Parvovirus infection is one of the most common enteric diseases affecting dogs in Egypt. A total number of 49 dogs younger than six month old, suffering from severe watery to bloody diarrhea, vomiting and lethargy were examined to confirm the infection with parvovirus through detection of the viral antigen serologically by FASTest® PARVO Card Test and genetically by using specific primers for both conventional and nested polymerase chain reaction (PCR). Serological assay confirmed the infection in 81.63% (40/49) of the examined animals, while the molecular techniques confirmed the infection in 81.63% (40/49) by the conventional PCR and in 97.96% (48/49) by the nested PCR. Sequencing revealed that CPV2a and CPV2b serotypes are circulating in the Egyptian field. The obtained sequences were submitted to the GenBank under the following accession MG764314, MG764315, MG930487 and MG930488. Serotype CPV2b showed some mutation in different sites

AL-Hosary, Amira A. T. A.

English

Parvovirus infection is one of the most common enteric diseases affecting dogs in Egypt. A total number of 49 dogs younger than six month old, suffering from severe watery to bloody diarrhea, vomiting and lethargy were examined to confirm the infection with parvovirus through detection of the viral antigen serologically by FASTestÂ® PARVO Card Test and genetically by using specific primers for both conventional and nested polymerase chain reaction (PCR). Serological assay confirmed the infection in 81.63% (40/49) of the examined animals, while the molecular techniques confirmed the infection in 81.63% (40/49) by the conventional PCR and in 97.96% (48/49) by the nested PCR. Sequencing revealed that CPV2a and CPV2b serotypes are circulating in the Egyptian field. The obtained sequences were submitted to the GenBank under the following accession MG764314, MG764315, MG930487 and MG930488. Serotype CPV2b showed some mutation in different sites

Journal of Advanced Veterinary Research (University Assiut, Egypt)

IntroductionCanine Parvovirus (CPV) infection was identified during the1970s then becomes endemic worldwide (Decaro and Buon-avoglia, 2012). It is a non-enveloped single strand DNA virusaffecting dogs especially young puppies; some strains of thisvirus have zoonotic importance especially in children(Makhlouf et al., 2017). It has two different strains, namelyCPV-1 and CPV-2; both are characterized by high evolutionrate. Canine parvovirus 2 has three variants (CPV-2a, CPV-2band CPV-2c) (Shackelton et al., 2005). The infection has twoforms (i) the intestinal form, which is the most common; it ischaracterized clinically by vomiting, bloody diarrhea, anorexiaand weight loss. (ii) Cardiac form is less common and usuallyaffects the cardiac muscles of very young puppies and endedwith death (Streck et al., 2009).This virus causes a highly contagious disease because dur-ing the enteric form the infected dogs usually shed the virusin their stools and contaminate the environment. Susceptibledog takes the infection through direct or indirect contact. Thisinfection characterized by high morbidity up to 100% and casefatality up to 10% (Appel, 1978; Appel et al., 1979; Chollom etal., 2013).This study aimed to compare between serological and mo-lecular detection of Parvovirus infection in household dogswith special reference to the molecular characterization andphylogenetic analysis of the circulating serotypes in Egypt.Materials and methodsAnimals, clinical examination and samplingForty nine dogs aged between one and six months suffer-ing from bloody diarrhea and vomiting were sampled for sero-logical and molecular confirmation of parvovirus infection.Clinical examination was carried out according to Cote (2011).Fecal sample was collected from each dog and divided to twoaliquots, one was used for serological detection of the virusantigen and the second was stored at -20ºC until used (Coles,1986).This work and the used procedures were performed ac-*Corresponding author: Amira A.T.A. AL-HosaryE-mail address: d.miravet@hotmail.comJournal of Advanced Veterinary Research Volume 8, Issue 4 (2018) 79-83Journal of Advanced Veterinary Researchhttp://advetresearch.com/index.php/avr/indexInfectious Diseases, Department of Animal Medicine, Faculty of Veterinary Medicine, Assiut University, Assiut, 71526, Egypt.ABSTRACTISSN: 2090-6277/2090-6269/ © 2018 JAVR. All rights reserved.Detection and Molecular Characterization of Parvovirus Serotypes inEgyptAmira A.T.A. AL-HosaryParvovirus infection is one of the most common enteric diseases affecting dogs in Egypt. A total numberof 49 dogs younger than six month old, suffering from severe watery to bloody diarrhea, vomiting andlethargy were examined to confirm the infection with parvovirus through detection of the viral antigenserologically by FASTest® PARVO Card Test and genetically by using specific primers for both conven-tional and nested polymerase chain reaction (PCR). Serological assay confirmed the infection in 81.63%(40/49) of the examined animals, while the molecular techniques confirmed the infection in 81.63%(40/49) by the conventional PCR and in 97.96% (48/49) by the nested PCR. Sequencing revealed thatCPV2a and CPV2b serotypes are circulating in the Egyptian field. The obtained sequences were sub-mitted to the GenBank under the following accession MG764314, MG764315, MG930487 andMG930488. Serotype CPV2b showed some mutation in different sites. ARTICLE INFOOriginal ResearchReceived:26 August 2018Accepted:25 September 2018Keywords:ParvovirusSerological fastest, PCR,EgyptJ. Adv. Vet. Res. (2018), 8 (4),79-83cording to the ethical standards of Assiut University and Vet-erinary authorities in Assiut province, Egypt.Serological diagnosisFASTest® PARVO Card Test Kit by Vetlab Supplies Ltd, UKwas used for direct detection of parvovirus specific antigenaccording to manufacture instructions. FASTest® PARVO Cardcontains two monoclonal antibodies. The mobile monoclonalantibodies bound to colloidal gold particles located in theconjugate pad; the second is immobile and located on the testline. The positive result was demonstrated by detection ofantigen-antibody-complex that forms pink-purple test line inthe test zone.Molecular diagnosisDNA was extracted from the fecal samples using ZYMOResearch Quick-DNATM Universal Kit (ZYMO Research, Co.,USA, Cat. No D4068 & D4069) for solid tissue and special sam-ples according to the manufacture instruction.Conventional PCR was performed in total volume of 25µlusing pCPV2ab specific primers pCPV2ab forward primer (5’-GAA GAG TGG TTG TAA ATA ATT-3’) and pCPV2ab reverseprimer (5’-CCT ATA TCA CCA AAG TTA GTA G-3’) that amplifiesof 681 bp fragment. Nested PCR carried out using pCPV2Nforward primer (5’-TGAGCTGCATTTAGTTAGTTTTGA-3’) andpCPV2N reverse primer (5’-TGTTTGCCATGTATGTGTTAGTCT-3’)was used for amplification of a 442 bp fragment using con-ventional PCR product as template. PCR and nPCR productswere electrophoresed in 1.5%  agarose gel stained with ethid-ium bromide to visualize the specific bands under UV trans-illuminator (Pereira et al., 2000).Sequencing and phylogenetic analysesFour positive samples were subjected to the same PCR re-action in 50 µl total volume then purified by using ZYMO Re-search DNA Clean & Concentrator™-5 kits (ZYMO Research,Co, USA) before sequencing in both directions using the sameprimers and Dideoxy chain termination method by BigDye®Terminator kit (Applied Biosystems, USA) according to themanufacturer’s instructions. Sequence reactions were analyzedby PRISM 310 genetic analyzer (Applied Biosystems, USA) atthe Molecular Biology Unit, Assiut University, Assiut, Egypt. The obtained sequences were assembled and subjected toBLAST search for similarity then each sequence was translatedto its open reading frames (amino acids) using ExPASy trans-lation tool (https://www.expasy.org/). The percent of identityand similarity between the obtained sequences and other se-quences on the GenBank was calculated by using the DNAS-TAR's “MegAlign” sequence alignment software. Phylogenetictree was constructed by using neighbor joining method onMega 6.0 program with Kimura two parameters and bootstrap1000, Canine Distemper partial sequence (AB025271) wasused as an out-group (Kimura, 1980). Translated amino acidsof CPV2b sequences were subjected to multiple alignmentswith previously detected Egyptian isolates on the GenBankdatabase using Clustal omega, EMBL-EBI.Statistical analysisThe obtained data were compared using Chi Square test(SPSS, Chicago, USA) at 5% threshold value. ResultsDogs younger than six months old suffered from severewatery to bloody diarrhea, vomiting and lethargy were in-fected with parvovirus. The infection was confirmed by theFASTest® PARVO Card Test and conventional PCR in 81.63%(40/49) of the examined dogs. While nPCR confirmed the in-fection in 97.96% (48/49) of the examined samples with sig-nificant difference (P ≤ 0.005) (Figs. 1 and 2). The four sequenced samples were assembled and submit-ted to the GenBank (Accession numbers: MG764314,MG764315, MG930487 and MG930488). In the phylogeneticanalysis these sequences were clustered into two clades onefor CPV2a and the second one for CPV2b. MG764314 andMG764315 sequences of CPV2a serotype are more related toeach other and showed 100% of identity. On the other hand,MG930487 and MG930488 amplicons were more related toCPV2b serotype and recorded 99.5% of identity. In respect tothe previous sequences recorded in Egypt, the identity was98.7 up to 99.5% and 98.3 up to 100% with other sequencesfrom different localities all over the world, respectively. Multi-ple alignments of CPV2b amino acids showed some mutationin different sites (Figs. 3, 4 and 5).80Amira A.T.A. AL-Hosary /Journal of Advanced Veterinary Research 8 (4) (2018) 79-83Fig. 1. Agarose Gel (M) DNA ladder 100 bp, lanes (1: 9) pos-itive samples and specific bands at 681 bp. Lanes (10) Posi-tive control samples.Fig. 2. Agarose Gel (M) DNA ladder 100 bp, lanes (1:6 & 8)positive and specific bands at 442 bp. Lanes (7) negative sam-ple and Lanes (9, 10) Positive and negative control samples.DiscussionCanine parvovirus infection is a common destructive en-teric viral disease leading to high morbidity and mortality ratesin unvaccinated puppies as well as in neglected untreated in-fected dogs (Decaro et al., 2006). The virus is transmitted fromthe diseased to susceptible dogs through direct or indirectcontact due to heavy shedding of the virus in feces and theexcretion of infected dogs that leads to high opportunity ofcontamination in food staff, water and fomites (Zhao et al.,2011). The virus shedding continue for some time after recov-ery and the animals considered as asymptomatic convalescentcarriers (Clegg et al., 2012). Canine parvovirus infection wascharacterized by severe dehydration, leathery, vomiting andfoul-smelling watery to bloody diarrhea because of erosivegastritis and enteritis, these findings come in agreement withAmthal, (2014) and Al-Hosary (2016), who reported similarclinical signs in dogs infected with parvovirus. FASTest®PARVO Card Test is a cheap, fast, accurate test for detectionof the virus antigen in clinically infected dogs. The test de-tected the real infection in 81.63% of the examined dogs. So,it is a recommended approach for confirmation of parvovirusinfection in dogs. Early and accurate diagnosis helps the dis-eased animals to recovery before any complication like severeanemia and dehydration, which are fetal in young animals ac-cording to Al-Hosary (2016).Amira A.T.A. AL-Hosary /Journal of Advanced Veterinary Research 8 (4) (2018) 79-83Fig. 3. Phylogenetic analyses for the Egyptian isolates against reference sequences of canine parvovirus.Fig. 4. Similarity and divergence between the Egyptian and reference sequences of canine parvovirus serotypes, the upperright values are the percentage of identity and the lower left values are the percentage divergences between sequences.81Molecular techniques were more sensitive and specific di-agnostic tool, especially nPCR. It confirmed the infection in97.96% of the examined animals, while serology and conven-tional PCR confirmed the infection in only 81.63% of the ex-amined cases. This finding may be attributed to the ability ofnPCR to detect the lowest amount of viral particles accordingto previous studies by Hirasawa et al. (1994); Mochizuki et al.(1996) and Kumar et al. (2010), who confirmed that nPCR isthe most sensitive test to detect the viral particles. According to the author’s knowledge, the current study isthe first report that provides data about the molecular char-acterization of the circulated parvovirus serotypes in Egypt,the obtained results confirmed that there are two differentvariants of CPV2 serotypes circulating in the Egyptian field thatinclude CPV2a and CPV2b. The Egyptian strains were clusteredinto two clades. The first clade incudes CPV2a isolates, whichare closely related to Chinese isolates, while the second cladeincludes the isolates of CPV2b, which were closely related topreviously isolated serotypes from Cairo, Egypt (Amthal, 2014)and serotypes isolated from Slemani Province, Iraq (Sheikh etal., 2017). CPV2b is the main circulated serotype in Egypt andwas recorded in some previous reports. Detection and molec-ular characterization of CPV2a and its close relationship withChinese serotypes for the first time during this study may beattributed to lack of studies that concerned on molecular char-acterization of the Egyptian circulating serotypes in generaland especially in Egypt. Also, this new finding suggested thatthis virus serotype (CPV2a) may be introduced from China toEgypt through contaminated dogs, contaminated premiseslike dog toys, plates for feeding and/or some feed stuff, whichserve as source for indirect infection because the virus has theability to be pathogenic under suitable environmental condi-tions during up to five months (Jacobs et al., 1980). Analysisand alignment of the translated amino acids confirmed theoccurrence of mutations in the Egyptian circulated isolates ofCPV2b obtained during this study in comparison to the previ-ously recorded sequences of the same serotype in Egypt.These findings confirmed that CPV2b circulated in differentregions in Egypt has unstable DNA genome and in agreementwith previous study by Miranda and Thompson, (2016), whoreported some mutation in CPV2 variants. Viral protein 2 (VP2)is one of the major structural protein encodes the viral capsidprotein and responsible for the antigenicity and involved inthe host immune response. So, any mutations will result inmore pathogenicity of the virus (Lin et al., 2014). Previousstudies reported that CPV is characterized by a high geneticsubstitution rate like that observed in RNA viruses and thischaracter is responsible for continuous antigenic evolutionand rapid displacement by new antigenic variants (Decaro etal., 2007a,b). This finding should be considered especiallywhen establish vaccination protocol against this infection be-cause these mutations perforate animal’s immunity and maybe lead to outbreaks. ConclusionCanine parvovirus (CPV) is one of the most common dis-eases affecting dogs in Egypt; especially, young unvaccinateddogs. Fast sero-diagnostic kit is a recommended tool for rapidroutine diagnosis while sophisticated technique like molecularassay is recommended for wide scale and epidemiologicalstudies specially to detect the infection in carrier animals.There are two serotypes of CPV circulating in Egypt includeCPV2a and CPV2b. CPV2b characterized with continuous anti-genic evolution, which affect the efficacy of vaccination inEgypt.AcknowledgementThe author would like to express thanks to Dr. AhmedMakram Radwan, owner of Jockey Veterinary Clinic, Mallawy,EL-Minia, Egypt, for his great help in sampling during thisstudy. ReferencesAl-Hosary, A.A.T., 2016. 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Detection and Molecular Characterization of Parvovirus Serotypes in Egypt

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Detection and Molecular Characterization of Parvovirus Serotypes in Egypt

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Journal of Advanced Veterinary Research (University Assiut, Egypt)