Journal of Feline Medicine and Surgery (2011) 13, 231e236doi:10.1016/j.jfms.2010.11.012

Canine parvovirus type 2c infection in a kittenassociated with intracranial abscess and convulsions

Nicola Decaro DVM, PhD*, Costantina Desario DVM, PhD, Francesca Amorisco DVM,Michele Losurdo DVM, PhD, Maria Loredana Colaianni DVM, Maria Fiorella Greco DVM,Canio Buonavoglia DVM

Department of Veterinary PublicHealth, Faculty of VeterinaryMedicine of Bari, Strada perCasamassima km 3, 70010Valenzano, Bari, Italy

*Corresponding author. Tel: þ39-00804679843. E-mail: n.decaro@veterinari

1098-612X/10/040231+06 $36.00/0

A case of canine parvovirus type 2c (CPV-2c) infection in a 3-month-old feralkitten with a cerebral abscess and neurological disease is reported. The catdisplayed ataxia and convulsions together with signs of gastroenteritis andprofound alteration of the total and differential white blood cell counts. Aparvovirus strain was detected by a TaqMan assay in the blood and faeces of theaffected kitten, which was characterised as CPV by means of molecular assaysbut did not react with any of the CPV type-specific probes. By sequence andphylogenetic analyses of the VP2-protein gene, the CPV-2c strain displayeda non-coding mutation in the probe-binding region. Although the role of CPV-2cin this particular case is unclear, it is possible that it predisposed the kitten to theclinical signs seen. Continuous surveillance is needed to monitor futurespreading of this CPV-2c mutant, and any associated clinical signs, in the dogand cat population.

Date accepted: 18 November 2010 � 2010 ISFM and AAFP. Published by Elsevier Ltd. All rights reserved.

Feline panleukopenia (FPL) is a contagious dis-ease of cats which is traditionally caused byfeline panleukopenia virus (FPLV), a member

of the genus Parvovirus closely related to canine par-vovirus type 2 (CPV-2) and other parvoviruses ofcarnivores.1,2 Carnivore parvoviruses are small, non-enveloped, single-stranded DNA viruses that arevery prone to genetic evolution. However, whileFPLV since its first identification in 1920 has not un-dergone significant changes in the antigenic and bio-logical properties,3 CPV-2 has progressively evolvedunder partly positive selection, giving rise to antigenicvariants that replaced the original type and possessdifferent biological and antigenic properties.4e6 Threeantigenic variants of CPV-2 are currently known,namely CPV-2a, CPV-2b and CPV-2c, that have com-pletely replaced the original type 2, still used inmost commercial vaccines.7,8 In comparison to theoriginal type 2, the antigenic variants are more aggres-sive and have re-gained the ability to replicate in vivoin the feline host.9,10 FPL outbreaks caused by CPV-2aand CPV-2b have been described worldwide.11,12 Incontrast, cases of CPV-2c infection in cats have been

804679832; Fax: þ39-a.uniba.it

� 2010 ISFM an

reported only in Italy12,13 and there is a single reportof overt disease associated to this antigenic variant.12

FPL is usually characterised by profound leukopeniainvolving both lymphocytes and polymorphocytes andacute gastroenteritis with fever (40e41.5�C), abdominalpain, vomiting and diarrhoea. In-utero and immediatepost-mortem infections may lead to the developmentof neurological disease as a consequence of virus coloni-sation of Purkinje cells of cerebellum, with newborn kit-tens displaying signs referable to cerebellar damagesuch as ataxia, circling and muscular weakness.1

In this paper, a CPV-2c infection is reported in a kit-ten with intracranial abscess and convulsions.

Materials and methods

Clinical case, physical, neurological and labora-tory investigations

On June 19, 2010, a European shorthair feral kitten,3 months of age, was presented at a veterinary clinicof Bari, Apulia region, Italy, with a soft subcutaneousswelling at the head from which a purulent materialwas oozing out. Anamnesis of the cat, including thevaccination history, was not known. At clinical exami-nation, an alopecic area, 4 cm of diameter, was evident

d AAFP. Published by Elsevier Ltd. All rights reserved.

232 N Decaro et al

in correspondence to the subcutaneous swelling. Thegeneral health conditions were good and the appetitewas preserved despite the presence of fever (39.8�C).The cat was discharged with a therapy consisting oforal administration of antibiotics (amoxicillineclavu-lanic acid 25 mg/kg q 12 h for 6 days). The day afterthe cat was again submitted to clinical examinationdue to the onset of neurological signs that consistedof ataxia and difficulty in standing and walking. Neu-rological examination revealed depressed posturalreactions in all limbs, whereas oculo-vestibular re-sponses, spinal reflexes, pain sensation and urinaryand faecal continence were normal. Fever (40.0�C)and anorexia were also observed. New antibioticswere administered (enrofloxacin 5 mg/kg q 24 h, clin-damycin 25 mg/kg q 12 h) in association with carpro-fen (2 mg/kg at q 48 h). Ethylenediamine tetraaceticacid (EDTA)-blood, serum and faecal samples were col-lected during the clinical examination for haematology,serum electrophoresis and immunochromatographictesting for detection of feline immunodeficiency virus(FIV) and feline leukemia virus (FeLV) (Snap FelineCombo Plus; Idexx Laboratories, Westbrook, Maine,USA) and CPV/FPLV (Snap; Canine Parvovirus Anti-gen Test, Idexx Laboratories) antigens.

Blood cell counts obtained from the EDTA-bloodsample showed a marked modification of white bloodcell (WBC) counts (28.5� 109 cells/l, reference inter-val (RI) 5.5e19.5� 109 cells/l), with lymphopenia(0.5� 109 cells/l, RI 1.5e7.0� 109 cells/l) and in-creased neutrophil counts (23.9� 109 cells/l, RI2.5e12.5� 109 cells/l). Reductions of haematocrit(23.9%, RI 30e45%) and platelets (79� 109 cells/l, RI175e600� 109 cells/l) were also observed. Serumelectrophoresis did not reveal any important modifi-cation of the serum proteins and FIV/FeLV testingwas negative, whereas the Snap Canine ParvovirusAntigen Test gave a positive result. In the subsequentdays, the cat underwent a progressive clinical im-provement, regaining appetite and a normal rectaltemperature and losing the neurological signs. OnJune 23, EDTA-blood, serum, nasal and faecal sampleswere collected and sent to our laboratory for furtherinvestigations. Five days later the kitten was presentedagain at the veterinary clinic with loss of appetite anddiarrhoea and an antibiotic therapy was started withsulfamethopyrazine (30 mg/kg q 24 h). On July 2, theappearance of convulsions was observed with morethan10 episodesperday. Bypost-contrast computed to-mography (CT), carried out using a Philips MX8000MultiSlice CT scanner, a focal area of markedly en-hanced signal intensity compatible with an intracranialabscess was evident in the left parietal region in closeproximity to the subcutaneous abscess. An adjacentarea of decreased signal intensity of possible oedema-tous origin was observed together with reduction ofthe third ventricle and deviation of the falx cerebri. Incontrast, there were no cerebellar abnormalities, andno signs of recent, or old, fractures of the cranial bones.Abscess and cerebrospinal fluid samples for cytology,

bacteriology and/or virology were not collected at theclinical examination nor during CT. Administration ofphenobarbital (2.5 mg/kg q 12 h) resulted in the rapidrecovery of the kitten and loss of the neurological signs.The abscesses underwent progressive adsorption lead-ing to complete recovery.

Molecular investigations

Nucleic acids were extracted from the nasal, faecal andEDTA-blood samples using the DNeasy Tissue Kit andQIAamp Viral RNAMini Kit (Qiagen, Milan, Italy) andstored at�70�C until their use. DNA extracts were sub-jected to TaqMan assay for detection of CPV/FPLV14

and to a minor groove binder probe (MGB) assay forrapid discrimination between true FPLV strains andthe antigenic variants of CPV-2.15 Parvovirus strainswere characterised as FPLV and CPV on the basis ofthe detected fluorescent reporter probe (VIC andFAM) signals, respectively. CPV-2 molecular character-isation was achieved by means of a panel of real-timepolymerase chain reaction (PCR) assays using MGBprobes able to identify the antigenic variants 2a, 2band 2c16 and to discriminate between CPV vaccineand field strains.17,18

Faecal RNA extracts were screened by either real-time or conventional reverse transcriptase PCR assaysspecific for carnivore coronaviruses19 and calicivi-ruses.20 The blood extracts were also used to detectthe proviral DNA of FIV21 and FeLV22 and the DNAof feline haemoplasmas.23 Feline herpesvirus andfeline calicivirus were searched for in the nasal swabextract.24

Virus isolation attempts

Parvovirus isolation was attempted as described previ-ously.25,26 Briefly, the parvovirus positive samples weretreated with antibiotics (penicillin 5000 IU/ml, strepto-mycin 2500 mg/ml, amphotericin B 10 mg/ml) at 37�Cfor 30 min and inoculated onto freshly trypsinisedCrandell feline kidney (CrFK) and canine A-72 cellsgrown in Dulbecco’s minimal essential medium con-taining 5% foetal calf serum. After an incubation periodof 5 days at 37�C, the cells were tested by an immuno-fluorescence (IF) assay using a dog serum positive forCPV-2 and a rabbit anti-dog IgG conjugated with fluo-rescein isothiocyanate (SigmaeAldrich, Milan, Italy).Three serial passages were carried out before virus iso-lation was considered unsuccessful.

Detection of CPV antibodies

Antibodies against CPV were searched for in the cat se-rum by means of a haemagglutination-inhibition (HI)test, as previously described.27,28 The test was per-formed at þ4�C in 96-well V-plates, using 10 haemag-glutinating units of CPV-2b antigen and 1% pigerythrocytes. Two-fold dilutions of each serum samplein phosphate buffered saline, starting from 1:10, were

233Canine parvovirus type 2c infection

tested. The HI titre was indicated as the highestserum dilution completely inhibiting viralhaemagglutination.

Sequence and phylogenetic analyses

The sequence of the full-length VP2 gene of the iden-tified parvovirus strain (300/10) was determinedthrough PCR amplification of three partially overlap-ping fragments, as previously described.3,6 The ob-tained sequences were assembled, edited andcompared with FPLV and CPV reference strains andwith a sequence database of parvovirus strains de-tected in Italy,3,6,12 using the BioEdit software pack-age, version 7.0.1 (www.mbio.ncsu.edu/BioEdit/bioedit.html). The VP2 gene sequence was depositedin the GenBank database under accession numberHQ025913.

The phylogenetic relationships were evaluated byusing Mega3;29 pair-wise genetic distances were calcu-lated by using the Kimura’s two-parameter model andphylogenetic trees were constructed by the neighbor-joining and maximum parsimony methods. A boot-strap analysis with 1000 replicates was undertaken toassess the confidence level of the branch pattern.Canine and feline strains used for tree constructionswere selected from previous studies.3,6,12

ResultsAll molecular assays for detection of the most com-mon pathogens of the cat gave negative results withthe exception of the parvovirus TaqMan assay, whichdetected low viral DNA titres of 7.43� 103/mg and2.50� 104/ml for the faecal and blood samples, re-spectively. Surprisingly, the parvovirus strain wascharacterised as CPV by means of the FPLV/CPVMGB probe assay and resulted as a CPV field strainby additional MGB probe assays. However, due tothe lack of reaction with any of the type-specificprobes of the 2a/2b and 2b/2c MGB probe assays,the strain was not fully characterised.

Virus isolation attempts on feline and caninecells were unsuccessful which is in agreementwith the low viral titres detected in clinical sam-ples. The HI test revealed the presence of CPV an-tibodies in the cat serum (titre of 1:640), whichconfirmed the exposure of the cat to a parvovirusstrain.

By sequence analysis of the full-length VP2-proteingene, strain 300/10 displayed residues typical of CPV-2 strains, Leu-87, Thr-101, Gly-300, Tyr-305, and Asp-375. The presence of codon GAA at position1276e1278 corresponding to residue E426 of the en-coded protein led us to type definitively the strainas CPV-2c. The change A1275G was detected in thebinding region of the type 2c-specific probe, which ac-counted for the absence of VIC fluorescence in the 2b/2c assay.16 This nucleotide change did not determinean amino acid substitution at residue 425 of the VP2

protein. The CPV-2c strain 300/10 of feline origin dis-played the highest nucleotide identity (99%) to Italiantype 2c strains displaying the same non-coding muta-tion at codon 425.6 By phylogeny obtained using theneighbor-joining method, strain 300/10 was found tofall in the same cluster as Italian type 2c CPVs(Fig 1), which was separate from that including a pre-vious CPV-2c strain isolated from a cat with FPL.12

The maximum parsimony method confirmed thetopology of the neighbor-joining tree.

DiscussionIn this paper, a CPV-2c infection in a 3-month-old feralkitten has been reported. Apart from the occurrence ofmarked lymphopenia, the haematological findingswere not typical of FPL as total WBC and neutrophilcounts were increased as a consequence of the concur-rent abscess, whereas FPL is usually characterised bya dramatic reduction of all leukocyte subsets. Clini-cally, the occurrence of diarrhoea was compatiblewith the parvoviral infection, whereas fever could beattributed to either FPL or suppurative process. Theprevalent sign in the cat was the appearance of neuro-logical disease which was characterised by ataxia andsubsequently by convulsions. FPL may cause neuro-logical disorders only in newborn kittens due to thevirus replication in Purkinje cells of the cerebellumsubsequent to perinatal infection. Thus, the majorfinding is represented by ataxia due to cerebellar hy-poplasia.1 In the present case, ataxia was observedonly in the early stage of the disease and was subse-quently followed by severe seizures. In addition, theCT scanning showed focal lesions compatible witha parietal abscess without any involvement of the cer-ebellum, so that the neurological signs could bemainly attributed to the suppuration rather than toa viral infection. However, the recovery of the cat to-gether with the lack of analyses on the cerebrospinalfluid prevented a definitive diagnosis for the neuro-logical disorders. Although the clinical history andvaccination status of the cat were unknown, basedon the low CPV-2c titres detected in the faeces andblood, it could be speculated that the cat was recover-ing from the infection when the neurological diseaseappeared. In this perspective, a preceding CPV-2c in-fection may have determined a general immunosup-pression which could have been responsible for theparietal-brain colonisation by pyogenes.

In recent years, FPL cases caused by the antigenic var-iants of CPV-2 have been repeatedly reported.11,12,30,31

However, to date there is a single report on clinical signsinduced in a cat by the new variant CPV-2c.12 Despitethe full vaccination against FPL through administrationof two doses of a classical FPLV vaccine, that cat wasaffected by overt clinical signs which included non-hae-morrhagic diarrhoea, depression and loss of appetitebut not alterations of the leukocyte counts. In the case re-ported in the present paper, there are no anamnesticdata about previous vaccinations as the feral kitten

CPV-2c/136/00-ItalyCPV-2c/40/07-Italy

CPV-2c/9/05-ItalyCPV-2c/cat234/08; G333/99-Germany; 382/06-Italy

CPV-2c/G7/97-GermanyCPV-2c/195/08-BelgiumCPV-2c/383/06-ItalyCPV-2c/243/07-Italy

CPV-2c/411/06-ItalyCPV-2b/311/04-Italy

CPV-2c/279/04-Italycat300/10

CPV-2c/327/06-Italy; 393/06-Italy; 43/07-ItalyCPV-2c/219/08-13-Italy

CPV-2c/56/00-ItalyCPV-2b/SAH vaccine

CPV-2b/42/05-49-ItalyCPV-2b/V209-Vietnam

CPV-2b/ HNI-1-18-VietnamCPV-2c/HNI-4-1-Vietnam

CPV-2b/29/97-ItalyCPV-2c/110/07-27-USA

CPV-2a/T25-TaiwanCPV-2c/Taiwan

CPV-2b/HN-3-ChinaCPV-2b/TWN1-Taiwan

CPV-2b-ZD1-ChinaCPV-2a/BJ069/O7-China

CPV-2a/BD2-ChinaCPV-2a/APD1-China

CPV-2a/17/08-Italy; 100/08-ItalyCPV-2a/cat11/08-Italy

CPV-2a/96/02-ItalyCPV-2b/162/97-Germany

CPV-2b/CPV39-USACPV-2b/46-PolandCPV-2a/CPV15-USA

CPV-2/CPVb-USACPV-2/Uttar Pradesh-India

FPLV/Tiger/PT06-Portugal; Lion/PT06-PortugalFPLV/134/04-2-Italy

FPLV/22/06-ItalyFPLV/134/04-1-Italy

FPLV/355/04-2-ItalyFPLV/GT-2-China

FPLV/198/01-ItalyFPLV/50/07-1-UK

FPLV/443/07-ItalyFPLV/Purevax vaccine

FPLV/490/07-UK50/07-2-UK

FPLV/97/06-11-UKFPLV/193/70-Austria

FPLV/97/06-10-UKFPLV/ARG02-Argentina

FPLV/CU-4-USAFPV-483-Japan

77

7065

75

64

4963

56

54

4153

5148

21

17

13

47

35

34

29

22

22

8

7

13

34

24

39

87

56

100

7064

5645

35

459

16

27

33

34

13

19

3935

0.001

A1275G

CPV-2c

Fig 1. Neighbor-joining tree based on the full-length VP2 gene sequences (1755 nucleotides) of FPLVs and CPVs. TheGenBank accession numbers for the reference strains used for phylogenetic tree construction are reported in previous papers.Canine and feline strains used for tree constructions were selected from previous studies.6,12,15 A statistical support was pro-vided by bootstrapping over 1000 replicates. The scale bar indicates the estimated numbers of nucleotide substitutions per site.

234 N Decaro et al

235Canine parvovirus type 2c infection

was found already ill. This further detection of CPV-2cin a cat confirms the circulation of the new canine vari-ant in the feline population, stressing again our recom-mendation to carefully evaluate the efficacy of classicalFPLV vaccine against the CPV-2 antigenic variant.

Importantly, the feline CPV-2c was not character-ised by the type-specific MGB probe assays as it dis-played a mutation in the probe-binding region thatprevented the correct hybridisation of the CPV-2c-spe-cific probe. Untypeable CPV-2c strains with the samenucleotide change as the feline strain 300/10 havebeen previously detected in dogs.6 Residue 425 ispart of a major antigenic site located over the three-fold spike of the CPV-2 capsid and considered to affectviral immunogenicity.32 Considering that this muta-tion is non-coding, the antigenicity of the virus isnot altered. Further studies are needed to determinethe distribution of this atypical CPV-2c in the canineand feline populations and any disease that mightbe associated with it.

AcknowledgementsThis work was supported by grants from Universityof Bari, Italy, Ricerca di Ateneo 2009, project ‘Epide-miologia molecolare del parvovirus del cane nei carni-vori domestici e selvatici’ and contribution to PRIN07,project ‘Infezione da parvovirus nei carnivori: aspettimolecolari, patogenetici ed immunologici’. We aregrateful to Dr Gianfranco Pastorelli (Veterinary ClinicSanta Fara, Bari, Italy) for providing anamnestic dataand clinical samples and to Dr Alice Casali(Veterinary Hospital Pingry, Bari, Italy) for the com-puted tomography scanning.

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