Veterinary Quarterly Isolation and characterization ...

This article was downloaded by: [39.158.130.9] On: 03 July 2015, At: 15:28 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: 5 Howick Place, London, SW1P 1WG

Veterinary Quarterly Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tveq20

Isolation and characterization of porcine circovirus type 2 from pigs showing signs of post‐weaning multisystemic wasting syndrome in the Netherlands f

b

c

a

G.J. Wellenberg , S. Pesch , F.W. Berndsen , P.J.G.M. Steverink , W. Hunneman d

a

d

b

e

, T.J.K. Van der Vorst , N.H.M.T. Peperkamp , V.F. Ohlinger , R. Schippers ,

J.T. Van Oirschot & M.F. de Jong

d

a

Department of Mammalian Virology , Institute for Animal Science and Health (IDLelystad) , PO Box 65, Lelystad, 8200 AB, the Netherlands b

BioScreen European Veterinary Disease Management Center GmbH, Minster , Germany c

Veterinary practice Diessen , Diessen, the Netherlands

d

Animal Health Service , the Netherlands

e

Boehringer Ingelheim Vetmedica , Alkmaar, the Netherlands

f

Department of Mammalian Virology , Institute for Animal Science and Health (ID‐Lelystad) , Edelhertweg 15, PO Box 65, Lelystad, 8200 AB, the Netherlands Phone: +31–320–238219 Fax: +31–320–238219 E-mail: Published online: 01 Nov 2011.

To cite this article: G.J. Wellenberg , S. Pesch , F.W. Berndsen , P.J.G.M. Steverink , W. Hunneman , T.J.K. Van der Vorst , N.H.M.T. Peperkamp , V.F. Ohlinger , R. Schippers , J.T. Van Oirschot & M.F. de Jong (2000) Isolation and characterization of porcine circovirus type 2 from pigs showing signs of post‐weaning multisystemic wasting syndrome in the Netherlands, Veterinary Quarterly, 22:3, 167-172, DOI: 10.1080/01652176.2000.9695049 To link to this article: http://dx.doi.org/10.1080/01652176.2000.9695049

PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions

fr.=: rm.l.Ir rl

Ink F: Oft WI Ina Oulll

11111\_2/1111

611L-1-

_

ISOLATION AND CHARACTERIZATION OF PORCINE CIRCOVIRUS TYPE 2 FROM PIGS SHOWING SIGNS OF POST-WEANING MULTISYSTEMIC WASTING SYNDROME IN THE NETHERLANDS G.J.Wellenberg*, S. Pesch2, F.W. Berndsen3, P.J.G.M. Steverink1, W. Hunneman4, T.J.K. Van der Vorst1, N.H.M.T. Peperkamp4, V.F. Ohlinger2, R. Schippers5, J.T. Van Oirschot, and M.F. de Jong4. Vet Quart 2000; 22: 167-72

Downloaded by [39.158.130.9] at 15:28 03 July 2015

Accepted for publication: February 14, 2000.

SUMMARY Pigs with wasting syndrome were examined for macroscopic and histopathological lesions, and for porcine circovirus (PCV). Histopathological lesions were comparable to those previously documented for post-weaning multisystemic wasting syndrome (PMWS). In addition, in seven out of ten examined PMWS-affected pigs focal-to-slight mononuclear meningitis and focal cerebral mononuclear infiltrates (4 out of 10) were observed. A virus was isolated from organs and sera from pigs showing wasting syndrome. An immunoperoxidase monolayer assay and an indirect immunofluorescence assay were performed on the infected PK-15 and Dulac cell cultures, respectively, and both assays indicated the presence of PCV type 2 (PCV2). The nested-polymerase chain reaction (nPCR) technique, based on the use of PCV2 specific oligonucleotides, revealed specific amplified products of 481 bp. Nucleotide sequence analysis of the entire genome of the Dutch PCV isolate 24657 NL showed a homology with known nucleotide sequences of porcine PCV type 1 (PCV1) and PCV2 isolates of 77.1% and >96%, respectively. This is the first report of the isolation and characterization of PCV2 in PMWS-affected pigs in the Netherlands. Keywords: PCV1, PCV2, PMWS, virus isolation, nucleotide sequence analyses. INTRODUCTION Post-weaning multisystemic wasting syndrome (PMWS) is an emerging disease in growing nursery and fattening pigs that is

being detected increasingly often in pig-producing countries throughout the world (1, 3, 9, 10, 16, 17). PMWS most often affects five to 12-week-old piglets, and is clinically characterized by a poor physical condition, progressive weight loss,

tachypnoea, dyspnoea, pallor and occasionally jaundice. Consistent gross pathological findings are bronchopneumonia and lymph node enlargement. Histopathological lesions in affected lymph nodes are characterized by lymphocyte depletion, lympho-histiocytic cellular infiltrations, and by the pre-

sence of multinucleated giant cells (4, 7, 8, 15). Under 1

Department of Mammalian Virology, Institute for Animal Science and Health (IDLelystad), PO Box 65, 8200 AB, Lelystad, the Netherlands.

2 BioScreen European Veterinary Disease Management Center GmbH, Minster, -1

' 5

Germany. Veterinary practice Diessen, Diessen, the Netherlands. Animal Health Service, the Netherlands. Boehringer Ingelheim Vetmedica, Alkmaar, the Netherlands.

Address for correspondence and reprint requests to GJ. Wellenberg M.Sc., B.Sc., Institute for Animal Science and Health (ID-Lelystad). Department of Mammalian Virology, Edelhertweg 15, PO Box 65, 8200 AB, Lelystad, the Netherlands. Telephone:

+31-320-238219, Fax: +31-320-238050. E-mail: [email protected]

Abbreviations: PCV1, porcine circovirus type 1; PCV2, porcine circovirus type 2; PMWS, post-weaning multisystemic wasting syndrome.

167

T II E

experimental conditions, a concurrent PCV2 and porcine parvovirus (PPV) infection induced symptoms in pigs comparable as those described for PMWS (2, 5). However, PCV2 in combination with other viruses, such as porcine reproductive and respiratory syndrome virus (PRRSV) has also been detected in PMWS-affected pigs (4, 9, 14).

Until now, PMWS has not been described in the Netherlands. The present report describes the first isolation and characterization of PCV2 in PMWS-affected pigs in the Netherlands. MATERIALS AND METHODS Farms and animals Pigs showing signs of wasting syndrome were sent in for necropsy. The pigs came from three different farms.

Clinical history

Farm 1 Three nine-week old pigs were sent in for further investigation. The animals came from a well-managed herd, containing 270 sows, and had a very good hygiene status. The pro-

blem started in August 1998 with symptoms such as respiratory distress, inappetence, unthrifteness, diarrhoea and arthritis. The wasting pigs became pale and jaundiced at a later stage of the disease. Symptoms started one to two weeks after weaning. The morbidity among the pigs was 15%, while the mortality was 7%. From August 1998 to July 1999, the morbidity ranged from 2 to 15% (average of 5%), and the mortality was 4.3%.

Farm 2 This herd obtained pigs for fattening from farm 1. The clinical problems started in October 1998. Within one to two weeks after arrival, apparently healthy pigs showed symptoms of respiratory distress, inappetence, and unthrifteness. The mortality in this herd was 6%. Seven pigs with a poor physical condition, four of unknown age and three animals of 11, 12 and 13 weeks of age respectively, were sent in for necropsy.

Farm 3 Fattening pigs showed growth retardation, poor clinical condition, and acute symptoms of respiratory distress and diarrhoea. In the month of October 1998 12-15 pigs died (herd of approximately 1000 fattening pigs). Two 16-week old pigs were presented for post mortem examination.

Collection of samples

At necropsy, pigs from all three farms were examined for gross pathological findings. The ileum, mesenteric lymph nodes, lungs, kidneys, and brains were sampled for histological examination. Additionally, inguinal lymph nodes,

VETERINARY QUARTERLY, VOL 22, No 3, JULY, 2000

I-1

P F-71

,H

saik r

,17--Th

AA

rillONIIN Aim rriu' IL.- Iry lido

spleen, and tonsils (pigs from farms 1 and 2), and colon (pigs

from farms 2 and 3), were collected. Parts of the liver, spleen, ileum, kidneys, brain, tonsil, mesenteric lymph nodes, and inguinal lymph nodes of three pigs from farms 1 and 2 were sampled at necropsy for virus isolation on PK-15

cells and for nested-polymerase chain reaction (nPCR). Mesenteric lymph nodes from two pigs from farm 3 were sampled for virus isolation on PK-15 cells and for nPCR. The tissue samples were stored at -70°C prior to processing. Sera collected from other PMWS-affected pigs from farm 1

(not sent in for necropsy) were used for virus isolation on Dulac cells as described below.

Histology For histological examination, parts of all tissue samples were fixed in 10% neutral buffered formalin, in paraffin embedded, sectioned at 5 pm, and stained with haematoxylin and eosin (HE). Virus isolation

Small blocks of organs and tissues from each pig were Downloaded by [39.158.130.9] at 15:28 03 July 2015

pooled and a 10% tissue suspension was prepared in Earle's

minimal essential medium (EMEM). These suspensions were homogenized and subsequently clarified by centrifugation at 1500 x g for 10 min (4°C). The supernatant was used for virus isolation. Virus isolation was performed as described by Allan et al. (1) with minor modifications. Briefly, 1 ml of the supernatant was incubated with 4 ml of PK-15 cell suspension (106 cells/m1) in EMEM (containing 15% normal

calf serum, 0.5% antibiotic mix, and 1% glutamine). The mixtures were incubated in F25 tissue culture flasks at 37°C for 5 hours (5% CO2). The resulting semi-confluent mono-

layers were treated with 300 mM D-glucosamine-HCL (Serva, Heidelberg, Germany) (18) and incubated at 37°C for 30 min (5% CO2). The semi-confluent cell monolayer was washed twice with EMEM to remove the D-glucosamine-HCI. A volume of 5 ml of EMEM was added to the flask, and then the incubation was continued for a further 4872 h at 37°C (5% CO2). Cell cultures were frozen and thawed 3 times. Each inoculum/cell lysate obtained after the freeze/thaw cycle was passaged twice by adding 2 ml of the appropriate inoculum/cell lysate to PK-15 cell suspensions (106 cells/m1), and seeded on F25-flasks. After each passage, the freshly seeded flasks were incubated for 24 hours, treated with D-glucosamine-HC1, and further incubated as described above. After three passages, monolayers of PK-15 cells were screened for PCV antigens by using the immunoperoxidase monolayer assay (IPMA) technique. Therefore, sixwells tissue culture plates were seeded with 1 ml of frozen and thawed inoculum/cell suspension and mixed with 4 ml of PK-15 cells, seeded, and incubated for 24 hours. The cells were treated with D-glucosamine-HC1 as described above. Cell medium was removed 24 hours after the D-glucosamine-HCI treatment and cells were fixed after drying and freezing (-20°C) with 4% of cold formaldehyde for one hour. Screening of fixed cells for PCV antigens was carried out using pooled sera, collected from five PMWS-affected pigs from two PCV2 nPCR positive herds. This serum pool contained no antibodies against PPV and PRRSV. The, cell cultures were incubated with a 1:10 dilution of this porcine serum pool. The IPMA was further performed as described by Wellenberg et al. (19), using a HRPO-labelled rabbit antiswine conjugate (Dakopatt, Glostrup, Denmark). Inoculated PK-15 cell cultures were also stained with a rabbit anti-

1 68

PCV1 antiserum to check for the presence of PCV1, antigens. A PK-15 cell culture, continuously infected with PCV1

served as PCV1 positive control. Non-inoculated PCVfree

PK-15 cells served as negative cell control and a serum sample collected from a specified pathogen free (SPF) pig served as negative serum control. In parallel, sera of PMWS-affected pigs from farm 1 were inoculated on semi-confluent Dulac cell monolayers in 24well cell culture plates using EMEM (containing 5% fetal calf serum, non-essential amino acids, and 0.15% antibiotics) as cell culture medium. Dulac cells were screened for PCV antigens by indirect immunofluorescence assay (IFA) using porcine sera collected from sows experimentally infected with PCV2 and FITC conjugated goat-anti-swine (Fab) (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA 19390, USA). Pre-infection sera of sows that were experimentally infected with PCV2 were used as negative control sera (pre-sera). Polymerase chain reaction DNA extraction Total DNA samples were extracted from control cells (PK-15 cells), PCV1 infected PK-15 cells, supernatants of pooled organ and tissue suspensions, and inoculated PK-15 cells (after the first and the third passage). DNA extraction was performed by column chromatography using the QIAamp Blood and Tissue kit (QIAGEN, Westburg, the Netherlands). For DNA extraction, a volume of 200 iil was used. Cell lysis, digestion with proteinase K, DNA precipitation, and elution of DNA were performed as recommended by the manufacturer. The DNA extraction products were stored at 20°C.

PCV specific nPCR Two primer pairs were designed for the detection of PCV genomes by nPCR. Primers 4F and 9R are able to produce amplification products from DNA of PK-15 cells infected with

PCV1, as well as from DNA of PMWS-affected pigs (6). The primer pair, which has been used for the specific amplification of PCV2 genomic sequences, has been described by Ellis et al. (5). A volume of 5 ttl of the total DNA extract was used for PCR amplification. The first PCR round was carried out in a total volume of 50 1.d, containing 1 x PCR buffer minus magne-

sium chloride, 5.0 mM magnesium chloride, 5 mM dNTP mix, 30 pmol of each primer (4F and 9R) and 2.0 U Taq polymerase. After denaturation at 95 °C for one minute, the reac-

tion mixtures were subjected to thermal cycling in a DNA Thermal Cycler (Perkin Elmer Cetus, Norwalk, USA) for 35 cycles each of 94°C for 1 min, 58°C for one minute, and 72°C for 1.5 min. A time delay of 7 min at 72°C was included prior

to a 4°C soak. Nested PCR was performed with 5 IA of the first PCR round products. The nPCR was performed as described for the first PCR round but with the PCV2-specific oligonucleotides, an annealing temperature of 54 °C, and the mixture was subjected to 25 thermal cycles. Five 1,11 of nPCR

products were analysed by horizontal agarose (1.25%) gel electrophoresis. Gels were stained with 1 [tg/m1 ethidium bromide and photographed under UV light. Specificity of the PCR products was checked by sequence analysis. As controls for the PCV-specific nPCR were used: non-inoculated PCV-

free PK-15 cells as negative cell control, distilled water. (H20) as negative template control, and PCV1-infected PK15 cells as PCV1 positive control. PCV2 negative and posi-

tive porcine lymphocyte fractions were used as quality

THE VETERINARY QUARTERLY, VOL

22, No 3, JULY, 2000

OM% rib IURI Ma 111

r,

WirluMa a irrialus _ checks of the performed nPCR runs. To avoid false-positive results, classical precautions for PCR were strictly followed (11).

Sequence analysis Sequence analysis of the entire PCV genome was performed on DNA extractions obtained from porcine lungtissue (pig from farm 2), from monolayers inoculated with the tissue suspension of the same lung (sequence identified as 24657 NL), and from control cell monolayers (non-inoculated monolayer) Using the QIAamp blood and tissue kit (Qiagen LTD, Hilden, Germany). Oligonucleotide primers were derived from nucleotide sequences of PCV2 isolates available in GenBank/NCBI (accession codes AF055391 (USA-1) and AF 055394 (France1) ) (13). All the selected primers were used in several separated PCRs, yielding several overlapping fragments that covered the entire PCV genome. M13-s and as-tails were incorporated at the 5' end of the nested primers to facilitate the direct sequencing of PCR-products by fluorescence-labelled M13-sequen-

cing primers (MWG Biotech, Ebersberg, Germany). Nucle-


otide sequencing was performed in sense and antisense directions using a LI-COR DNA Analyzer GENE READIR 4200 (MWG Biotech, Ebersberg, Germany), according to the recommendations of the manufacturer. Nucleotide sequences were aligned and analysed by DiscoverIR, vs1.0 (LI-COR, Inc,

Lincoln, USA). To run non-cross contaminating PCRs for diagnosis and sequencing a RoboAmp 4200 (MWG Biotech, Ebersberg, Germany) was used for automated pipetting and cycling.

Figure 1. Haematoxylin-eosin staining of a section of an enlarged mesenteric lymph node from a PMWS-affected pig from farm 3. Note the multinucleated giant cells (arrows). Bar = 50 Ion.

there were many multinucleated giant cells. In seven out of eight kidneys there was dispersed focal mononuclear cell infiltration and additionally multinucleated giant cells in one kidney. Moreover, in two kidneys exudative and membranoproliferative glomerulopathy were present.

In lung tissue perivascular and peribronchial mononuclear cuffing was present in six out of ten samples examined. In three other PMWS-affected pigs, distinct dispersed thickening of alveolar septa by mononuclear cells was noticed. In all lungs, alveolar collapse, an increased number of alveolar macrophages, and a few multinucleated giant cells were observed.

RESULTS

Gross pathology For pigs of all three farms, the most prominent findings at post mortem examination were poor physical condition, reduced consistency of intestinal contents particularly of the large intestines (six pigs from farms 1 and 2; two pigs from farm 3), and multiple enlarged lymph nodes. Additionally, catarrhal bronchopneumonia of the apical lobes (two pigs from farm 2; two pigs from farm 3) and nephritis (two pigs from farm 1; three pigs from farm 2; two pigs from farm 3),

were observed. Occasionally, abscesses of the pituitary gland, chronic peritonitis with multiple intra-abdominal abscesses, chronic pleuropericarditis, and severe oesophagogastric ulceration with anaemia were found. Histology For pigs of all three farms, the most consistent findings were observed in lymph nodes, kidneys, lungs, and brains. In all examined lymph nodes, both mesenteric and inguinal, and in ten out of eleven examined gut-associated lymphatic tissue, an increased number of histiocytic cells was present with a varying number of multinucleated giant cells. The number and size of the multinucleated giant cells varied considerably from a few double nucleated cells to a large number of multinucleated giant cells (Figure 1). Histiocytic cells were often present in the centre of lymphoid follicles as varying sized

clusters of cells, sometimes mimicking epitheloid cells. Histiocytic cells were sometimes clustered as elongated cells. In two out of eleven examined mesenteric lymph nodes, focal necrosis of lymphoid tissue was present. In five out of six examined splenic tissues, an increased number of histiocytic cells was noticed in association with the presence of multinucleated giant cells. In two out of four tonsils, histiocytic cells were present and, additionally, in one tonsil

169

Focal-to-slight mononuclear meningitis, mostly perivascular, was present in seven out of ten samples of brain tissue. In four brain tissue samples, focal cerebral mononuclear infiltrates were observed. In two out of five colon samples, a mixed infiltrate ofmononuclear cells and eosinophils was present. Virus isolation

After inoculation of pooled organ suspensions from pigs from all three farms and the sera from pigs from farm 1, no cytopathogenic effect was observed in PK-15 and Dulac cell cultures, respectively, after three passages. However, immu-

noperoxidase (IPMA) staining of the formaldehyde-fixed preparations of inoculated PK-15 cells, using pooled sera from PMWS-affected pigs, revealed intense staining of mainly the nuclei after inoculation with pooled organ homogenates from pigs of all three farms (Figure 2). Immunoperoxidase staining was not observed in non-inoculated PK15 cells, in the inoculated PK-15 cell culture incubated with the rabbit anti-PCV1 antiserum, or when SPF control serum was used. Immunofluorescent staining of Dulac cells, inoculated with sera from PMWS affected pigs from farm 1, was observed by IFA using porcine antisera to PCV2. No immunofluorescent staining was observed by IFA using pre-sera as controls. PCR A clear fragment of 481 bp was detected by PCV2 nPCR on DNA extracted from suspensions of all six pooled organ homogenates from pigs from farms 1 and 2 and in homogenates of the lymph nodes from two PMWS-affected pigs from farm 3. A fragment of 481 bp was also observed by nPCR on DNA extracts prepared from PK-15 cell cultures, inoculated with pooled organ homogenates from PMWS-affected pigs from

THE VETERINARY QUARTERLY, VOL 22, No 3, JULY, 2000

-'` P

42A-Ii4111411111liall

#14

ma ria Paw Oft ails

111_1k

Ilk I V I I MIMI. r'" /air Lis ri Ii0 .

all three farms. Nested-PCR revealed PCV2 specific fragments after the first and the third passage (Figure 3). The second passage was not tested for PCV-specific genomic se-

quences. No PCV2-specific fragments of 481 bp were


observed after amplification of DNA extracted from non-inoculated PCV-free PK-15 cells, distilled water, and PCV1-infected PK-15 cells (Figure 3).

Sequence analysis Sequence analysis of the entire genome of the PCV tissue culture isolate 24657 NL (isolated from the lung tissue from a pig from farm 2) showed 96.2% homology with the published sequence of the isolate PCV2 USA-1 (13). However, only 77.1% homology was found with PCV1 (13). The sequence of the entire genome of PCV tissue culture isolate 24657 NL was exactly the same as the sequence of the PCV derived from the original lung tissue. No PCV genomic sequences were detected in the control cell monolayer (non-inoculated). The total genomic length of isolate 24567 NL was 1,767 nucleotides. Only two nucleotide differences, at locations 1167 and 1547, were found after comparison with the European PCV2 isolates 24567 NL and France-1. At both locations, C and T were sequenced for isolate 24657 NL and France-1, respectively. Both differences were located in the open reading frame 2. The genomic sequence of PCV2 isolate 24657 NL showed one deletion at position 1042 when compared to isolate USA-1. Nucleotide sequence accession number: The GenBank/NCBI accession number of the Dutch PCV2 DNA sequence is AF 201897.

bronchopneumonia and multiple enlarged lymph nodes. Histopathological findings, attributable to PMWS, were found in lungs, lymphatic tissue, and kidneys, which are the major affected organs in PMWS (8, 15). In this study, histio-

cytic infiltrates were observed in affected lymph nodes or lymph tissues, and an increased number of histiocytic cells as well as a varying number of multinucleated giant cells. Comparable microscopic lesions with histiocytic infiltrates and large multinucleated giant cells have been described by others (5, 15). In this study, however, a focal to slight mononuclear meningitis (7 out of 10) and focal cerebral mononuclear infiltrates (4 out of 10) were observed in PMWS-affec-

ted pigs. Although lesions in PMWS-affected pigs might occur in a wide range of tissues, including the brain (10), mononuclear meningitis with cerebral mononuclear infiltrates has not been described as typical for PMWS. This finding

warrants further investigation of brain tissues for the presence of PCV2. The presence of PCV2 in PMWS-affected pigs was confir-

med by virus isolation with IPMA or IFA, nPCR and sequence analysis. Virus isolation, in combination with IPMA or IFA, revealed the presence of PCV2 in organs and tissues from PMWS-affected pigs from all three farms. IPMA indi-

cated the presence of PCV2 in infected PK-15 cells. However, the IPMA was based on porcine sera collected from PMWS-affected pigs, and therefore this staining could also be the result of antibody binding to viruses other than PCV2. The staining pattern, in particular the intense nuclear staining of infected cells, was similar to the pattern described by Allan et al. (1). The presence of PCV1 was excluded because no PCV1 antigens were detected by IPMA in simultaneously inoculated PK-15 cells. Although, the IPMA indica-

DISCUSSION This is the first report on the isolation and characterization of PCV2 in pigs showing signs of PMWS in the Netherlands. The clinical signs, as described in pigs from all three farms, were consistent with PMWS. In particular, the clinical problems in farm 1 and, to a minor extent, in farms 2 and 3, were

ted the presence of PCV2 antigens in pigs from all three farms, this was still not the evidence for the presence of PCV2. Therefore an IFA was performed to determine the presence of PCV2 antigens in PMWS-affected pigs. This IFA, which was based on the use of porcine sera collected from sows experimentally infected with PCV2, further un-

characteristic for PMWS, for example, pneumonia, poor

derlined the presence of PCV2 in PMWS-affected pigs from farm 1. The IFA was not performed on samples from farms 2

physical condition, skin pallor, and jaundice (1, 4, 7, 8, 16). Besides the clinical symptoms of PMWS, macroscopic le-

sions indicative of PMWS were observed, for example, iiv-aa-,- ire., .,i.t..:

i.. rp'..,..;.:;4 ,'

,..:

1,-,:.,,

.

-

.i,,,

, . !..

.,

17,11,-,-.s1.70,--;;X ;I:...1,719'..,

..

'

'7'

0,

-,.,

,., ,

.

4,

yi ,

ro-.

r4)..

.4. Nei

r

.....

.7

.

''

,'"

'i

genic cross-reactivity between the PCV isolates from PMWS affected pigs with the PK-15 PCV isolate has been described (1), no cross-reactivity of the PCV1 antiserum

el.

"

''.,.,.`"g:41,6-,-,!..ov.-:

'II.

d

"":,

,

-*t.'!''):-1.4

c'r,

". T.44, t

I.,(?7 t, .

. A' A..: 44,4.4-.

'''//;t" .7

:

'

' 1

,543.z.;,.. r::

..

14

'

6

f

,

4.

.,11

\.

't '1:1'11.

,

.

1149

.1::

rivw 4.

*4'

-;

calf 5

Figure 2. Immunoperoxidase monolayer staining of PK-15 cell culture after inoculation with organ homogenates from PMWS-affected pigs from farm 1. IPMA, using pooled sera from PMWS-affected pigs, revealed intense nuclear staining of some cells. Negative reactions are characterized by absence of colour.

170 -

Additional tests, such as nPCR, sequence analysis, or in situ hybridization (pigs from farm 3; as described below), were performed to further demonstrate the presence of PCV2 in pigs from farms 2 and 3. Although, a certain degree of anti-

-cN.`4,°.:,' '4` ...' ' 4 , -:,44 -, ,.... ,.:. :'. .: L.1, ...:,...) '_,..,..--. .,, .,?4. . . - *.: -,-.i; .,,

.4.,

t" ,

,''

e -04',;.-..;', ,,", '2'.:,. -:-' :,.. , ... #,I. ;:. 1 ,:.! t.,. .: i o ,,,. , ,q,

.

:It A .17' :V: ;7( ).

.

.

'V:

1

kliA),7:" ;

;

,,....:

.

,Y, - ,

,,. :. *, :

,

...,v...??..

d4......k,..

'..

'i1,14:i., .,,,;--,k7-,- ,A ,.i'-1,..., , , z.' -. . .,. . trolti: ',.,., : , .; 4.f%''. '(i44. 7,-'f :.., -,,t4i ... , 4. ,

)

em

,;:i' .!.'

.',

..,,..,,..f.:3,i 17 i;.,

.. .

.

i.....:,',., .1;4_,74..t.T.,,,:r, :.47,:-.:,;,.. .

.

.,),44-:-.:

..

and 3, because of lack of this more specific antiserum.

with PCV2, or porcine PCV2 antiserum with the PCV isolate from PK-15 cells was observed by IPMA. This suggests that the antigenic homology islimited. Others also suggested that there are significant antigenic differences between the PCV from PK-15 cells and field isolates of PCV-like viruses from pigs showing signs of PMWS (3, 4).

On the basis of the results of nPCR and sequence analysis, the PCV isolate was characterized as PCV2. Inoculated PK15 and Dulac cell cultures contained PCV2 specific genomic sequences by nPCR and, additionally, PCV2-specific nPCR products were obtained from pooled organ suspensions prepared from pigs from all three farms. These data indicate successful isolation of PCV2 from pooled organ suspensions. Sequence analysis of the entire genome of PCV cell culture isolate 24657 NL showed a high degree of homology with other described PCV2 isolates (>96%). Additionally,


1

elk

PRI rish

sa I

1

2

3

4

rib IP° 111% elk IrnIlitry Li Irk 117 Oft

f WV% I VP I id 111111111116

5

6

7

8

9

10

one pig was positive for Brachyspira innocens (farm 2). The fact that some pathogenic bacteria were isolated in combination with PCV2 from lesions of PMWS-affected pigs could be the result of a primary PCV2 infection. The role of PCV2 in the pathogenesis of PMWS is still uncertain and Koch's postulates still remain to be fulfilled in

establishing PCV2 as the primary aetiological agent in

Ef

481 bp-o-

PMWS. Further experiments need to be performed to determine the detrimental effects of PCV2 for pig breeding herds and to elucidate its role in the pathogenesis of PMWS. An epidemiological survey of PCV2 in Dutch herds is scheduled, in order to determine the prevalence of PCV2 and hence the spread of the virus among Dutch pigs.

,,

°

r 11

ACKNOWLEDGEMENTS

11kft

The authors would like to thank Dr. J. Vos, and Dr. N. Stockh8fe-Zurwieden

for pathological and histological examination, Dr. Segalés for the in-situ hy-

bridization technique, Dr. Babiuk and Dr. E. Bibbons for providing the


.1

Figure 3. PCV2 nPCR amplification fragments of 481 bp from an organ homogenate from a PMWS-affected pig, and inoculated PK-15 cell cultures. Lane 1/10: Marker, 100 bp ladder; Lane 2: H20 (negative) control; Lane 3: uninfected PK-15 cell (negative) control; Lane 4: PCV1 infected PK-15 cell culture (PCV1 positive control); Lane 5: Organ homogenate PMWS-affected pig from farm 1; Lane 6: PK-15 cell culture passage 1 (inoculated with organ homogenate PMWS-affected pig from farm 1); Lane 7: PK-15 cell culture passage 3 (inoculated with organ homogenate PMWS-affected pig from farm 1); Lane 8: PCV2 negative controls (porcine lymphocyte fraction obtained from bioScreen, Germany); Lane 9:

PCV1 antiserum, Dr. F.A.M. Rijsewijk and S.B.E. Verschuren for technical advice and assistance. We thank Mrs. Dreier and Mr. Wolters for excellent technical assistance. -

REFERENCES 1.

Allan GM, McNeilly F, Kennedy S, Daft B, Clarke EG, Ellis JA, Haines DM, Meehan BM, and Adair BM. Isolation of porcine circovi-

rus-like viruses from pigs with a wasting disease in the USA and 2.

PCV2 positive controls (porcine lymphocyte fraction obtained from

Europe. J Vet Diagn Invest 1998; 10: 3-10. Allan GM, Kennedy S, McFeilly F, Foster JC, Ellis JA, Krakowka SJ, Meehan BM, and Adair BM. Experimental reproduction of severe

wasting disease by co-infection of pigs with porcine circovirus and

bioScreen, Germany).

porcine parvovirus. J Comp Path 1999; 121: 1-11.

the genomic size (1,767 nucleotides) correlated with that of known PCV2 isolates (13) and was identical to that of the PCV2 isolate France-1, whereas non-pathogenic PCV1 genomes are 1,759 nucleotides in length (6, 12, 13). The PCV2 genomic sequences of DNA extracted from the original or-

3.

Allan GM, McNeilly F, Meehan BM, Kennedy S, Mackie DP, Ellis JA, Clarke EG, Espuna E, Saubi N, Riera P, Botner A, and Charreyre CE. Isolation and characterisation of circoviruses from pigs with wasting syndromes in Spain, Denmark, and Northern Ireland. Vet Micr

4.

Ellis JA, Hassard L, Clark EG, Harding J, Allan G, Willson P,

1999; 66: 115-23. Strokappe J, Martin K, McNeilly F, Meehan B, Todd D, and Haines D.

gan material as well as after cell culture were identical. These results also indicate successful isolation and characterization of PCV2 from pigs from farm 2. In addition, the presence of PCV2 in PMWS affected-pigs was also indicated by the in situ hybridization technique (data not shown). The in

Isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome. Can Vet J 1998; 39: 44-51. 5.

situ hybridization technique, based on the use of a PCV2 single-stranded oligonucleotide DNA probe, was performed

6.

on sections of mesenteric lymph nodes from the two pigs from farm 3 (16), and PCV2 nucleic acids were detected. PMWS is an emerging disease in swine herds in many coun-

tries of Europe and North America. PCV2, in combination with the detection of PCV antigens and/or genomic sequences, has been consistently isolated from lesions of PMWS-affected pigs and these findings support the hypothesis that PCV2 could play a role in the aetiology of PMWS (1, 3, 4). Under experimental conditions, a concurrent PCV2 and PPV infection induced symptoms in pigs comparable to those described for PMWS (2, 5). However, other viral pathogens in combination with PCV2 have been detected in PMWS-affected pigs. In recent studies, PMWS-affected pigs were shown to be also positive for PRRSV (4, 9, 14). In our study (data not shown), PRRSV (European strain) was also detected in most

pigs of farms 1 and 2 (four out of six pigs), suggesting that most of the pigs were infected with PRRSV at the same time as they were infected with PCV2. The interrelationship between PRRSV and PCV2 and their possible synergetic action in the induction of PMWS need to be clarified. In this study (data not shown), only occasionally were pathogenic bacteria, e.g. Streptococcus suis, Haemophilus parasuis or Pasteurella multocida, isolated from the lungs; the intestine of

171

7.

8.

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, and McNeilly F. Reproduction of lesions of postweaning multisystemic wasting syndrome in gnotobiotic piglets. J Vet Diagn Invest 1999; 11: 3-14. Hamel AL, Lin LL, and Nayar GPS. Nucleotide sequence of porcine circovirus associated with postweaning multisystemic wasting syndrome in pigs. J Virol 1998; 72: 5262-7. Harding JCS. Post-weaning multisystemic wasting syndrome (PMWS); preliminary epidemiology and clinical presentation. Proceedings Am Assoc Swine Pract 1997; 28: 503. Harding JCS, and Clark EG. Recognizing and diagnosing postweaning

multisystemic wasting syndrome (PMWS). Swine Health and 9.

Production 1997; 5: 201-3. Hinrichs U, Ohlinger VF, Pesch S. LiWang, Tegeler R, Delbeck FEJ,

and Wendt M. Erster Nachweis einer Infection mit dem porzinen 10.

11. 12.

13.

14.

15.

Circovirus Typ 2 in Deutschland. Tierärztl Umsch 1999; 54: 255-8. Kennedy S, Allan G, McNeilly F, Adair BM, Hughes A, and Spillane P. Porcine circovirus infection in Northern Ireland. Vet Rec 1998; 143: 495-6. Kwok S, and Higuchi R. Avoiding false positives with PCR. Nature 1989; 339: 237-8. Mankertz A, Persson F, Mankertz J, Blaess G, and Buhk HJ. Mapping and characterization of the origin of DNA replication of porcine circovirus. J Virol 1997; 71: 2562-6. Meehan BM, McNeilly FM, Todd D, Kennedy S, V, Ellis JA, Hassard LE, Clark EG, Haines DM, and Allan GM. Characterization of novel circovirus DNAs associated with wasting syndromes in pigs. J Gen Virol 1998; 79: 2171-9. Ohlinger VF, Pesch S, Bischoff C, and LiWang. Kiimmernde Ferkel und Läufer- auch and PMWS denken. Schweinezucht und Schweinemast. 1999; 3: 9-11.

Rosell C, Segalés J, Plana-Duran J, Balasch M, Rodriguez-Arrioja GM, Kennedy S, Allan GM, McNeilly F, Latimer KS, and Domingo

THE VETERINARY QUARTERLY, VOL 22, No 3 , JULY, 2000

0111

III aft 9 Ili /7,

lift A rib

eau

iv% dm

rs

M. Pathological, immunohistochemical, and insitu hybridization studies of natural cases of postweaning multisystemic wasting syndrome (PMWS) in pigs. J Comp Path 1999; 120: 59-78. 16.

Segales J, Sitjar M, Domingo M, Dee S, Del Pozo M, Noval R,

Sacristan C, De Las Heras A, Ferro A, and Latimer KS. First report of post-weaning multisystemic wasting syndrome in pigs in Spain. Vet Rec 1997;141: 600-1. 17. Spillane P, Kennedy S, Meehan B, and Allan G. Porcine circovirus in-

fection in the Republic of Ireland. Vet Rec 1998; 143: 511-2. Tischner I, Peters D, Rasch R, and Pociuli S. Replication of porcine circovirus: induction by glucosamine and cell cycle dependence. Arch Virol 1987; 96: 39-57. 19. Wellenberg GJ, Rooij EMA van, Maissan J, and Oirschot JT van. Evaluation of newly developed immunoperoxidase monolayer assay for detection of antibodies against bovine herpesvirus 4. CDLI 1999: 447-51. 18.

CEREBELLAR CORTICAL ABIOTROPHY IN TWO PORTUGUESE PODENCO LITTERMATES S.E. van Tongeren1,2, 1. K. van Vonderen3, J.J. van Nes3, and T.S.G.A.M. van den Ingh1 Vet Quart 2000; 22: 172-4


Accepted for publication: November 10, 1999,

SUMMARY

CASE PRESENTATION

Cerebellar cortical abiotrophy in two Portuguese Po-

The two affected roughcoated, medium-sized Portuguese Podenco pups were presented to the Department of Clinical

denco littermates is reported and discussed. The disease is characterized by progressive cerebellar ataxia with an early onset of two to three weeks. Extensive loss, degeneration, and necrosis of Purkinje cells particularly involved the cerebellar hemispheres. An autosomal recessive pattern of inheritance is suspected.

Key words: Dog, cerebellar cortical abiotrophy, Portuguese podenco. INTRODUCTION Cerebellar cortical abiotrophies have been described in most

domestic animal species and in a few rodents and primates (11). This disease has been investigated most extensively in dogs, and breeds with cerebellar abiotrophy reported include Kerry Blue Terrier, Gordon Setter, Rough-Coated Collie, Australian Kelpie, Airedale, Bernese Running dog, Finnish Harrier, Brittany Spaniel, Border Collie, Beagle, Wire Fox Terrier, Irish Setter, Labrador Retriever, Golden Retriever, Great Dane, and Rhodesian Ridgeback (2, 3, 5, 6, 7, 9, 10, 11, 12, 13, 14). In several of these breeds a genetiC basis has been established or proposed, with an autosomal recessive mode of inheritance, affecting both sexes equally. Cerebellar abiotrophy is characterized by loss, degeneration, and necrosis of Purkinje cells followed by a decrease in the number of granule cell neurons and shrinkage of the molecular layer. The animals are usually born without any neurological complaints and then develop cerebellar deficits, which progressively worsen. The age of onset and progression of the clinical signs vary markedly and are consistent for the disorder in each particular breed. Cerebellar abiotrophy has not been described in Portuguese Podenco dogs before. This case report documents a case of two littermates of this breed with cerebellar cortical abiotrophy. Department of Veterinary Pathology, University Utrecht, PO Box 80158, 3508 TD Utrecht, the Netherlands. 2 Correspondence: Drs. S.E. van Tongeren. Department of Veterinary Pathology, University Utrecht, PO Box 80158, 3508 TD Utrecht, the Netherlands. Department of Clinical Sciences of Companion Animals, University Utrecht, PO Box 80154, 3508 TD Utrecht, the Netherlands. t

1 72

Sciences of Companion Animals of Utrecht University at the age of 13 weeks. The dogs, one male and one female, came from a litter of four. The dogs developed cerebellar deficits which were progressive from the age of two to three weeks. The male pup had problems with walking (loss of balance, ataxia) and developed head and intention tremors; the female pup was not able to walk but crawled like a swimmer and also developed head tremors. Both animals were not able to eat themselves and were hand fed by the owner. However, they were attentive and responsive, notifying the owner of the need to defecate; only eye contact was difficult to establish. There was considerable growth retardation. The other two littermates (male and female) and the parents were clinically normal. The sire and dam were half-siblings. They were both imported from the same kennel in Portugal and had not been mated before. CLINICAL FINDINGS Clinical examination revealed that both dogs were in a moderate physical condition. The male dog was alert, while the

female dog showed decreased mental awareness. Ataxia of head, trunk and limbs, intention tremor, and atactic eye movements were present in both dogs. The ataxia was quite severe, preventing the dogs from walking or even standing upright. Cranial and spinal reflexes were present, while postural reactions could not be judged reliably due to the ataxia. The clinical conclusion for both dogs was a congenital cerebellar disease. The sopor in the female dog indicated addi-

tional involvement of the cerebrum. Further diagnostic measures were not taken as the prognosis was poor, and the owner wished to determine whether a genetic abnormality was the cause of the problem. PATHOMORPHOLOCIGAL FINDINGS Material and Methods The necropsy was performed immediately following euthanasia. Tissue samples of the brain, spinal cord, and skin of the head were fixed promptly in 10% neutral-buffered for-


I