Jul 2, 2003 - new syndrome has been associated with porcine circovirus type 2 (PCV2) (2). However PMWS is sporadic in contrast with the widespread ...
4th International Symposium on Emerging and Re-emerging Pig Diseases – Rome June 29th – July 2nd, 2003
COMPARATIVE SEROLOGICAL AND VIROLOGICAL STUDY OF HERDS WITH AND WITHOUT POSTWEANING MULTISYSTEMIC WASTING SYNDROME (PMWS) 1
1
2
R. Larochelle , R. Magar , S. D’Allaire 1
Laboratoire d’hygiène vétérinaire et alimentaire, Agence canadienne d’inspection des aliments, Saint-Hyacinthe, Canada; 2 Département de sciences cliniques, Faculté de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, Canada PMWS, etiology, PCV2, PRRSV
Introduction The postweaning multisytemic wasting syndrome (PMWS) was identified as a new condition in 1997 in Western Canada and is characterized by wasting, dyspnea, lymph node hypertrophy and sometimes diarrhea and jaundice (4). This new syndrome has been associated with porcine circovirus type 2 (PCV2) (2). However PMWS is sporadic in contrast with the widespread PCV2 infection in swine and PCV2 has been present in the swine population since at least 1985 in Canada (9). In addition, field studies have demonstrated that PCV2 is not always associated with clinical signs and lesions of PMWS (7, 8). Although generally only mild to moderate lesions have been reproduced experimentally in young pigs following inoculation with PCV2 (3, 10), severe disease has been demonstrated following co-infections with porcine parvovirus (PPV) (1) and porcine reproductive and respiratory syndrome virus (PRRSV) (5) and following immunostimulations models (6). While cofactors such as coinfecting pathogens and immunostimulation have been suggested based on experimental studies, the exact mechanisms triggering PMWS in the field still are unresolved and very few field studies in herds with and without PMWS have been published. The present study was undertaken to determine whether there were differences in the kinetics of PCV2 infection and in the genomic sequences of PCV2 strains identified in PMWS and non-PMWS herds from Québec. Other factors possibly related to the triggering of PMWS were also investigated, namely the presence of PRRSV and PPV and the use of immunomodulating agents such as vaccines.
source of piglets. In 4 of 5 PMWS-positive herds (A, B, C, D) clinical signs were first observed between 9 and 15 weeks of age, whereas in one herd (E) they were observed at 6-7 weeks of age (Table I). Clinical signs were observed either in late nursery (C, E) or early grower stage (A, B, D), usually between 3 and 6 weeks following the transfer of the animals in the respective stage. In addition 4 of 5 PMWS herds were PRRSV-positive. Aside from vaccination against atrophic rhinitis in herd C, no vaccination was performed in piglets. Vaccination against PRRS was practised in sows of herds A, B, C and D. In herds without PMWS clinical signs, PRRSV was present in herd N but herd P was free of PRRSV. In herd N, sows were vaccinated for PRRS and piglets, for Haemophilus parasuis at 6 and 9 weeks of age. Serological results obtained in herd A showed the presence of PCV2 antibodies in all piglets at 3 weeks of age. The number of PCV2 antibody positive pigs decreased until 11 weeks followed by an increase at 15 weeks, all pigs being positive by 19 and 23 weeks of age. A similar serological profile to that of herd A was obtained for 5 of the other 6 herds of the study, that is 3 PMWS herds (B, C, D) and the 2 non-PMWS herds (N, P). In all these 5 herds the number of pigs with PCV2 antibodies decreased from 3 to 11 weeks and increased from 15 to 23 weeks in spite of the fact that 2 herds did not have PMWS (Table I). When titrations were performed on sera from herd A (PMWS), a decrease in the level of antibodies followed by a seroconversion was clearly demonstrated (Figure 1). Initially low to moderate titers (1/801/640) observed at 3 weeks decreased, and 9 of 10 pigs became seronegative by 11 weeks. Then seroconversion to PCV2 was observed in 6 pigs at 15 weeks (1/160-1/1280). By 19 weeks all pigs had seroconverted and high antibody titers were obtained (1/5120-1/20480) and remained high in all pigs at 23 weeks of age. The general serological profile obtained in a non-PMWS herd (P) was similar to the one obtained in herd A except that at week 15, titers were greater (Figure 1).
Materials and methods A total of 7 herds from Québec were studied, 5 of these with clinical signs of PMWS (A - E) and 2 without (N, P). Sixty blood samples were collected from pigs of each herd at approximately 4 week intervals from 3 to 23 weeks of age. Blood samples from pigs of herd A were collected from the same 10 pigs over time (cohort study), whereas in the other herds, 10 pigs in the different age groups were sampled at one time (cross-sectional study). Detection of antibodies to PCV2 and titrations were performed by indirect immunofluorescence as previously described (9). Antibodies to PCV2 were titrated in sera from 2 herds with PMWS (A, E) and one herd without PMWS (P). Level of antibodies to PRRSV was determined by a commercial enzyme-linked immunosorbent assay. For PCR, DNA was extracted from 200 µl of serum and the presence of PCV2 nucleic acid was determined using PCV2specific primers previously described (7). For detection of PRRSV, RNA was extracted from 250 µl of serum. PCR was carried out using pairs of primers amplifying ORF5 and ORF7. The presence of PPV DNA was detected by PCR using specific primers amplifying a region coding for the nonstructural protein gene (protocol and primers provided by Dr. P. Tijssen, IAF/INRS, Laval, Canada). The PCV2 strains detected in each herd were sequenced over the entire genome. Several pairs of primers were designed and used to generate overlapping PCR amplified fragments (8).
Figure 1. Comparative PCV2 serological profiles and PCV2 PCR detection in herds A (PMWS) and P (non-PMWS). 100000
8
10000
6
1000
4
100
2
10
0
1 3
7 11 Farm A - PCR
15
Farm A - Serology
19 23 Farm P - PCR Farm P - Serology
Mean PCV2antibody titers
Number of PCV2 PCR-positive pigs
10
Age (weeks)
In herd E, in which clinical signs were observed at 6 to 7 weeks of age, a different pattern of PCV2 infection occurred. At 3 weeks of age, 8 of 10 pigs demonstrated low to moderate antibody titers, similar to those obtained for herds A and P at this timepoint (P>0.05). By 7 weeks, 9 of 10 serum samples were positive and an increase in titers suggested a seroconversion in this herd at this timepoint. All 10 pigs sampled at 11, 15, 19 and 23 weeks demonstrated a strong PCV2 antibody-positive reaction by indirect immunofluorescence.
Results All herds were farrow-to-finish operations with only one
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4th International Symposium on Emerging and Re-emerging Pig Diseases – Rome June 29th – July 2nd, 2003
Table 1. Characteristics of each herd. Age (weeks) Herd PMWS status
PRRSV status
Vaccination in piglets
A
+
+
none
PRRSV vaccination in sows +
PMWS clinical signs 12-13
B
+
+
none
+
15-16
15
11-19
11
7-11
negative
C
+
+
rhinitis
+
9-11
15
11-23
11
3-11
negative
D
+
+
none
+
12-14
15
15-23
11
11-19
15
E
+
-
none
-
6-7
7
7-19
negative
N
-
+
H. parasuis
+
none
15
15-19
11
P
-
-
none
-
none
15
15-23
negative
Antibody kinetics for PRRSV in the 5 herds positive to PRRSV (A, B, C, D, N) followed the same general profiles as for PCV2 except that seroconversion to PRRSV preceeded that to PCV2 by one month (Table I). Most pigs at 3 weeks of age demonstrated the presence of PRRSV antibodies whereas at 7 weeks of age few pigs were seropositive and S/P ratios were lower. At 11 weeks most pigs were seropositive and all pigs remained seropositive thereafter. In all herds, either PMWS or non-PMWS, PCV2 could be identified by PCR in serum samples collected at certain ages only (Table I). In PMWS herds, the highest rate of PCV2 detection was generally first observed at the time of PCV2 seroconversion, that is at 7 weeks of age for herd E and at 15 weeks of age for the other herds (A, B, C, D). PCV2 could still be detected in many sera (24/50) collected at 23 weeks of age. In both non-PMWS herds (N, P), PCV2 nucleic acid was detected mainly at 15-19 weeks of age, and at 23 weeks of age 5/20 sera were still PCV2-positive. The total number of PCV2-positive serum samples was greater in PMWS herds (43%) than in non-PMWS herds (25%) and furthermore the PCR signal was generally stronger. In all 5 PRRSV-positive herds, the highest rate of virus detection in sera varied between 7 and 11 weeks of age, 4 weeks before the first detection of PCV2 in these sera. In contrast with PCV2, PRRSV was detected in pigs of 3 weeks of age in one herd. Both PCV2 and PRRSV could be detected by PCR in the serum of a few pigs in the PMWS herds as well as in a non-PMWS herd. PPV nucleic acid was detected in a few sera from only 3 of 7 herds, including a nonPMWS herd (N). Detection of PPV occurred mainly at 19 weeks of age, which was after the onset of PMWS in affected herds. Sequence analysis of each of the 7 PCV2 strains showed that the complete genome of each strain was 1768 nt in length. PCV2 strains identified in all herds except herd D were closely related, displaying 99.4 to 100% nucleotide sequence identity. The PCV2 strains of 3 herds, herds B and C and the non-PMWS herd N, demonstrated 100% nucleotide identity. The complete homology between these PCV2 strains could not be related to a common geographical area, source of piglets or of breeding animals. PCV2 strains from herds P (non-PMWS) and E (PMWS) presented 99.8% homology with those of herds B, C and N. In the present study, kinetics of PCV2 infection in both PMWS and non-PMWS herds were found similar in most cases. The triggering of PMWS in the herds could not be linked to either co-infections with PRRSV or PPV nor to the use of a specific immunostimulant such as vaccines or to particular genomic differences between the PCV2 strains identified. Additional field studies are needed to better understand risk factors
PCV2 PCV2 in seroserum conversion (PCR) 15 15-23
PRRSV PRRSV in PPV in seroserum serum conversion (PCR) (PCR) 11 11-15 19
negative negative 11-15
19-23
negative negative
associated with PMWS and the triggering mechanisms of this disease. References 1. Allan G, Kennedy S, McNeilly F, Foster J, Ellis J, Krakowka S, Meehan B, Adair B. 1999. Experimental reproduction of severe wasting disease by co-infection of pigs with porcine circovirus and porcine parvovirus. J. Comp. Pathol. 121: 1-11. 2. Ellis J, Hassard L, Clark E, Harding J, Allan G, Willson P, Strokappe J, Martin K, McNeilly F, Meehan B, Todd D, Haines D. 1998. Isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome. Can. Vet. J. 39: 44-51. 3. Ellis J, Krakowka S, Lairmore M, Haines D, Bratanich A, Clark E, Allan G, Konoby C, Hassard L, Meehan B, Martin K, Harding J, Kennedy S, McNeilly F. 1999. Reproduction of lesions of postweaning multisystemic wasting syndrome in gnotobiotic piglets. J. Vet. Diagn. Invest. 11: 3-14. 4. Harding JCS, Clark EG. 1997. Recognizing and diagnosing postweaning multisystemic wasting syndrome (PMWS). Swine Health Prod. 5: 201-203. 5. Harms PA, Sorden SD, Halbur PG, Bolin SR, Lager M, Morosov I, Paul PS. 2001. Experimental reproduction of severe disease in CD/CD pigs concurrently infected with type 2 porcine circovirus and porcine reproductive and respiratory syndrome virus. Vet. Pathol. 38: 528-539. 6. Krakowka S, Ellis JA, McNeilly F, Ringler S, Rings DM, Allan G. 2001. Activation of the immune system is the pivotal event in the production of wasting disease in pigs infected with porcine circovirus-2 (PCV2). Vet. Pathol. 38: 31-42. 7. Larochelle R, Antaya M, Morin M, Magar R. 1999. Typing of porcine circovirus in clinical specimens by multiplex PCR. J. Virol. Methods 80: 69-75. 8. Larochelle R, Magar R, D’Allaire. S. 2002. Genetic characterization and phylogenetic analysis of porcine circovirus type 2 (PCV2) strains from cases presenting various clinical conditions. Virus Research 90: 101-112. 9. Magar R, Müller P, Larochelle R. 2000. Retrospective serological survey of antibodies to porcine circovirus type 1 and type 2. Can. J. Vet. Res. 64: 184-186. 10. Magar R, Larochelle R, Thibault S, Lamontagne L. 2000. Experimental transmission of porcine circovirus type 2 (PCV2) in weaned pigs : a sequential study. J. Comp. Pathol. 123: 258-269.
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