Molecular epidemiology of canine parvovirus in Morocco

    Molecular epidemiology of canine parvovirus in Morocco Nadia Amrani, Costantina Desario, Ahlam Kadiri, Alessandra Cavalli, Jaouad Berrada, Khalil Zro, Ghizlane Sebbar, Maria Loredana Colaianni, Antonio Parisi, Gabriella Elia, Canio Buonavoglia, Jamal Malik, Nicola Decaro PII: DOI: Reference:

S1567-1348(16)30131-9 doi: 10.1016/j.meegid.2016.04.005 MEEGID 2703

To appear in: Received date: Revised date: Accepted date:

26 December 2015 2 April 2016 6 April 2016

Please cite this article as: Amrani, Nadia, Desario, Costantina, Kadiri, Ahlam, Cavalli, Alessandra, Berrada, Jaouad, Zro, Khalil, Sebbar, Ghizlane, Colaianni, Maria Loredana, Parisi, Antonio, Elia, Gabriella, Buonavoglia, Canio, Malik, Jamal, Decaro, Nicola, Molecular epidemiology of canine parvovirus in Morocco, (2016), doi: 10.1016/j.meegid.2016.04.005

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Title: Molecular epidemiology of canine parvovirus in Morocco

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Authors: Nadia Amrani,a* Costantina Desario,b Ahlam Kadiri,a Alessandra Cavalli,b Jaouad Berrada,a Khalil Zro,d Ghizlane Sebbar,d Maria Loredana Colaianni,c Antonio Parisi,c Gabriella Elia,b Canio Buonavoglia,b,c Jamal Malik,d Nicola Decarob

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Affiliations: a, Microbiology, Immunology and Infectious Diseases Unit, Department of Veterinary Pathology and Public Health, Hassan II Agronomic and Veterinary Institute, Rabat, Morocco b, Department of Veterinary Medicine, University of Bari, Italy c, Istituto Zooprofilattico Sperimentale di Puglia e Basilicata, Foggia, Italy d, Society of Veterinary Pharmaceutical and Biological Productions (Biopharma), Rabat, Morocco

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*Corresponding author at: Nadia Amrani, Microbiology, Immunology and Infectious Diseases Unit, Department of Veterinary Pathology and Public Health, Hassan II Agronomic and Veterinary Institute, PO box 6202 Rabat, Morocco, Tel: +212 537675539 Fax: +212 537776796 Email address: [email protected]

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Abstract Since emerged in the mid-1970’s, canine parvovirus 2 (CPV-2) has evolved giving rise to new antigenic variants termed CPV-2a, CPV-2b and CPV-2c, which have completely replaced the original strain and had variously distribution worldwide. In Africa limited data are available on epidemiological prevalence of these new types. Hence, the aim of the present study was to determine circulating variants in Morocco. Through TaqMan-based real-time PCR assay, 91 samples, collected from symptomatic dogs originating from various cities between 2011 and 2015, were diagnosed. Positive specimens were characterised by means of minor groove binder (MGB) probe PCR. The results showed that all samples but one (98.9%) were CPV positive, of which 1 (1.1%) was characterised as CPV-2a, 43 (47.7%) as CPV-2b and 39 (43.3%) as CPV-2c. Interestingly, a co-infection with CPV-2b and CPV-2c was detected in 4 (4.4%) samples and 3 (3.3%) samples were not characterised. Sequencing of the full VP2 gene revealed these 3 uncharacterised strains as CPV-2c, displaying a change G4068A responsible for the replacement of aspartic acid with asparagine at residue 427, impacting the MGB probe binding. In this work we provide a better understanding of the current status of prevailing CPV strains in northern Africa.

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Key words: Canine parvovirus, Morocco, antigenic variants, real-time PCR

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1. Introduction Canine parvovirus (CPV) is the most dreadful enteropathogen causative of fatal disease in puppies and adult dogs worldwide (Decaro and Buonavoglia, 2012). CPV is a non-enveloped, single-stranded DNA virus that has been recently included in the same viral species, Carnivore protoparvovirus 1, along with feline panleukopenia virus (FPLV) and other parvoviruses of wild carnivores (Cotmore et al., 2014). Since its emergence in the mid-1970s likely as a host variant of FPLV through adaptation to an intermediate wild-carnivore host, CPV has further evolved giving rise to three antigenic variants, namely CPV-2a, CPV-2b (Hoelzer and Parrish, 2010) and the most recent CPV-2c (Buonavoglia et al., 2001). These variants show a high pathogenic potential and the ability to replicate in cats (Decaro and Buonavoglia, 2012). The geographic distribution of CPV antigenic variants is heterogeneous, with a prevalence of CPV-2b in United States, followed by CPV-2c and CPV-2a (Hong et al., 2007). In Mexico, only CPV-2c was typed (Pedrozaroldán et al., 2015). The three variants are circulating in South America with a higher frequency of CPV-2c in Ecuador (Aldaz et al., 2013), Argentina (Gallo et al., 2015), Brazil (Pinto et al., 2012) and Uruguay. Interestingly, the occurrence of CPV-2a was gradually reported and increased since 2011 being the main strain in Uruguay (Maya et al., 2013). The variant CPV-2a was the predominant in New Zealand with a prevalence of 98.5% beside the original strain CPV-2 detected at a small proportion (1.5%) (Ohneiser et al., 2015). In Japan, CPV-2b was the most prevailing strain followed by CPV-2a at a lesser frequency (Soma et al., 2013). In Taiwan a co-circulation of CPV-2a and CPV-2b at the same rate was reported (Lin et al., 2014). In China and Korea CPV-2a was the predominant variant followed by CPV-2b (Jeoung et al., 2008; Yi et al., 2014) and a recently circulation of CPV-2c was identified in northeast China (Geng et al., 2015). In India the three variants were identified with predominance of CPV-2b (Nandi et al., 2010).Whereas, in a recent study CPV-2c was not found (Mittal et al., 2014). Predominance of CPV-2a in European countries, Bulgaria (Filipov et al., 2011), Italy, UK, Spain, Germany, France, Belgium and Hungary, followed by CPV-2c and CPV-2b was reported (Decaro et al., 2007). Whereas, in

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Portugal, CPV-2c was the dominant variant, followed by CPV-2b and CPV2a at a smaller proportion (Miranda et al., 2016). In Africa, there are only few reports elucidating the CPV type distribution in the continent. While both CPV-2a and 2b were detected in South Africa and Namibia (Dogonyaro et al., 2013; Steinel et al., 1998), only CPV-2a was reported in Nigeria (Dogonyaro et al., 2013). A recent study from Tunisia reported a high prevalence of CPV-2c in this country (Touihri et al., 2009). However, there is no other study that was carried out in northern Africa. In Morocco, during the last years, a high number of suspected outbreaks of CPV infection were reported by breeders and veterinarians in vaccinated and unvaccinated dogs with lethargy, diarrhea and/or vomiting, but only few cases were confirmed by a laboratory diagnosis, which was based on hemagglutination (HA) or virus isolation (VI) assays (unpublished results). These assays have been recognised to be poorly sensitive, resulting in a high proportion of false-negative results (Decaro et al., 2005b; Desario et al., 2005). Molecular assays, based on the detection of the viral nucleic acid, represent the gold standard for a sensitive diagnosis of CPV infection (Desario et al., 2005). In addition to gel-based PCR tests, real-time PCR (qPCR) assays were developed for a more sensitive, specific and reproducible diagnosis (Decaro et al., 2005b; Wilkes et al., 2015). These assays were successfully used for rapid prediction of the antigenic type by means of type-specific minor groove binder (MGB) probes labelled with different fluorophores (Decaro et al., 2006b). MGB probe assays were also set up for discrimination between vaccine and field viruses (Decaro et al., 2006a, 2006c), which could be useful in the case of occurrence of post-vaccinal gastroenteritis. The aim of the present paper is to report the results of an epidemiological investigation for CPV carried out on dogs with diarrhea in Morocco using traditional and molecular tools.

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2. Material and methods 2.1. Sampling A total of 91 specimens (86 fecal samples and 5 pools of organs, consisting of liver, spleen and myocardium) were collected from sick and/or autopsied dogs of different breeds received at the university veterinary clinic in Rabat or at private veterinary clinics localised in Khemisset, Machraa Belakssiri, Rabat, Sale, Temara, Casablanca, Settat and Marrakech during years 2011-2015. These animals, had displayed pronounced clinical signs compatible with CPV infection, including hemorrhagic diarrhea. Details about nature and year of collection of the tested samples, as well as about age, breed, sex, origin and vaccination status of the sampled dogs are reported in Table 1. Clinical samples were homogenized in phosphate buffered-saline (PBS, pH 7.4) in a 10% w/v proportion and then clarified by brief centrifugation. The supernatants were stored at -80°C until use. 2.2. HA testing HA screening was conducted according to the method described by Carmichael et al., (1980) using a 1% PBS suspension of freshly prepared porcine erythrocytes. Serial dilutions of each specimen were tested twice in order to ensure accuracy. The viral titre was expressed as the highest sample dilution giving complete hemagglutination. 2.3. DNA preparation DNA extraction from faecal samples was carried out by boiling 200µl of homogenate supernatants for 10min, chilling and dilution at 1:10 in water, as useful treatment to ensure inactivation of Taq DNA polymerase inhibitors (Schunck et al., 1995). DNA extraction from organs was achieved using a commercial kit (DNeasy® Kit, QIAGEN), following the manufacturer’s instructions. 2.4. TaqMan real-time PCR Real time PCR was conducted according to the method developed by Decaro et al., (2005), using primers and probe which allows for detection of a 93-bp amplicon (Table 2). The 25-µl final PCR mixture consisted of

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12.5µl of Master mix (BioRad Laboratories), 600 nM of each CPV primer 555For and 555Rev, 200 nM of probe CPV-Pb and 10 µl of template DNA, and nuclease-free water. The thermal cycle was performed in a CFX96TM BioRad (BioRad laboratories) and consisted of activation of iTaq DNA polymerase at 95°C for 3 min, followed by 45 cycles of denaturation at 95°C for 10 s and primer annealing and extension at 60°C for 30s. Data were analysed by the BioRad CFX Manager Software (BioRad Laboratories). 2.5. MGB probe assays for CPV characterization CPV positive samples were characterised by real-time PCR assays using primers and fluorescently labelled MGB probes specific for types 2a/2b and 2b/2c, following the method developed by Decaro et al., (2006b). Briefly, each positive DNA template was tested in two reaction mixtures, containing primers and probes specific to detect type 2a/2b or 2b/2c (Table 2). The reaction was carried out to obtain a final mixture of 25µl consisting of 12.5 µl of Master Mix (BioRad Laboratories), 900 nM of specific primers, 200 nM of probe, 10 µl of template DNA, and free-nuclease water. Reactions were cycled under the same thermal conditions as previously detailed for generic real-time PCR. 2.6. Conventional PCR In order to identify uncharacterized strains, conventional PCR assays using three sets of primers designed to amplify overlapping sequences covering the full length of the capsid protein VP2 gene (Table 2) were carried out (Buonavoglia et al., 2001; Decaro et al., 2008). PCR amplifications were conducted in 25-µl final volume containing 2.5 µl of MgCl2, 2.5 µl of buffer, 4 µl of dNTPs, 0.25 µl of Taq polymerase (Takara Biotechnology Co. Ltd), 1 mM of forward and reverse primers, 10 µl of DNA, and nuclease-free water. The thermal conditions were as follows: after an initial denaturation at 94°C for 10 min, 40 cycles of 94°C for 30 sec, 55°C for 1 min and 72°C for 1 min were performed, with final extension at 72°C for 10 min. PCR products were analysed by electrophoresis on a 1.5% agarose gel containing a fluorescent nucleic acid marker (GelRed™ ) for DNA visualization under fluorescent light. The amplicons were subjected to direct sequencing in both directions. Sequence reactions were carried out using the BigDye 3.1 Ready reaction mix (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. The obtained nucleotide sequences were then analyzed using Geneious 9.0.4 (http://www.geneious.com) and compared with VP2 gene sequences of reference carnivore protoparvoviruses retrieved from the GenBank database (accession numbers are reported in parentheses): FPLV strain CU-4 (M24004), CPV-2 strain CPV-b (M38245), CPV-2a strain CPV15 (M24003), CPV-2b strain CPV39 (M74849) and CPV-2c strain 136/00 (FJ005195).

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3. Results 3.1. CPV detection by HA and real-time PCR assays HA test detected 41 (45.1%) positive and 50 (54.9%) negative samples, whereas all samples but one (98.9%) tested positive by the TaqMan-based real-time PCR assay. Viral loads ranged from 9.7 × 103 to 2 × 1010 DNA copy numbers mg-1 of sample. The HA-negative samples that tested positive by real-time PCR contained discrete viral titers, ranging from 1.5 × 104 to 4.6 × 109 DNA copy numbers mg-1 of sample. Considering real-time PCR as the gold standard, puppies under 6 months displayed the highest CPV detection rates, accounting for 85.5% of the positive animals. Fifty-three (58.8%) of the positive dogs were male and 31 (34.4%) were female. Seventy-seven dogs (85.5%) belonged to large-sized breeds and only 8 (8.8%) animals were small-sized. In addition, 48 (53.3%) CPV-infected dogs had been never vaccinated and 37 (41.1%) dogs had received at least one dose of CPV vaccine (Table 3). Information about sex, age, breed and vaccination status was not available for 6, 5, 5 and 5 animals, respectively. 3.2. CPV characterisation by MGB probe assays By using the MGB probe technology, the detected CPV strains were characterised as CPV-2b (n = 43, 47.7%), CPV-2c (n = 39, 43.3%)or CPV-

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2a (n = 1, 1.1%). Interestingly, co-infection with CPV-2b and CPV-2c was detected in 4 (4.4%) samples. Additionally, by this test 3 CPV strains (3.3%) were not characterised (Table 4). The single CPV-2a strain was recovered from a sample collected in 2012. In 2013 CPV-2c was the predominant variant, whereas in 2014 the frequency of detection of CPV-2b increased, with this variant being predominant in 2015 (Fig. 1).

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3.3. Sequence analysis of the uncharacterized strains The VP2 gene sequence of the 3 uncharacterised strains was determined through PCR amplification of overlapping fragments and direct sequencing of the PCR products. Sequence analysis showed a 100% nucleotide identity between these strains, which exhibited the same key residues of the CPV variants and was characterized as CPV-2c on the basis of the presence of codon GAA at position 4062-4064 of the viral genome, accounting for a glutamic acid residue at position 426 of the encoded protein. However, all these uncharacterised strains displayed the change G4068A which was responsible for the replacement of aspartic acid with asparagine at residue 427 of the VP2 protein. Thus, the nucleotide change accounted for the absence of binding of the CPV-2c specific probe to the target region. Another singleton non-synonymous substitution occurred at nucleotide position C2827G, generating the amino acid change from proline to arginine at residue 13 of the encoded VP2 protein. The synonymous substitution A4391G was also observed. By comparing the complete VP2 gene sequence of the CPV-2c uncharacterized strains with that of reference strains FPLV CU4, CPV-2 CPVb, CPV-2a CPV15, CPV-2b CPV39 and CPV-2c 136/00, nucleotide identities were 98.40%, 99.31%, 99.54%, 99.54%, and 99.77%, respectively.

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4. Discussion By HA testing of fecal samples from dogs with diarrhea in Morocco, more than one half of the tested samples (54.9%) were negative. In contrast, screening by real-time PCR showed that all HA-negative samples but one were positive and also contained considerable amounts of CPV DNA. These findings are in line with those reported previously by other authors (Decaro et al., 2006b, 2005a; Desario et al., 2005). However, in spite of the low sensitivity, HA is still the reference test for CPV diagnosis in labs that are not equipped with PCR-based platforms. To date, only few studies have reported the CPV epidemiology in the African country (Dogonyaro et al., 2013; Steinel et al., 1998; Touihri et al., 2009). The current study provides some useful information about the distribution of the CPV variants in Morocco throughout the last 5 years, accounting for a co-circulation of types 2b and 2c, whereas only one specimen tested positive for CPV-2a. This was in contrast with the results of a previous study carried out in Tunisia, where all the three variants were found to circulate approximately with the same frequency (Touihri et al., 2009). In South Africa and Namibia, CPV-2a was detected at high rates together with CPV-2b and it was the unique CPV variant detected in Nigeria so far (Dogonyaro et al., 2013). Noteworthy, three CPV strains that were not characterized by the MGB probe assays displayed an unexpected mutation at position 4068 of the viral genome, which prevented the binding of the CPV-2c specific probe. Mutations affecting the probe-binding region were already known in type 2c CPVs, but they were synonymous, thus not altering the virus antigenicity (Decaro et al., 2013). In contrast, change G4068A reported in this study determined an amino acid substitution at residue 427, which is close the unique change encountered between CPV-2a, CPV-2b and CPV-2c. However, to what extent this change may alter the antigenic profile of the virus is presently not known. Another important finding is that 4 samples were found to contain more than one CPV strains, namely CPV-2b and CPV-2c. Coinfections with different CPV variants were previously reported in Italy (Battilani et al., 2007) and Portugal (João Vieira et al., 2008), but the clinical and epidemiological implications of such coinfections are not yet

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clear. It is well known that the canine variants have regained the ability to infected cats, causing clinical signs resembling to feline panleukopenia (Decaro et al., 2011, 2010), but it was surprising that CPV/FPLV coinfections were observed in the feline species (Battilani et al., 2013, 2011). In conclusion, the present study contributes to a better understanding of the CPV evolution and variant distribution in northern Africa, thus aiming at a correct planning of vaccination protocols against this severe infection of domestic carnivores.

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Acknowledgements We wish to thank Professor Ouafaa Fassi Fihri for providing facilities to conduct this work.

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Lin, C., Chien, C., Chiou, M., Chueh, L., Hung, M., Hsu, H., 2014. Genetic characterization of type 2a canine parvoviruses from Taiwan reveals the emergence of an Ile324 mutation in VP2. Virol. J. 11, 1– 7. doi:10.1186/1743-422X-11-39 Maya, L., Calleros, L., Francia, L., Hernández, M., Iraola, G., Panzera, Y., Sosa, K., Pérez, R., 2013. Phylodynamics analysis of canine parvovirus in Uruguay: evidence of two successive invasions by different variants. Arch. Virol. 158, 1133–41. doi:10.1007/s00705-012-1591-5 Miranda, C., Parrish, C.R., Thompson, G., 2016. Epidemiological evolution of canine parvovirus in the Portuguese domestic dog population. Vet. Microbiol. 183, 37–42. doi:10.1016/j.vetmic.2015.11.037 Mittal, M., Chakravarti, S., Mohapatra, J.K., Chug, P.K., Dubey, R., Upmanuyu, V., Narwal, P.S., Kumar, A., Churamani, C.P., Kanwar, N.S., 2014. Molecular typing of canine parvovirus strains circulating from 2008 to 2012 in an organized kennel in India reveals the possibility of vaccination failure. Infect. Genet. Evol. 23, 1–6. doi:10.1016/j.meegid.2014.01.015 Nandi, S., Chidri, S., Kumar, M., Chauhan, R.S., 2010. Occurrence of canine parvovirus type 2c in the dogs with haemorrhagic enteritis in India. Res. Vet. Sci. 88, 169–171. doi:10.1016/j.rvsc.2009.05.018 Ohneiser, S.A., Hills, S.F., Cave, N.J., Passmore, D., Dunowska, M., 2015. Canine parvoviruses in New Zealand form a monophyletic group distinct from the viruses circulating in other parts of the world. Vet. Microbiol. doi:10.1016/j.vetmic.2015.05.017 Pedroza-roldán, C., Páez-magallan, V., Charles-niño, C., Elizondoquiroga, D., Cervantes-mireles, R.L. De, López-amezcua, M.A., 2015. Genotyping of Canine parvovirus in western Mexico. doi:10.1177/1040638714559969 Pinto, L.D., Streck, A.F., Gonçalves, K.R., Souza, C.K., Corbellini, Â.O., Corbellini, L.G., Canal, C.W., 2012. Typing of canine parvovirus strains circulating in Brazil between 2008 and 2010. Virus Res. 165, 29–33. doi:10.1016/j.virusres.2012.01.001 Schunck, B., Kraft, W., Truyen, U., 1995. A simple touch-down polymerase chain reaction for the detection of canine parvovirus and feline panleukopenia virus in feces. J. Virol. Methods 55, 427–33. Soma, T., Taharaguchi, S., Ohinata, T., Ishii, H., Hara, M., 2013. Analysis of the VP2 protein gene of canine parvovirus strains from affected dogs in Japan. Res. Vet. Sci. 94, 368–371. doi:10.1016/j.rvsc.2012.09.013 Steinel, A., Venter, E.H., Van Vuuren, M., Parrish, C.R., Truyen, U., 1998. Antigenic and genetic analysis of canine parvoviruses in southern Africa. Onderstepoort J. Vet. Res. 65, 239–42. Touihri, L., Bouzid, I., Daoud, R., Desario, C., El Goulli, A.F., Decaro, N., Ghorbel, A., Buonavoglia, C., Bahloul, C., 2009. Molecular characterization of canine parvovirus-2 variants circulating in Tunisia. Virus Genes 38, 249–58. doi:10.1007/s11262008-0314-1 Wilkes, R.P., Lee, P.-Y.A., Tsai, Y.-L., Tsai, C.-F., Chang, H.-H., Chang, H.-F.G., Wang, H.-T.T., 2015. An insulated isothermal PCR method on a field-deployable device for rapid and sensitive detection of canine parvovirus type 2 at points of need. J. Virol. Methods 220, 35–8. doi:10.1016/j.jviromet.2015.04.007 Yi, L., Tong, M., Cheng, Y., Song, W., Cheng, S., 2014. Phylogenetic Analysis of Canine Parvovirus VP2 Gene in China 1–8. doi:10.1111/tbed.12268

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Figure and Tables: Figure 1: CPV variants identified during 2011-2015. detected variants each year is indicated above the bars

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Table 1: Nature of samples, year of collection, age, breed, sex, and vaccination status of tested dogs

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Sample

Year

Age(month)

Breed

Sex

Vaccination

Origin

Strain

Alg-11 S10-11 Dark11 Parvo 3 Parvo 4 P01-13 P02-13 P03-13 P04-13 P05-13 P06-13 P09-13 P1-13 P2-13 P3-13 P4-13 P5-13 P6-13 P7-13 P8-13 P9-13 P10-13 P11-13 P12-13 P13-13 P14-13 P15-13 P16-13 P5-14 P6-14 P7-14 P8-14 P9-14 P10-14 P11-14 P12-14 P13-14 P14-14 P15-14 P16-14 P17-14 P18-14 P1-15 P2-15 P3-15 P4-15

I I S I I RS RS I RS Mc,S RS S I I RS I I I I L,S,Mc L I RS RS RS RS RS I I RS RS RS I RS RS I I I RS RS RS RS RS RS RS RS

2011 2011 2011 2012 2012 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2015 2015 2015 2015

NA 2 NA 1 NA 3 4 6 6 3 2 6 4 3 3 3 3 3 3 3 3 3 3 6 5 5 5 5 6 4 4 4 3 2 3 4 2 3 3 3 3 1 7 3 5 7

NA R NA R NA P LR LR GR MB P MB MB MB MB MB MB MB MB MB MB MB MB MB MB GS GS St. G R R H St. G C BF H C LA GS GS GS GS MB MB GS GS GS

NA F NA F NA F M F F F M NA M M F F M M M F F F M F F M M M M F M F F M F M M M M M F M M M M M

NA UV NA UV NA V V V V V UV UV UV UV UV UV UV UV UV UV UV UV UV UV UV V V UV V UV V V V V V V V V V V V UV UV V UV V

Marrakech Rabat NA Rabat Rabat Rabat Rabat Rabat Rabat Rabat Rabat Rabat Khemisset Khemisset Khemisset Khemisset Khemisset Khemisset Khemisset Khemisset Khemisset Khemisset Khemisset Khemisset Khemisset Rabat Rabat Rabat Rabat Casablanca Casablanca Casablanca Casablanca Casablanca Settat Casablanca Casablanca Casablanca Casablanca Casablanca Casablanca Rabat Rabat Rabat Rabat Rabat

NC 2c 2c 2c 2a 2c 2c 2c 2c 2c 2c 2c 2c 2c 2b 2c 2c 2c 2c 2c 2c 2c 2c 2c 2c NC NC 2c 2c 2c 2c 2c 2c 2c 2b 2b 2b 2c 2c 2b+2c 2b 2c 2b 2c 2b 2c

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Rabat 2b 6 MB M UV Machraa 2b Belaksiri P7-15 RS 2015 7 MB M UV Machraa 2c Belaksiri P8-15 RS 2015 7 L M UV Rabat 2b P9-15 RS 2015 6 MB M UV Rabat 2b P10-15 RS 2015 3 MB M UV Rabat 2b P11-15 RS 2015 5 GS M UV Rabat 2b P12-15 RS 2015 NA NA NA NA NA 2b P13-15 RS 2015 2 GS M V Rabat 2b P14-15 RS 2015 3 P F UV Rabat 2b P15-15 RS 2015 24 ASD F V Rabat 2c P16-15 RS 2015 8 BS F V Rabat 2b P17-15 RS 2015 4 GS F V Rabat 2b P18-15 RS 2015 3 GS M UV Rabat 2b P19-15 RS 2015 5 MB F UV Rabat N P20-15 RS 2015 5 P F V Rabat 2b P21-15 RS 2015 1 R M V Rabat 2b+2c P22-15 RS 2015 4 GS M UV Rabat 2c P23-15 RS 2015 NA NA NA NA Temara 2c P24-15 RS 2015 4 R M V Rabat 2c P25-15 RS 2015 5 St. G M UV Rabat 2b P26-15 RS 2015 5 St. G M UV Rabat 2b+2c P27-15 RS 2015 5 St. G M UV Rabat 2b P28-15 RS 2015 5 St. G M UV Rabat 2b P29-15 RS 2015 5 MB M UV Rabat 2b P30-15 RS 2015 2 LR F V Temara 2b P31-15 RS 2015 2 LR M V Temara 2b P32-15 RS 2015 3 LR F V Temara 2b P33-15 RS 2015 2 LR M V Temara 2b P34-15 RS 2015 3 MB M UV Rabat 2b P35-15 RS 2015 3 MB M UV Rabat 2b P36-15 RS 2015 4 MB F UV Rabat 2b P37-15 RS 2015 4 LR M UV Rabat 2b P38-15 RS 2015 4 MB M UV Rabat 2b P39-15 RS 2015 2 LR F V Temara 2b P40-15 RS 2015 2 St. G M V Temara 2b P41-15 RS 2015 2 St. G M V Temara 2b P42-15 RS 2015 3 MB M UV Rabat 2b P43-15 RS 2015 3 MB M UV Rabat 2b P44-15 RS 2015 2 BS F UV Temara 2b+2c P45-15 RS 2015 2 BS F UV Temara 2b P46-15 RS 2015 2 BS F UV Temara 2b P47-15 RS 2015 7 MB M UV Rabat 2b P48-15 RS 2015 4 BS M UV Rabat 2b P49-15 RS 2015 1 R F V Sale 2b M: Male ; F: Female ; I: Intestine ; L: Liver ; S: Spleen ; Mc: Myocardium ; RS: Rectal swabs ; V: Vaccinated ; UV: Unvaccinated ; NA: No information available ; ASD: Atlas Shepherd Dog ; BS: Belgian

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Shepherd ; BF: bichon frise ; C: Chihuahua ; GR: Golden retriever ; GS: German shepherd ; H: Husky ; LA: Lhassa Apso ; LR: Labrador Retriever ; MB: Mixed breed ; P: Poodle ; R: Rottweiler ; St. G: Saint-Germain Pointing Dog ;N: Negative; NC: Not characterised by MGB technology

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Table 2: Nucleotide sequences, position and amplicon size of primers and probes used in the current study Primer/pro be

Sequence

Position

Generic Real-time PCRA MGB 2a/2bB

CPV-For CPV-Rev CPV-Pb CPVa/b-For CPVa/b-Rev CPVa-Pb CPVb1-Pb

AAACAGGAATTAACTATACTAATATATTTA AAATTTGACCATTTGGATAAACT FAM−TGGTCCTTTAACTGCATTAAATAATGTACC−TAMRA AGGAAGATATCCAGAAGGAGATTGGA CCAATTGGATCTGTTGGTAGCAATACA VIC−CTTCCTGTAACAAATGATA−MGB FAM−CTTCCTGTAACAGATGATA−MGB

MGB 2b/2cB

CPVb/c-For

GAAGATATCCAGAAGGAGATTGGATTCA

CPVb/c-Rev

ATGCAGTTAAAGGACCATAAGTATTAAATATATTAGTATAGTTAATTC

4014-4035E 4176-4198E 4143-4172E 17191744F,G 17851811F,G 1765-1783F 1765-1783G 1721-1748G 1155-1182H 1823-1870G 1257-1304H

CPVb2-Pb

FAM−CCTGTAACAGATGATAAT−MGB

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1202-1219H CPVc-Pb VIC−CCTGTAACAGAAGATAAT−MGB 4003–4022 E 583 Convention 555-ForC CAGGAAGATATCCAGAAGGA C 555-Rev 4561–4585 E al GGTGCTAGTTGATATGTAATAAACA C D PCR 3381-For 3381–3400 E 717 CCATGGAAACCAACCATACC D 4116-Rev 4093–4116 E AGTTAATTCCTGTTTTACCTCCAA D 2655-For 2655-2675 E 837 AAAAAGAGACAATCTTGCACCA D 3511-Rev 3489–3511 E TGAACATCATCTGGATCTGTACC A: Decaro et al., 2005b ; B: Decaro et al., 2006b ; C: Buonavoglia et al., 2001; D: Decaro et al., 2008 ; E: Oligonucleotide positions are referred to the sequence of CPV-2 (original type) strain CPV-b (Genbank accession no. M38245); F: Oligonucleotide positions are referred to the sequence of CPV-2a strain CPV-15 (Genbank accession no. M24003); G: Oligonucleotide positions are referred to the sequence of CPV-2b strain CPV-39 (Genbank accession no. M74849); H: Oligonucleotide positions are referred to the sequence of CPV-2c strain 56/00 (Genbank accession no. AY380577).

Table 3: Results of generic real-time PCR assay by vaccination status Vaccination status Vaccinated Unvaccinated Not Available Total

Positive samples 37 (41.1%) 48 (53.3%) 5 (5.6%) 90 (98.9%)

Negative samples 0 1 (1.1%) 0 1 (1.09%)

Total 37 (40.6%) 49 (53.8%) 5 (5.5%) 91 (100%)

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Table 4: CPV variants detected in Morocco

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CPV-2c

1 (1.1%)

43 (47.7%)

39 (43.3%)

CPV-2b and 2c 4 (4.4%)

Not characterised 3 (3.3%)

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Total of positive samples

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Highlights:

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- Samples were collected from 91 dogs received in Moroccan veterinary clinics with symptoms predictive of canine parvovirus infection.

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- Ninety samples were diagnosed CPV positive. MGB Characterisation identified 43 samples as CPV-2b, 39 as CPV-2c and 1 CPV-2a. A Co-infection with CPV-2b and CPV-2c was

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-VP2 gene sequencing identified uncharacterised samples as CPV-2c with the mutation

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