Results
Full-length CPV VP2 sequences were amplified from 79 faecal samples collected at 20 veterinary practices in 5 Australian states (Figure 1). The median age at diagnosis was 4 months (range 1 to 96 months). Of dogs for which complete vaccination data were available, 49.1% (26/53) were incompletely vaccinated, 47.2% (25/53) were unvaccinated, and 3.77% (2/53) were completely vaccinated. The two dogs that developed parvoviral enteritis despite being completely vaccinated were a 20-month-old female Cavoodle that developed parvoviral enteritis 8 months after its first annual booster vaccination, and a 3-year-old male Staffordshire Bull Terrier that developed disease 5 months after receiving an annual booster vaccination (Table 2).
There were 44 dogs for which outcomes were known, of which 52.3% recovered (23/44), 16 (36.4%) died despite treatment and 5 (11.4%) were euthanised without treatment. CPV VP2 sequence analysis detected CPV-2b (426D) in 43/79 (54.4%) samples, CPV-2a (426N) in 34/79 (43.1%) and CPV-2 in 2/79 (2.5%). The two isolates identified as CPV-2 were from dogs with unknown vaccination histories.
On phylogenetic analysis the 79 CPV strains fell into six clades (Figure 2). The largest clade (Figure 2, Clade 1) comprised CPV-2b viruses. In addition to 426-Asp, most viruses in Clade 1 contain one novel (5-Gly) and two well-recognised (267-Tyr and 297-Ala) non-synonymous mutations compared to “classical” strains of CPV-2 (e.g. strain “N” and strain “CPV-b”), which are typically characterised by 267-Phe, 297-Phe and 5-Ala (Table 3). The majority of Clade 1 viruses originate from dogs in adjacent rural regions in central west and far west NSW (Figure 1, Table 2). Clade 1 also contains several viruses from dogs from Western Australia, all of which had a novel mutation 262-Thr compared to 262-Ala in classical CPV-2.
Clade 2, the second largest clade is also the most heterogeneous, comprising 16 CPV-2a viruses, and 5 CPV-2b viruses (Figure 1) and contains viruses from all 5 States sampled in the study. In addition to the 5-Gly, 267-Tyr and 297-Tyr aa residues described for Clade 1 viruses, unique aa mutations 564-Asn and 568-Ala were present all but one of the viruses in this clade (Table 2).
Clade 3 contains most of the viruses sequenced from Victoria in 2015. All of the viruses in this clade contain both a novel (21-Ala) and a well-recognised (324-Ile) mutation compared to “classical” CPV-2 (324-Tyr, 21-Thr). With the exception of one CPV-2b (426-Asp) virus, this clade comprises only CPV-2a viruses (426-Asn).
Viruses in Clade 4, all collected in 2019, are characterised by a novel mutation (321-Lys) compared to “classical” CPV-2 (321-Asn). Three, from dogs in Northwest NSW have identical VP2 sequences to an attenuated CPV-2b vaccine strain (“SAH”), except for residue 570-Lys, which is glutamate in the vaccine strain. The dogs were from a large breeding establishment and two had been vaccinated with the same SAH strain, while one, an unvaccinated puppy, died at 7 weeks of age after an 8-day history of lethargy, dehydration, vomiting and haemorrhagic diarrhoea. Similarly, one VP2 sequence from a dog in Western Australia differed by only one aa (413-Asn) to the SAH vaccine strain (413-Asp) (Figure 1, Table 2). The vaccination history of this dog was unknown.
Clade 5 contains CPV-2a viruses collected from dogs in metropolitan Sydney between 2016 and 2018, which, with the exception of 297-Ala, did not harbour the 5-Gly, 21-Ala and 267-Tyr mutations commonly present in other Australian CPV-2a viruses. Clade 6 contained two CPV-2 VP2 sequences from dogs with unknown vaccination history.