Short Communication

A Comparison of the Lymphocyte Subpopulations ofPigs Experimentally Infected with Porcine Circovirus 2and/or Parvovirus

JUNGHYUN KIM and CHANHEE CHAE

Department of Veterinary Pathology, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University,Suwon 441-744, Kyounggi-Do, Republic of Korea

KEYWORDS:EYWORDS: Lymphocyte subpopulation; porcine circovirus; porcine parvovirus.

Porcine circovirus (PCV) has been classified in thenew family Circoviridae which share the distinctivegenomic structure of a covalently closed, circular,negative-sense, single-stranded DNA molecule(Meehan et al., 1997). Two types of PCV have beendetected and characterized, and were subsequentlynamed PCV1 and PCV2 (Meehan et al., 1998). PCV1has been recognized as the contaminant of a por-cine cell line (PK-15) for more than 25 years (Ti-scher et al., 1974). PCV2 has less than 80% nucleicacid homology with PCV1, and is associated withpostweaning multisystemic wasting syndrome(PMWS) (Hamel et al., 1998; Kim et al., 2002a).

PMWS is a porcine disease that affects mainlynursery and early growing pigs, and is characterizedclinically by poor body condition, dyspnoea, pallorof the skin and occasional icterus (Choi & Chae,2000; Kim et al., 2002a). In previous experiments,gross and histopathological lesions similar to thoseof PMWS were observed in pigs inoculated withPCV2 (Allan et al., 1999; Kennedy et al., 2000;Krakowka et al., 2000; Magar et al., 2000). Co-infec-tion with PCV2 and porcine parvovirus (PPV) in-duced lesions of greater severity and ensuing clinicaldisease (Allan et al., 1999; Ellis et al., 1999; Kennedyet al., 2000; Krakowka et al., 2000). PCV2/PPVco-infection has been demonstrated in a signifi-

cant proportion of cases of PMWS in pigs in Koreaand in Western Canada (Ellis et al., 2000; Kim et al.,2002a).

The objective of present study was to compare, byflow cytometry, alterations in lymphocyte subpopu-lations in both lymph nodes and peripheral bloodmononuclear cells (PBMC). It was hoped that thiswould afford a better understanding of the rela-tionship between PCV2 and PPV in pigs experi-mentally infected with either PCV2 or PPV orsimultaneously co-infected. The experiment wasapproved by the Institutional Animal Care and UseCommittee of Seoul National University.

Tissue culture-propagated PCV2 (strainSNUVR000463) and PPV (strain SNUVR000464)were used as the sources of viral inocula (Kim et al.,2002b). For inoculation, a PCV2 pool containing a1:2 � 105 tissue culture infective dose 50 ðTCID50Þ/mL and a PPV pool containing a 1:3 � 105 TCID50/mL was prepared as previously described (Krakowkaet al., 2000). Twenty-four colostrum-deprived con-ventional 28-day-old pigs were randomly dividedinto four groups. All pigs were seronegative for PCV,PPV, and porcine reproductive and respiratory syn-drome virus. Six pigs were inoculated with 1 mL of a1 in 20 dilution of the PCV2 pool while another sixpigs were inoculated with 1 mL of a 1 in 20 dilutionof the PPV pool. A further six pigs were inoculatedwith a mixture (2 mL) of an equal volume of a 1 in20 dilution of both the PCV2 and PPV pools. Sixnegative control pigs were inoculated with PCV-freePK-15 cell lysates. All experimental groups were heldin separate isolators (two pigs per isolator) and ex-

The Veterinary Journal 2003, 165, 325–329doi:10.1016/S1090-0233(02)00240-X

1090-0233/02/$ - see front matter � 2002 Elsevier Science Ltd. All rights reserved.

Correspondence to: Chanhee Chae, Department of VeterinaryPathology, College of Veterinary Medicine and School ofAgricultural Biotechnology, Seoul National University, Suwon441-744, Kyounggi-Do, South Korea. Tel.: +82-31-290-2736; fax:+82-31-294-4588; E-mail: swine@plaza.snu.ac.kr

amined at regular intervals. Two pigs from eachgroup were humanely killed at 10, 21, and 35 dayspostinoculation (dpi). Lymph node and bloodsamples were examined for lymphocyte subpopula-tions.

Superficial inguinal lymph nodes were collectedat necropsy. The lymphocyte subpopulations of thistissue were assessed, as they had previously beenfound to show consistent and intense labelling forboth PCV2 and PPV, and the development of his-topathological lesions at 10, 21, and 35 dpi (Kimet al., 2002b). Lymphocytes were isolated from thelymph node as described previously (Magar et al.,2000). Briefly, the lymph node was teased apartthrough a 70 lm cell stainer into cold culture me-dium (RPMI 1640 containing FBS 20%). Largeparticles were removed by sedimentation on acushion of 5 mL FBS for 5 min and the cells en-riched by passage through a Lymphocyte-H gradient(Cedarlane, Hornby). Cells at the interface werecollected, washed twice with culture medium, re-suspended in 1 mL of culture medium, and countedelectronically. Blood samples collected in tubescontaining EDTA as the anti-coagulant were used toisolate PBMC by a Ficoll–Hypaque gradient (specificdensity 1.077 g/mL).

Cell suspensions from lymph nodes and PBMCwere adjusted to a final concentration of 107 cells/mL in phosphate-buffered saline solution (PBSS)containing 1% bovine serum albumin and 0.1%sodium azide (FACS buffer). The cells were thenindirectly double-immunolabelled with monoclonalantibodies against porcine leucocyte CD2 (cloneMac80, IgG2a rat; Cedarlane), CD4 (clone PT90A,IgG2a mouse, VMRD), and CD8 (clone MIL12,IgG2a mouse; Cedarlane). After incubation for30 min followed by washing in FACS buffer, the cellswere incubated with the appropriate fluoresceinisothiocyanate (FITC)- or phycoerythrin (PE)-con-jugated goat anti-rat IgG (Caltag Lab) or with goatanti-mouse IgG (Cedarlane) antibodies. The B cellswere identified by double-labelling with FITC-con-jugated goat anti-swine IgG, rat anti-CD2 antibodies,and PE-conjugated anti-rat IgG. After washing inFACS buffer, labelled cells were fixed overnightwith 1% formaldehyde in PBS. Labelled cells werethen analyzed on a flow cytometer (FACSCAN,Becton–Dickinson). Ten thousand cells were ana-lyzed per sample using the Cell Quest (Becton–Dickinson) software. The percentage of CD2þCD4þ,CD2þCD8þ, and CD2�Igþ cell subsets were re-corded after multiparametric analysis. The Wilco-xon matched pairs signed rank test was used for the

comparison of two consecutive measurements with-in the same group, whereas the Kruskal–Wallis testwas used for comparison of the intergroup variabilitybetween two consecutive measurements.

Comparisons within each group showed that thepercentages of CD2þCD4þ cells in lymph nodes(Table I) and PBMC (Table II) decreased signifi-cantly ðP < 0:05Þ at 21 and 35 dpi compared tothose at 10 dpi. The percentages of CD2þCD8þ cellsin lymph nodes and PBMC increased significantlyðP < 0:05Þ at 21 dpi compared to those at 10 and35 dpi in the PCV2 and PPV co-infected pig group.The percentages of CD2�Igþ cells in lymph nodesand PBMC decreased significantly ðP < 0:05Þ at21 dpi compared to those at 10 and 35 dpi in thePCV2 and PPV co-infected pig group. There were nosignificant differences in the other two experimen-tal groups during the infection. The assessment oflymphocyte subpopulations between each group aresummarized in Tables I and II.

Increased levels of CD2þCD8þ cells and de-creased levels of both CD2�Igþ and CD2�Igþ cellswere observed in pigs co-infected with PCV2 andPPV. However, comparison of the proportion ofeach phenotype revealed that the reduction in ab-solute numbers of cells may be due to an increasednumber of other phenotypes. Conversely, the in-creased number of CD2þCD8þ cells may be due toan overall decrease the number of other phenotypesmeasured. Thus, even if the number of one phe-notype of cells remains unchanged, their propor-tion will differ by either the increase or reduction ofother phenotypes in the population. Further studymay be required in order to investigate the absolutenumber of lymphocytes for comparison betweenlymphocytes subpopulations.

An increase in CD2þCD8þ cells was observed inpigs co-infected with PCV2 and PPV. CD2þCD8þ

cells can be either natural killer (NK) cells, helper Tcells with the phenotype of CD2þCD4þCD8þ, asubpopulation of cdT cells (Yang & Parkhouse,1996), or cytotoxic T cells (Davis & Hamilton,1998). Although the nature of CD2þCD8þ cellscannot be identified definitely without CD3 analysis,substantial numbers of CD2þCD8þ cells are likely tobe NK cells in pigs of the age used in the presentstudy (Yang & Parkhouse, 1996). Moreover, NK cellsmay produce interferon-c which can be involved inviral inactivation (Trinchieri, 1995).

The first significant reduction in cell subpopula-tions we observed was in the CD2�Igþ (B cells)subset at 10 dpi. The next most significant decreasewas seen in the CD2þCD4þ cell subset at 21 dpi in

326 THE VETERINARY JOURNAL, 165, 3

pigs co-infected with PCV2 and PPV. However, asubset of porcine B cells express CD2 (Sinkora et al.,1998), therefore CD2�Igþ cells do not represent thetotal B cell population in pigs. These results were ingeneral agreement with those of previous studies

(Shibahara et al., 2000; Sarli et al., 2001; Segales et al.,2001). B cell depletion during dual viral infectionwas not mediated by the actual virus themselvessince the two types used in this study do not repli-cate in lymphocytes (Choi & Chae, 1999; Choi &

Table IIPercentages of lymphocyte subsets in peripheral blood mononuclear cells of pig experimentally infected

with porcine circovirus (PCV) 2 or porcine parvovirus (PPV) or both

Viruses DPI Cell subset percentages (mean� standard deviation)

CD2+CD4+ CD2+CD8+ CD2)Ig +

Control 34:97 � 1:03 46:4 � 1:75 20:95 � 1:77

PCV2 10 26:54 � 2:04 48:55 � 1:90 14:60 � 0:71

21 26:20 � 4:42 51:42 � 5:58 19:91 � 0:55

35 25:08 � 3:38 48:02 � 0:69 18:37 � 0:66

PPV 10 33:08 � 1:53 50:96 � 1:76 21:67 � 1:88

21 33:47 � 1:75 53:56 � 2:46 22:51 � 0:84

35 34:13 � 3:36 50:76 � 2:46 21:15 � 2:19

PCV2 & PPV 10 24:30 � 1:84a 52:92 � 3:14 17:38 � 0:74

21 16:74 � 1:07b;c 60:17 � 2:79b 12:22 � 2:10b;c

35 21:26 � 1:61b;a 59:57 � 0:81b 13:40 � 1:98

DPI, days postinoculation; Control, mean values form six uninfected pigs.a Significant difference from PPV-infected group (P < 0:05).b Significant difference within group (P < 0:05).c Significant difference from other groups (P < 0:05).

Table IPercentages of lymphocyte subsets in superficial inguinal lymph nodes of pig experimentally infected

with porcine circovirus (PCV)-2 or porcine parvovirus (PPV) or both

Viruses DPI Cell subset percentages (means� standard deviation)

CD2+CD4+ CD2+CD8+ CD2)Ig +

Control 47:47 � 2:21 16:12 � 0:59 17:90 � 1:27

PCV-2 10 44:02 � 1:87 17:72 � 2:01 16:55 � 0:93

21 44:12 � 2:23 18:44 � 1:01 14:77 � 2:07

35 41:99 � 1:71 15:84 � 1:22 17:07 � 0:52

PPV 10 46:15 � 1:34 16:24 � 1:75 21:61 � 2:26

21 50:51 � 2:11 17:03 � 2:47 19:99 � 1:43

35 44:03 � 2:44 16:75 � 3:45 18:46 � 0:79

PCV-2 & PPV 10 40:11 � 1:27 20:99 � 1:29 12:86 � 1:20a

21 29:74 � 2:18b;a 23:94 � 0:34b;c 10:97 � 0:95b;a

35 27:81 � 0:86b;c 20:16 � 3:51 14:82 � 4:32

DPI, days postinoculation; Control, mean values form six uninfected pigs.a Significant difference from PPV-infected group (P < 0:05).b Significant difference within group (P < 0:05).c Significant difference from other groups (P < 0:05).

LYMPHOCYTE SUBPOPULATIONS IN PIGS INFECTED WITH PCV2 AND PPV 327

Chae, 2000). B cell depletion may therefore becontrolled by other indirect mechanisms involvingvirus–host interaction. It seems likely that host fac-tors induced by the virus co-infection were involvedin B cell depletion. This would have consequencesin terms of the compromising the immunity of an-imals with challenged with concomitant or second-ary infections. It is known that farm pigs with PMWSoften carry secondary infection (Kim et al., 2002a).

This study demonstrated that a strong influx ofNK cells occurs in the lymph node and PBMC dur-ing co-infection with PCV2 and PPV. In contrast,PCV2 or PPV did not themselves induce a cellularimmune response in pigs. The synergistic mecha-nisms involved in the co-infection induced alter-ation of lymphocytes subpopulations are unknown.One possible explanation is that certain physiolog-ical stimuli induced in pigs co-infected with PCV2and PPV results in the increase NK cells. This syn-ergistic relationship should be the topic of furtherstudies.

ACKNOWLEDGEMENTS

The research reported here was supported by Min-istry of Agriculture, Forestry and Fisheries-SpecialGrants Research Program (MAFF-SGRP), and BrainKorea 21 Project, Republic of Korea.

REFERENCES

ALLANLLAN, G. M., KENNEDYENNEDY, S., MCNEILLYCNEILLY, F., FOSTEROSTER, J. C.,ELLISLLIS, J. A., KRAKOWKARAKOWKA, S. J., MEEHANEEHAN, B. M. & ADAIRDAIR, B.M. (1999). Experimental reproduction of severe wast-ing disease by co-infection of pigs with porcinecircovirus and porcine parvovirus. Journal of Compara-tive Pathology 121, 1–11.

CHOIHOI, C. & CHAEHAE, C. (2000). Distribution of porcineparvovirus in porcine circovirus-2-infected pigs withpostweaning multisystemic wasting syndrome as shownby in-situ hybridization. Journal of Comparative Pathology123, 302–5.

CHOIHOI, C. & CHAEHAE, C. (1999). In-situ hybridization for thedetection of porcine circovirus in pigs with postwea-ning multisystemic wasting syndrome. Journal of Com-parative Pathology 121, 265–70.

DAVISAVIS, W. C. & HAMILTONAMILTON, M. J. (1998). Comparison of theunique characteristics of the immune system in differ-ent species of mammals. Veterinary Immunology andImmunopathology 63, 7–13.

ELLISLLIS, J. A., BRATANICHRATANICH, A., CLARKLARK, E. G., ALLANLLAN, G.,MEEHANEEHAN, B., HAINESAINES, D. M., HARDINGARDING, J., WESTEST, K. H.,KRAKOWKARAKOWKA, S., KONOBYONOBY, C., HASSARDASSARD, L., MARTINARTIN, K. &MCNEILLYCNEILLY, F. (2000). Coinfection by porcine circovi-ruses and porcine parvovirus in pigs with naturallyacquired postweaning multisystemic wasting syndrome.Journal of Veterinary Diagnostic Investigation 12, 21–7.

ELLISLLIS, J., KRAKOWKARAKOWKA, S., LAIRMOREAIRMORE, M., HAINESAINES, D., BRATA-RATA-

NICHNICH, A., CLARKLARK, E., ALLANLLAN, G., KONOBYONOBY, C., HASSARDASSARD, L.,MEEHANEEHAN, B., MARTINARTIN, K., HARDINGARDING, J., KENNEDYENNEDY, S. &MCNEILLYCNEILLY, F. (1999). Reproduction of lesions of post-weaning multisystemic wasting syndrome in gnotobi-otic piglets. Journal of Veterinary Diagnostic Investigation11, 3–14.

HAMELAMEL, A. L., LININ, L. L. & NAYARAYAR, G. P. S. (1998).Nucleotide sequence of porcine circovirus associatedwith postweaning multisystemic wasting syndrome inpigs. Journal of Virology 72, 5262–7.

KENNEDYENNEDY, S., MOFFETTOFFETT, D., MCNEILLYCNEILLY, F., MEEHANEEHAN, B., ELLISLLIS,J., KRAKOWKARAKOWKA, S. & ALLANLLAN, G. M. (2000). Reproductionof lesions of postweaning multisystemic wasting syn-drome by infection of conventional pigs with porcinecircovirus type 2 alone or in combination with porcineparvovirus. Journal of Comparative Pathology 122, 9–24.

KIMIM, J., CHOIHOI, C. & CHAEHAE, C. (2002a). Pathogenesis ofpostweaning multisystemic wasting syndrome repro-duced by co-infection with Korean isolates of porcinecircovirus-2 and porcine parvovirus. Journal of Compar-ative Pathology (in press).

KIMIM, J., CHUNGHUNG, H.-K., JUNGUNG, T., CHOHO, W.-S., CHOIHOI, C. &CHAEHAE, C. (2002b). Postweaning multisystemic wastingsyndrome of pigs in Korea: prevalence, microscopiclesions and coexisting microorganisms. Journal of Vet-erinary Medical Science 64, 57–62.

KRAKOWKARAKOWKA, S., ELLISLLIS, J. A., MEEHANEEHAN, B., KENNEDYENNEDY, S.,MCNEILLYCNEILLY, F. & ALLANLLAN, G. (2000). Viral wastingsyndrome of swine: experimental reproduction ofpostweaning multisystemic wasting syndrome in gno-tobiotic swine by coinfection with porcine circovirus 2and porcine parvovirus. Veterinary Pathology 37, 254–63.

MAGARAGAR, R., LAROCHELLEAROCHELLE, R., THIBAULTHIBAULT, S. & LAMONTAGNEAMONTAGNE,L. (2000). Experimental transmission of porcinecircovirus type 2 (PCV 2) in weaned pigs: a sequentialstudy. Journal of Comparative Pathology 123, 258–69.

MEEHANEEHAN, B. M., CREELANREELAN, J. L., MCNULTYCNULTY, M. S & TODDODD, D.(1997). Sequence of porcine circovirus DNA: affinitieswith plant circoviruses. Journal of General Virology 78,221–7.

MEEHANEEHAN, B. M., MCNEILLYCNEILLY, F., TODDODD, D., KENNEDYENNEDY, S.,JEWHURSTEWHURST, V. A, ELLISLLIS, J. A., HASSARDASSARD, L. E., CLARKLARK, E.G., HAINESAINES, D. M. & ALLANLLAN, G. M. (1998). Character-ization of novel circovirus DNAs associated with wast-ing syndromes in pigs. Journal of General Virology 79,2171–9.

SARLIARLI, G., MANDRIOLIANDRIOLI, L., LAURENTIAURENTI, M., SIDOLIIDOLI, L., CERATIERATI,C, ROLLAOLLA, G. & MARCATOARCATO, P. S. (2001). Immunohisto-chemical characterisation of the lymph node reactionin pig post-weaning multisystemic wasting syndrome.Veterinary Immunology and Immunopathology 83, 53–67.

SEGALESEGALES, J., ALONSOLONSO, F., ROSELLOSELL, C., PASTORASTOR, J., CHIANINIHIANINI, F.,CAMPOSAMPOS, E., LOPEZ-FUERTESOPEZ-FUERTES, L., QUINTANAUINTANA, J., RODRIGUEZ-ODRIGUEZ-

ARRIOJAARRIOJA, G., CALSAMIGLIAALSAMIGLIA, M., PUJOLSUJOLS, J., DOMINGUEZOMINGUEZ, J.& DOMINGOOMINGO, M. (2001). Changes in peripheral bloodleukocyte population in pigs with natural postweaningmultisystemic wasting syndrome (PMWS). VeterinaryImmunology and Immunopathology 81, 37–44.

SHIBAHARAHIBAHARA, T., SATOATO, K., ISHIKAWASHIKAWA, Y. & KADOTAADOTA, K. (2000).Porcine circovirus induces B lymphocytes depletion inpigs with wasting disease syndrome. Journal of VeterinaryMedical Science 62, 1125–31.

328 THE VETERINARY JOURNAL, 165, 3

SINKORAINKORA, J., REHAKOVAEHAKOVA, Z., SINKORAINKORA, M., CUKROWSKAUKROWSKA, B.,TLASKALOUA-LASKALOUA-HOGENOVAOGENOVA, BIANCHIIANCHI, A. T. & DE GEUSE GEUS, B.(1998). Expression of CD2 on porcine B lymphocytes.Immunology 95, 443–9.

TISCHERISCHER, I., RASCHASCH, R. & TOCHTERMANNOCHTERMANN, G. (1974). Char-acterization of papovavirus- and piconavirus-like parti-cles in permanent pig kidney cell lines. Zentralblatt furBakteriologie, Parasitenkunde, Infektionskrankheitenund Hygiene. Erste Abteilung Originale. Reihe A 226, 153–67.

TRINCHIERIRINCHIERI, G. (1995). Natural killer cells wear differenthats: effector cells of innate resistance and regulatorycells of adaptive immunity and of hematopoiesis.Seminars in Immunology 7, 83–8.

YANGANG, H. & PARKHOUSEARKHOUSE, R. M. E. (1996). Phenotypicclassification of porcine lymphocyte subpopulations inblood and lymphoid tissues. Immunology 89, 76–83.

(Accepted for publication 20 July 2002)

LYMPHOCYTE SUBPOPULATIONS IN PIGS INFECTED WITH PCV2 AND PPV 329