Canine parvovirus type 2b is the most prevalent genomic variant strain found in parvovirus antigen positive diarrheic dog feces samples across Canada

In 1978 an emerging virus, called canine parvovirus (CPV) was identified in the dog population; this virus was subsequently named CPV type 2 (CPV-2). The CPV-2 was distinct from the well-known parvovirus, canine minute virus (CnMV), which was formerly known as canine parvovirus type 1 (1). The CPV-2 virus induced hemorrhagic enteritis, severe diarrhea, vomiting, and leukopenia associated with a high mortality in infected dogs and spread into non-immune dog populations worldwide causing a pandemic. Furthermore, CPV-2 was responsible for myocarditis in puppies. Today, the prevalence of CPV antibodies in adult dogs is high due to vaccination and/or natural infection causing a high protective immunity status. Nevertheless, non-immune puppies that are 6 wk to 6 mo old are susceptible to CPV infection when there is loss of passive protection from maternal-derived antibodies (MDA), which are able to protect puppies against myocarditis (2).

The CPV-2 virus belongs to the virus family Parvoviridae, subfamily Parvovirinae (which infect vertebrates) and genus Parvovirus (3). A small non-enveloped single-stranded DNA virus, COV-2 is about 25 nm in size (1). The CPV-2 genome is 5.2 kilo bases (kb) long and possesses at least 2 major open reading frames (ORFs) (4). In the conventional orientation, the right-hand ORF encodes the viral capsid proteins VP1 and VP2, which are the main antigens that induce protective antibodies (5–7). The left-hand ORF encodes the non-structural proteins NS1 and NS2 (8). Since the emergence of CPV-2, several viral genomic variants have emerged due to a surprisingly high substitution rate in the CPV genome, similar to those of RNA viruses (1,9). At least 4 genomic variants have been described and officially recognized: the initial emerging CPV was named CPV-2, and was followed shortly after by CPV-2a in 1979, CPV-2b in 1984, and CPV-2c in 2000 (1). These CPV variants are differentiated through single nucleotide polymorphism (SNP) located at the VP2 gene (10). The prevalence of CPV variants is geographically restricted and has evolved over time (1,10). To our knowledge, the prevalence of CPV genomic variants in Canada is not known. Thus, the main objective of the present study was to determine the prevalence of CPV genomic variants within the Canadian diarrheic dog population.

Canadian veterinarian volunteers submitted feces from diarrheic animals that were previously confirmed to be positive for CPV antigen after local testing by any commercially available capture antigen assay such as the SNAP^® Parvo Test (IDEXX Laboratories, Westbrook, Maine, USA). The CPV vaccination status of diarrheic animals was requested and fecal samples of dogs that were known to be recently vaccinated (within the last 30 d) were excluded from the study. It has been recently reported that animals that are vaccinated with a live attenuated vaccine may shed the vaccine strain for up to 21 d post-vaccination, as established by CPV real-time polymerase chain reaction (qPCR) (11). Thus, a delay of 30 d post-vaccination for CPV genotyping was sufficient to circumvent the effects of vaccine strains.

Samples from 49 cases were included in this study. These samples were submitted over a 3-year period (from September 27, 2012 until August 21, 2015) and originated from 7 provinces (Table 1). Genotyping of CPV variants was performed by the concomitant analysis of 3 multiplex qPCR assays using minor groove binder (MGB) probes technology as previously described (12,13) but with minor modifications. These MGB probes allow SNP discrimination of the ORF encoding the VP2 protein and therefore allow genotyping of strains. Briefly, about 1 g of feces was suspended in 5 mL of phosphate-buffered saline (PBS). Nucleic acid extraction was performed on supernatants or directly on vaccine formulations (Duramune^®Max Pv and Nobivac^®Canine 3-DAPv which contain the CPV-2b and CPV-2 variants, respectively, as positive controls) with either the QIAamp cador Pathogen Mini Kit (Qiagen, Toronto, Ontario) or the BioSprint 96 One-For-All vet kit (Qiagen) according to the manufacturer’s instructions. Thereafter, the 3 multiplex qPCR assays were carried out in a 25-μL volume containing 5 μL template or control DNA, 12.5 μL of QuantiTect Probe PCR buffer 2× (Qiagen), 3 pmol of TET-labeled probe, and 1.25 μL of a 20× mixture consisting of forward and reverse primers and the FAM-labeled probe. The qPCR assays were performed on either a SmartCycler (Cepheid, Sunnyvale, California, USA) or a Rotor-Gene (Qiagen) thermal cycler. The first multiplex qPCR assay targeted the change from adenine to guanine at position 4062 (SNP A4062G in GenBank reference strain M38245) that distinquishes between CPV-2a and 2b variants, while the second multiplex qPCR assay targeted the thymine to adenine change at position 4064 (SNP T4064A) that differentiates CPV-2b and 2c variants. As previously reported, the first multiplex qPCR assay could not properly discriminate CPV-2 and 2a variants. Thus, a third multiplex qPCR assay targeting SNP T3088C was developed to ensure discrimination between the CPV-2 variant and all 3 other variants. Taken together, these 3 multiplex qPCR assays allow reliable and fast CPV genotyping results.

Table 1.

Prevalence of canine parvovirus type 2 genomic variants in feces of diarrheic dogs

Province	Number of submitted feces samplesa	Genomic variants % (n)
		CPV-2	CPV-2a	CPV-2b	CPV-2c
Nova Scotia	3	0.0 (0)	0.0 (0)	100.0 (3)	0.0 (0)
New Brunswick	1	0.0 (0)	0.0 (0)	100.0 (1)	0.0 (0)
Quebec	10	0.0 (0)	10.0 (1)	90.0 (9)	0.0 (0)
Ontario	13	0.0 (0)	0.0 (0)	92.3 (12)	7.7 (1)
Saskatchewan	1	0.0 (0)	0.0 (0)	100.0 (1)	0.0 (0)
Alberta	7	0.0 (0)	14.3 (1)	85.7 (6)	0.0 (0)
British Columbia	14	0.0 (0)	14.3 (2)	85.7 (12)	0.0 (0)
Total	49	0.0 (0)	8.2 (4)	89.8 (44)	2.0 (1)

^aSamples were submitted from CPV antigen positive diarrheic animals starting September 27, 2012 to August 21, 2015.

As shown in Table 1, the CPV-2b variant was the most prevalent in all 7 provinces from which the samples were obtained. The prevalence of CPV-2b between the different provinces varied from 85.7% to 100%. Overall, the Canadian CPV-2b prevalence was 89.8%. This finding was surprising because CPV-2c was reported to be the most prevalent variant in the United States (US) since 2007, varying between 48.1 and 73.5% (14,15). In contrast, only 1 sample (originating from Ontario) was positive for CPV-2c variant in the current study (Table 1). The CPV-2c virus has an overall Canadian prevalence of 2%, which is much lower than its prevalence in the United States. Four samples were positive for the CPV-2a variant and originated from 3 provinces: Alberta, British Columbia, and Quebec. Thus, CPV-2a has an overall Canadian prevalence of 8.2% (Table 1). As expected, no CPV-2 variant was detected, as it is considered to be extinct (Table 1).

Vaccination could significantly affect the genotyping of CPV as attenuated vaccine strains could be shed in feces (11); information on vaccination status was therefore requested for the submitted samples. Unfortunately, the vaccination status of 7 animals (i.e., 14.3% of the fecal samples) was unknown (data not shown). Even if those 7 cases are excluded from our analyses, the CPV-2b variant remained the most prevalent variant found in diarrheic dogs with a value of 90.5% (data not shown). Interestingly, 19 diarrheic animals were reported by veterinarians to be vaccinated and at least 3 different commercial vaccines had been used to vaccinate these animals (data not shown). Only 1 of these animals (5.3%), however, seems to have been vaccinated according to the equivalent guidelines of the American Animal Hospital Association (AAHA) and the World Small Animal Veterinary Association (WSAVA). These guidelines are: “Puppies with poor MDA may be vulnerable (and capable of responding to vaccination) at an earlier age, while others may possess MDA at such high titres that they are incapable of responding to vaccination until ≥ 12 weeks of age. No single primary vaccination policy will therefore cover all possible situations. Thus, puppies should be vaccinated every 3–4 weeks between the ages of 6 and 16 weeks (e.g., at 6, 10, and 14 weeks, or 8, 12, and 16 weeks). To minimize the risk of MDA interference with vaccination, the final dose of the initial series should be administered between 14 and 16 weeks of age, regardless of the product used” (16,17). Thus, at least 83.7% (n = 41) of the diarrheic animals of this study were not or were improperly vaccinated (data not shown) because they received only 1 or 2 doses or the last puppy booster was given before 14 wk of age. Three of the vaccinated animals received 3 injections of vaccine as recommended by AAHA and WSAVA but surprisingly, the last booster injection was given before 14 wk of age (data not shown).

Canadian veterinarians should encourage pet owners to allow vaccination of their puppies following the AAHA and WSAVA guidelines. Several reports have demonstrated a very good efficacy of CPV vaccines in the context of homologous and heterologous challenges (10,17). Our results may have indicated a vaccination failure in 1 vaccinated animal but it might be explained by errors included in the clinical data that were reported by the owner and/or the veterinarian. Moreover, other co-infecting microorganisms (bacteria, parasites, viruses) could have contributed to the diarrhea in the clinical cases that were submitted (18). Unfortunately, the intestinal microbiota of diarrheic animals was not part of this study and, therefore, it was not investigated. In conclusion, by far the most prevalent CPV variant found in the feces of Canadian diarrheic dogs is CPV-2b.