Diagnosis of post-weaning multisystemic wasting syndrome in pigs in Brazil caused by porcine circovirus type 2

Diagnóstico da síndrome da refugagem multissistêmica em suínos no Brasil causada pelo circovírus suíno tipo 2

VETERINARY MEDICINE

Diagnosis of post-weaning multisystemic wasting syndrome in pigs in Brazil caused by porcine circovirus type 2

Diagnóstico da síndrome da refugagem multissistêmica em suínos no Brasil causada pelo circovírus suíno tipo 2

J.R. Ciacci-Zanella; N. Morés

Embrapa Suínos e Aves Caixa Postal 21 89700-000 - Concórdia, SC

ABSTRACT

This report describes the first preliminary characterization of porcine circovirus type 2 (PCV2) isolates from pigs affected with post-weaning multisystemic wasting syndrome (PMWS) in Brazil. Diseased pigs were examined at necropsy and by histopathology. Macroscopic and microscopic analyses revealed lesions reported to be typical of PMWS, which included, respectively, emaciation, enlargement of lymph nodes, thymus atrophy and interstitial pneumonia, and granulomatous lymphadenitis with syncytial cells, among others. Using nested polymerase chain reaction (PCR) or imunoperoxidase it was possible to detected DNA or antigen of PCV2, respectively. The PCR' s amplified fragment could be differentiated from PCV1 and PCV2 from one another by restriction fragment length polymorphism (RFLP) analysis. PCV2 DNA was detected in 70% (14/20) of samples of pigs with clinical signs and lesions associated with PMWS. This study shows that PCV2 is associated with lesions and symptoms indicative of PMWS in pigs. It is also shown that the Brazilian PCV2 isolates may have variation in their genome.

Keywords: swine, porcine circovirus, post-weaning multisystemic wasting syndrome, preliminary characterization, PCV2

RESUMO

Este trabalho descreve a primeira caracterização preliminar de isolados de circovírus suíno tipo 2 (PCV2) a partir de órgãos de suínos acometidos pela síndrome da refugagem multissistêmica (SRM) no Brasil. Leitões doentes foram examinados à necropsia e por histopatologia. Análises macroscópicas e microscópicas demonstraram lesões típicas de SRM, respectivamente, emagrecimento, aumento do volume dos linfonodos, atrofia de timo e pneumonia intersticial, e linfadenite granulomatosa com células sinciciais, entre outras. A presença de antígeno ou DNA do PCV2 foi demonstrada por imunoperoxidase ou reação da polimerase em cadeia "nested" (nested PCR), respectivamente. Foi possível diferenciar PCV1 e PCV2 por análises de polimorfismo de fragmento de restrição (RFLP) do fragmento amplificado da PCR. O DNA do PCV2 foi detectado em 70% (14/20) das amostras obtidas de suíno com sintomatologia clínica e lesões associadas com SRM. Este estudo apresenta a associação de PCV2 com lesões e sinais clínicos de SRM em suínos e também indica que os isolados do PCV2 brasileiros podem apresentar variações genômicas.

Palavras-chave: suíno, circovirus suíno, síndrome da refugagem multissistêmica, caracterização, PCV2

INTRODUCTION

Circoviruses are small viruses, non-enveloped, icosahedral and are different from other animal viruses because their genome is composed of circular single stranded DNA (Tischer et al., 1974, 1982). Moreover, the genome of circuviruses is one of the smallest among animal viruses, being 1,76 kb (Todd, 2000). Members of the family Circoviridae include the chicken anemia virus, the psittacine beak and feather disease virus and the porcine circovirus (Todd, 2000). Two porcine circoviruses (PCV) have already been identified, PCV type 1 or PCV1, a persistent contaminant of the continuous tissue culture cells (PK-15, porcine kidney cells) which does not cause clinical symptoms in swine and PCV type 2 or PCV2, which has been associated with the occurrence of post weaning multisystemic wasting syndrome (PMWS) (Allan, Ellis, 2000). A common

feature of this family of viruses in animals is the association with illnesses that cause injuries in lymphoid tissues and the imunosuppression (Todd, 2000).

PMWS in pigs was first identified in Canada in 1991 and nowadays has a worldwide distribution (Allan et al., 1998; Ellis et al., 1998; Morozov et al., 1998). It is characterized by a progressive weight loss, respiratory signs, enlargement of lymph nodes and jaundice (Allan et al., 1998). Pathological lesions include inflammations in several organs, such as lymphadenopathy, interstitial pneumonia, hepatitis, interstitial nephritis and pancreatitis. (Ellis et al., 1998; Harding et al., 1998). PCV2 antigens and nucleic acids have been demonstrated in tissues of pigs with PMWS (Alan et al., 1998; Ellis et al., 1998; Kuipel et al., 1998; Kennedy et al., 2000; Larochelle et al., 1999; 2000a; 2000b). The accepted case definition of PMWS is based on three major features: a) clinical disease, b) gross and histological lesions, c) most importantly, the association of PCV2 with these lesions.

Porcine circovirus infection or PMWS has been considered an emerging disease. The prevalence of the virus in Brazil is still unknown but it may be disseminated in Brazilian swine herds. PCV2 is not a new virus, but a recently identified virus (Ellis, Allan, 2000). Many studies indicate that diseases or syndromes associated with PCV2 have affected swine herds worldwide for at least 15 years. For the last 4 years, the diseases associated to PCV2 have caused important economical impact to swine production worldwide. There are no available vaccines or effective treatment. Just control measures associated with the improvement of sanitary and management practices are recommended presently (Guilmoto, Wessel-Robert, 2000).

In this paper the association of PCV2 with cases of wasting disease in pigs in Brazil was investigated. Weaned pigs originated from swine farms where gradual wasting in the nursery and grower units had occurred were submitted for examination. PCV2 was isolated and its specific antigens were detected by immunohistochemistry in organs of pigs affected with PMWS and also in PK15 cells inoculated with those organs suspensions. This paper also reports the detection of PCV2 DNA by nested PCR from organs of pigs affected with PCV2. RFLP (restriction fragment length polymorphism) was used to specifically identify the PCV2 amplified fragments. In addition to recognizing PCV2 nucleic acid, the RFLP also provided a preliminary characterization of different Brazilian PCV2 isolates. Thus, the objectives of this work were to establish a diagnostic methodology and to investigate the occurrence of PCV2 in pigs which presented symptoms of PMWS.

MATERIAL AND METHODS

Twenty, 5 to 12-week-old piglets from eight different farms of the Southern Region of Brazil were studied. They were all live pigs submitted to diagnostic by necropsy at the Animal Health Laboratory at Embrapa Suínos e Aves, Concórdia, SC. These piglets were originated from different farms which had an ongoing history of progressive emaciation in pre-fattening piglets. Diarrhea and gastric ulcers were not always frequent but emaciation and low weight gain were, and these usually culminated in death of 5 to 10% of the piglets at this stage.

All of the swine brought to diagnosis were thoroughly examined. The pigs were euthanized by electrocution and a complete necropsy was performed. Samples of tissues, including brain, lungs, lymph nodes (superficial inguinal and mesenteric), tonsils, kidneys, liver, intestines, spleen and thymus were fixed in 10% buffered formalin and were embedded in paraffin, sectioned at 5µm thick and stained with hematoxylin and eosin for light microscopical examination.

For viral isolation lung, tonsil, lymph nodes, among others were collected at necropsy. Tissues were pooled and homogenized in F10 (Cultilab, Campinas, SP, Brazil) and 199¹ media containing penicillin (100U/µl) and streptomycin (100µg/ml) to make approximately a 10% suspension. The sample was clarified, filtered through a 0.22µm pore - size filter, and used as an inoculum. Viral isolation was carried out in PK-15 cell culture shown by PCR to be free of PCV1 contamination. Flasks of 25cm³ of semi-confluent PK-15 cells were inoculated with 1ml of the organ suspension and incubated for 1h at 37°C in CO₂ atmosphere for virus adsorption. Then, 4ml of fresh complete medium (F10 or 199 media supplemented with 10% FCS and antibiotics) was added, and the flasks were incubated for an additional 4h. Cells were treated with 300mM D-glucosamine for 30min and then incubated in fresh complete medium for a further 24h (Ellis et al., 1998). Inoculated cells were split every two days and treated with D-glucosamine 24h after each passage. After four passages, infected cells were removed from the flasks with trypsin treatment, washed briefly with PBS, resuspended in 0,5 ml complete growth medium and placed on slides (previously treated with 10% poly-L-lysine) for 10min in the hood or until dried. The monolayer of cells were fixed with cold methanol for 1min and analyzed by imunoperoxidase with polyclonal porcine anti-PCV2 serum.

After fixation, cells were washed in PBS, water, treated with protease XIV (Sigma - Aldrich, St Louis, MO, USA) (0.5mg/ml) for 30min at 37°C. The cells were washed with distilled water and blocked with 30 volumes of H₂O₂ 1% in distilled water for 15min at 37°C. Following this treatment, the slides were washed three times for 5min with PBS and incubated at 37°C for 30min. The cells were further blocked with normal rabbit serum for 30min at 37°C. After washing the cells briefly with PBS, they were incubated with primary polyclonal porcine anti PCV2 serum (1:10 dilution in PBS, kindly provided by Dr. Bruce Broderson, University of Nebraska Lincoln, USA) for 2h at 37°C. Cultures were washed three times with PBS for 5min and immunostained with a 1:50 dilution of rabbit anti-pig IgG conjugated with peroxidase² at 37°C for 1h. Cells were then washed with PBS three times, incubated with AEC (3-amino 9-ethyl-carbazole - hydrogen peroxide substrate)² solution for 5min at room temperature, and counterstained with Meyer' s hematoxylin for 10sec. Slides were then mounted with an aqueous mounting medium and examined microscopically. Negative control procedures included the omission of primary antiserum.

DNA was isolated from swine organs (lung, tonsil, lymph nodes, among others) previously kept frozen at -80°C. Tissues were minced, digested overnight at 55°C in 5ml of TEN buffer per gram of tissue (10mM Tris, 1mM EDTA, 100mM NaCl) containing 0,5% SDS and 0.1mg proteinase K. DNA was extracted twice with phenol: chloroform: isoamyl alcohol (25:24:1), precipitated with 10M ammonium acetate and cold 100% ethanol (twice the final volume) and kept at 20°C for 2h. The DNA was washed in 70% ethanol, air dried and resuspended in TE (10mM Tris, 1mM EDTA, pH 7,4) (Sambrook et al., 1989).

PCR amplifications were carried out as described by Hamel et al. (2000). The primers used were designed from conserved sequences among PCV isolates of PCV1 and PCV2 and two reactions were performed, first PCR outer and later the PCR nested. The PCR outer produced an amplified fragment of 534 bp, which corresponded to nucleotide positions 771-797 (5' -GGT GGA ACT GTA CCT TTT TTG GCC CGC-3' ) and 1304-1282 (5' -CTC CTC CCG CCA TAC AAT CCC CC-3' ) respectively. Following this reaction, another PCR (nested) was performed and the internal primers used corresponded to the nucleotides 831-859 (5' -GAA TGG TAC TCC TCA ACT GCT GTC CCA GC -3' ) and 1268-1242 (5' -CCA CTC CCG TTA ATT CAC ACC CAA ACC-3' ) of PCV2, and produced an amplified fragment of 438bp. Reaction mixes were made up in ultra pure Millipore water containing 0.8mM dNTP mix (all four), 0.3mM each primer, 10U/ ml of Taq DNA polymerase (Promega, USA) and Taq DNA polymerase buffer (1,5 mM MgCl₂)³. Aliquots of reaction mix were dispensed in 0.5ml polypropylene microcentrifuge tubes and 1µg of the each DNA sample was added to its PCR reaction tube, mixed, microcentrifuged and placed in the thermocycler (Eppendorf Mastercycler Gradient, Germany). The amplifications (PCR outer and nested) occurred with an initial step of 4min at 95°C, followed by 35 cycles of 1min at 90°C, 1min at 50°C and 2min at 70°C. At the end of the program, an additional incubation of 70°C for 10min was done. In order to perform the nested PCR, 1µl of the outer PCR reaction was added to its appropriate tube containing the set of primers NEST (nucleotides 831-1268). Gel electrophoresis was carried out in 1% agarose gel in TBE buffer.

Positive samples were re-amplified by nested PCR and the amplified product was digested with restriction enzymes KpnI (New England Biolabs, USA) or HinfI³ for 4h at 37°C, according to manufacturers recommendations. The digested samples were then separated by electrophoresis in 1% agarose gel in TBE buffer and photographed.

RESULTS AND DISCUSSION

In order to establish a diagnostic methodology for PCV2 and confirm its association with cases of PMWS, 20 wasted pigs from different farms were examined, materials were collected and submitted to laboratory tests. To verify whether wasted pigs had PMWS, it was necessary to combine the data on clinical disease (signs), macroscopic and microscopic lesions and the presence of the agent (PCV2) in affected organs. Table 1 summarizes the results of this study.

Table 1
- Click to relarge

At necropsy, enlargement and paleness of lymph nodes were the most obvious lesions in all pigs examined. Extreme emaciation, non-collapsed lungs, hypotrophy of the thymus and connective tissue edema were also consistent findings. Gastric ulcer was present in one pig but was not a frequent finding.

Microscopic lesions attributable to PMWS were found in lymphoid organs (including lymph nodes, tonsil, Peyer' s patches and spleen), liver, kidney and lungs. Varying degrees of lymphocellular depletion, affecting both lymphoid follicles and parafollicular zones, and progressive multifocal to diffuse infiltration of lymphoid tissue by large histocytic cells were the characteristic lesions. Syncytial cells were seen frequently, especially in the lymph nodes and Peyer' s patches. Cytoplasmic inclusions in histocytic cells were also observed in Peyer' s patches and tonsil as well as necrotic lymphoid cells. Lung lesions included multifocal interstitial pneumonia with variable dissemination over the organ. Other changes included infiltration of inflammatory cells (histocytes) and severe bronchitis. The most common liver lesion was lymph-histocytic infiltration of portal zones, to variable degrees of intensity. Multifocal necrosis of single hepatocytes was also observed. In the kidney, a mild multifocal interstitial nephritis with discrete vasculitis, especially on the renal cortex area was present. In the brain discrete multifocal gliosis and mononuclear meningoencephalitis was also observed. Variation in intensity and distribution of lesions in target organs in cases of PMWS probably depends on the stage of the disease in the affected pig (Rosell et al., 1999).

PK-15 cells inoculated with swine organ suspensions and treated with D-glucosamine were passaged four times and no cytopathic effect (CPE) was observed. This finding was observed previously by other investigators (Ellis and Allan, 2000). However, additional PCR analyses performed in those inoculated cells showed the presence of PCV2 DNA following four passages (results not shown), suggesting the infection of PK15 cells by PCV2 present in the inoculum.

Immunoperoxidase staining in PK-15 monolayers was accomplished in order to detect PCV2 antigen in cells inoculated with tissue homogenates of pigs affected with PMWS. PCV2 antigen was detected in six out of 20 pigs with PMWS symptoms. In all positive cells examined immunolabeling was most frequently found in the cytoplam of the cells. The same was observed when immunofluorescence labelling was performed (results not shown). Although the specificity of our reagents is not complete, the immunoperoxidase examination used a swine polyclonal serum generated from a pig from the United States and it may not recognize all Brazilian PCV2 strains, our system was able to detect positive cells inoculated with organ samples from six different pigs affected with PCV2, and all of those were also positive by PCR and by histology analysis.

Swine organs samples were also analyzed by PCR in order to amplify a sequence of PCV2 DNA present in those tissues. This test amplifies sequences of PCV2 and also of PCV1. PCR amplified products were analyzed by RFLP using restriction enzymes. This test, which is based on a Canadian classification (Hamel et al., 2000), identifies PCV1, PCV2A, B, C, D or E, using the enzymes KpnI or HinfI. Thus, it is possible to identify PCV2 and to differentiate it from PCV1. Among all 20 positive samples, 14 contained PCV2 DNA. Although PCV2 DNA was not amplified from some wasted pigs (6/20), the PCR and histopathology exams together gave the most consistent results. The absence of PCV2 DNA amplification from six samples of wasted pigs may be explained by the lack of viral DNA in those samples, due to the stage of the disease, but mostly due to the negativity of the other results. Wasted pigs are not necessarily pigs with PMWS and there are many other pathogenic agents which cause wasting in post-weaning pigs (Allan et al., 1998; Ellis et al., 1998; Kuipel et al., 1998).

Fig. 1 shows a PCR assay for PCV that was carried out on routine clinical specimens. All samples were from pigs from three different farms affected with PMWS and the presence of microscopic lesions and PCV2 antigen were already confirmed by histopathology and imunoperoxidase tests, respectively. Fig. 1 also shows the results of restriction analysis using KpnI or HinfI of the amplified fragment from nested PCR. The enzyme KpnI did not cut the PCV2' fragment but did cut PCV1 into two smaller fragments, of 333 and 102 base pairs (bp). It was possible to identify which PCV was amplified in the sample. On the other hand, the enzyme HinfI cuts the PCV1 amplicon into two fragments, 269 and 166bp, and the sequence amplified from PCV2 into several different sizes, which categorizes PCV2 into five different profiles-PCV2A (198, 164, 38, 38bp), PCV2B and PCV2C (274, 164bp), PCV2D (236, 164, 38bp), PCV2E (181, 164, 38, 38, 17bp) (Hamel et al., 2000).

The digestion profile of all three samples was similar. However there was a small variation of the sizes of digested amplified fragments of the sample 2 (line 6), in comparison with samples 1 and 3, which may suggest a genetic diversity among them and may indicate differences among these PCV2 isolates The other eight samples also presented a differentiated electrophoresis profile (data not shown). This study is a preliminary characterization of Brazilian PCV2 isolates. Nucleotide sequencing and aligning of isolates will provide a more informative analysis.

Lines 1 and 11 are molecular weight markers of 100bp ladder and 1 kb, respectively. Sample 1 (lines 2, 3 and 4), sample 2 (lines 5, 6 and 7) and sample 3 (lines 8, 9 and 10) were obtained from organs of pigs with PMWS from three different farms which had a history of progressive emaciation in the grower unit. Lines 2, 5, and 8 are undigested nested PCR products (438bp). Lines 4, 7 and 10 are amplified products digested with the enzyme KpnI. Lines 3, 6 and 9 are amplified products digested with the enzyme HinfI.

The isolation of PCV2 from pigs affected with wasting diseases, in addition to the demonstration of PCV2 antigen and nucleic acid in close association with lesions in affected animals, supports the diagnosis of PCV2 as an etiologic agent of PMWS in pigs in Brazil.

Recebido para publicação em 19 de setembro de 2002

Recebido para publicação, após modificações, em 10 de abril de 2003

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Publication Dates

• Publication in this collection
  13 Jan 2004
• Date of issue
  Oct 2003

History

• Reviewed
  10 Apr 2003
• Received
  19 Sept 2002