stillborn and mummies Total born and stillborn,no significant di

43、fferences between PCV3 positive and negative litters PCV3-positive litters had a higher numbers of mummies than PCV3-negative litters(0.52:0.19,P 0.05)VariablesOR95%CIPParityFirst parityReferenceSecond parity1.9621.2323.1250.05Yi Wang,Zhen Yang,unpublished dataPCV3 dynamic in farrowing roomsDays aft

44、er farrowingPCV3 positivePCV3 negativeTotal littersPositivity22P11112313483%4.690.19671033113477%14983613473%21983613473%uLitters PCV3 detection,udder wipe samplesuDay 1,7,14,21,no differences uThe detection rate of PCV3 was the highest on Day 1 after farrowing and dropped in the subsequent sampling

45、sYi Wang,Zhen Yang,unpublished dataDiscussion and limitationuPCV3 detection rate in farrowing rooms was highuProcessing fluids and udder wipe samples had a higher detection sensitivity than pooled umbilical bloods with a fair concordanceuPCV3 detection rate may be associated with higher mummified li

46、tters and high paritiesuPCV3 dynamics in the farrowing rooms were still unclearuFuture perspective:Large scale perspective studies on PCV3 dynamics in sow farms and downstream nursery and finishersuDr.Yangs PCV3 project-A call for collaboration and industry funds Y 15720804413 AcknowledgmentsFunding

47、 support:Jiangsu Natural Science Foundation#BK20210401Swine Health Information Center grant 18-201Zhen Yang Start UP fundUS-China Joint DVM Scholarship ProgramChinese Scholarship Council My students:Yi Wang Qiyang ZhangJiamao YangChenglin HouNAU swine infectious disease team:Ping Jiang Xianwei WangY

48、ufeng LiJuan Bai Gongguan LiuXing LiuDanchen YangYanni GaoHuanshan GroupKangning ZhaoYuetian LiZekun BaoCollege of Veterinary Medicine,Nanjing Agricultural UniversityCollege of Veterinary Medicine,University of MinnesotaVeterinary Diagnostic Laboratories,University of Minnesota Leman Swine Conference Veterinary Veterinary Diagnostic Diagnostic LaboratoryLaboratory VeterinaryVeterinary PopulationPopulation MedicineMedicineUMN swine group:Albert RoviraDouglas Marthaler Montserrat TorremorellKimberly VanderWaalTodd KnutsonFangzhou ChenFabio VannucciSunil KumarThank you!