Canine parvovirus (CPV) is a major pathogen causing severe gastroenteritis in dogs. Since its emergence, CPV has undergone continuous evolution, leading to the predominance of variants such as CPV-2a, CPV-2b, and CPV-2c. To characterize the genetic features and evolutionary trends of CPV-2 at a regional level, 775 fecal samples were collected from domestic and stray dogs with suspected CPV-2 infection in Shanghai between 2016 and 2025. The overall positivity rate was 23.2% (180/775); incidence was substantially higher in stray dogs (30.2%) than in domestic dogs (15.9%). Thirty-one CPV-2 strains were successfully isolated. Temporal analysis revealed a pronounced genotype shift: isolates from 2016 to 2020 were predominantly New CPV-2a, whereas CPV-2c became the dominant genotype from 2021 through 2025. Sequence analysis identified the polymorphism of VP2 gene and characteristic mutations F267Y, Y324I, N426E, Q370R and A440T in CPV-2c strains. A novel I447M mutation was detected in several isolates. Phylogenetic analysis showed that Shanghai isolates formed distinct clusters; CPV-2c strains were closely related to the Asian lineage. Structural modeling indicated that mutations at residues L87M, T101I, Y267F, A297S, G300A, Y305D, I324Y, Q370R, N426E, A440T, and I447M may alter the tertiary structure of the VP2 protein, potentially affecting antigenicity and receptor recognition. Collectively, these results demonstrate the complete genotype replacement of CPV-2 in Shanghai; CPV-2c is now predominant. Identification of the novel I447M mutation and structural analysis of key amino acid substitutions provide insight into CPV molecular evolution. These findings suggest that vaccines primarily based on older CPV-2 or CPV-2b genotypes offer suboptimal protection, highlighting the need for updated vaccine strategies targeting prevalent CPV-2c variants.

Toxoplasma gondii (T. gondii) is an important zoonotic pathogen which induces both acute and chronic toxoplasmosis. Timely diagnosis of T. gondii is crucial for effective disease management. Here, we present a pioneering approach using europium (III)-chelated nanoparticles (EuNPs) in a rapid lateral flow immunochromatographic test strip (ICTS) for detecting T. gondii antibodies in serum samples. By conjugating EuNPs with Staphylococcus aureus protein A, we efficiently captured T. gondii-specific antibodies, which bound to T. gondii antigens on the test line (T-line), generating a distinct fluorescent signal. Employing this novel method, we conducted an extensive epidemiological investigation of T. gondii infections among dogs and cats in Shanghai, China. This innovative ICTS allows for rapid results within 25 min, which include a qualitative result through naked-eye observation under an ultraviolet lamp and a quantitative one derived using a strip reader. With a detection limit of 1:6400 for dog positive serum and no cross-reactivity with other canine and feline pathogens, the EuNPs-ICTS demonstrated excellent consistency with standard enzyme-linked immunosorbent assay results for dogs (κ = 0.91) and cats (κ = 0.92). In addition, 20.38% of 996 dog serum samples and 14.18% of 416 cat serum samples revealed T. gondii antibodies, highlighting the efficacy of this approach. Our study presents a rapid, sensitive, specific, and reproducible EuNPs-ICTS, serving as a promising tool for on-the-spot diagnosis of T. gondii infections in dogs and cats.

Enterocytozoon bieneusi is reported to be a common microsporidian of humans and animals in various countries. However, scarce information on E. bieneusi has been recorded in cats ( Felis catus ) and dogs ( Canis familiaris ) in China. As such, we undertook molecular epidemiological investigation of E. bieneusi in cats and dogs in Shanghai, China. A total of 359 genomic DNAs were extracted from individual faecal samples from cats ( n = 59) and dogs ( n = 300), and then tested using a nested PCR-based sequencing approach employing internal transcribed spacer (ITS) of nuclear ribosomal DNA as the genetic marker. Enterocytozoon bieneusi was detected in 34 of all 359 (9.5%) faecal samples from cats (32.2%; 19/59) and dogs (5.0%; 15/300), including 24 stray cats and dogs (22.6%; 24/106), as well as ten household/raised cats and dogs (4.0%; 10/253). Correlation analyses revealed that E. bieneusi positive rates were significantly associated with stray cats and dogs ( P < 0.05). The analysis of ITS sequence data revealed the presentation of five known genotypes CD7, CHN-HD2, D, PtEb IX and Type IV and two novel genotypes D-like1 and PtEb IX-like1. Zoonotic genotype D was the predominant type with percentage of 61.8 (21/34). Phylogenetic analysis of ITS sequence data sets showed that genotypes D, D-like1 and Type IV clustered within Group 1, showing zoonotic potential. The others were assigned into Group 10 with host specificity. These findings suggested that cats and dogs in Shanghai harbor zoonotic genotype D of E. bieneusi and may have a significant risk for zoonotic transmission. Further insight into the epidemiology of E. bieneusi in animals, water and the environment from other areas in China will be important to have an informed position on the public health significance of microsporidiosis caused by this microbe.