RESULTS

Study population and epidemiological characteristics

A total of 200 fecal samples were collected from companion animals in and around Anand District, Gujarat, India, comprising 100 cat and 100 dog samples. Among the sampled animals, 117 (58.5%) showed clinical signs consistent with gastroenteritis, including diarrhea, vomiting, dehydration, anorexia, and abdominal discomfort, whereas 83 (41.5%) were apparently healthy at the time of sampling. Samples were obtained from the Veterinary Clinical Complex, private veterinary clinics, rescue organizations, and shelters, representing a heterogeneous companion animal population.

Animals aged 3–6 months constituted the majority of positive cases. The study population included 113 males (54 cats and 59 dogs) and 87 females (46 cats and 41 dogs). Breed distribution showed that non-descript and Persian cats were similarly represented, whereas Labrador Retrievers constituted the largest proportion among dogs, followed by German Shepherds.

Vaccination history showed that 62 animals were fully vaccinated, 57 were partially vaccinated, and 81 were unvaccinated. FPV detection was lower among fully vaccinated cats, whereas CPV infection was observed in dogs irrespective of vaccination status. Seasonal distribution showed higher positivity during summer and winter, suggesting potential environmental influences on viral persistence and transmission.

RAT detected FPV antigen in 10 (5.0%) samples and CPV antigen in 80 (40.0%) samples. Conventional PCR targeting the VP2 gene detected 39 positive samples (19.5%), comprising 11 feline and 28 canine samples. In contrast, probe-based qPCR detected 124 positive animals (62.0%), including 27 FPV-positive and 97 CPV-positive cases, demonstrating substantially higher sensitivity.

Molecular detection of FPV and CPV

Conventional PCR: Amplification targeting the VP2 gene produced the expected 681 bp amplicon, confirming the presence of parvoviral DNA in fecal samples. Of the 200 samples screened, 39 (19.5%) tested positive, including 11 cats (11%) and 28 dogs (28%).

The primers used in this assay targeted conserved regions of the VP2 gene and therefore detected both FPV and CPV. Consequently, although PCR confirmed the presence of parvoviral DNA, it could not differentiate between FPV and CPV infections. The amplified PCR products were resolved on 1% agarose gel, and a specific amplicon of approximately 681 bp was observed in positive samples, confirming the presence of parvoviral DNA (Figure 1).

Figure 1: Agarose gel electrophoresis image of VP2 gene polymerase chain reaction-amplified products of feline parvovirus. Lane W: 100 bp DNA ladder (Catalog #SM0243, Applied Biosystems™, Thermo Fisher Scientific Inc.); lanes S1–S6: Positive samples showing the target-specific amplicon of 681 bp; lane PC: Positive control; lane NTC: No-template control.

qPCR detection and differentiation: A probe-based multiplex qPCR assay targeting virus-specific regions of the VP2 gene was used to differentiate FPV and CPV infections. Among the 200 samples tested, 124 (62.0%) were positive for parvovirus, including 26 FPV-positive and 12 CPV-positive cats, and 85 CPV-positive and one FPV-positive dog.

Among feline samples, 26% were FPV-positive and 12% were CPV-positive, whereas one canine sample tested positive for FPV, indicating possible cross-species circulation of parvoviruses between feline and canine hosts. Among dogs, 85% tested positive for CPV, confirming widespread circulation within the canine population. The qPCR amplification plots for FPV, CPV, and the combined detection of FPV and CPV are presented in Figures 2, 3, and 4, respectively.

Comparative evaluation of diagnostic methods

Comparison of diagnostic methods revealed marked differences in detection rates. RAT detected only 5% of cases, whereas conventional PCR detected 19.5%. In contrast, qPCR detected 62% of infections, demonstrating higher analytical sensitivity.

Figure 2: Quantitative polymerase chain reaction amplification plot for feline parvovirus.

Figure 3: Quantitative polymerase chain reaction amplification plot for canine parvovirus.

Figure 4: Quantitative polymerase chain reaction amplification plot for feline parvovirus and canine parvovirus.

The detection pattern observed in this study was as follows:

RAT (5%) < PCR (19.5%) < qPCR (62%)

The comparative diagnostic performance of RAT, conventional PCR, and qPCR for the detection of FPV and CPV in cats and dogs is summarized in Table 2.

PCR primers described by Carreno et al. [12] target a conserved VP2 region and therefore detect parvoviral DNA without differentiating FPV and CPV. Differentiation of FPV and CPV was performed using the probe-based qPCR assay described by Decaro et al. [26].

Virus isolation

Virus isolation was attempted from nine molecularly confirmed parvovirus-positive samples using CRFK cell monolayers. Four samples produced characteristic CPE, including cell rounding, aggregation, granulation, and detachment, beginning between the third and fourth passages. These cultures eventually showed partial to complete destruction of the monolayer within 48–72 h.

PCR confirmation of culture supernatants produced the expected 681 bp VP2 amplicon, verifying viral replication in infected cell cultures. Overall, 44.44% of samples were successfully adapted to CRFK cells. The CPE observed during virus isolation in CRFK cell culture is shown in Figure 5, whereas PCR amplification of the isolated virus, showing the specific VP2 gene band, is shown in Figure 6.

Species | RAT FPV | RAT CPV | PCR FPV/CPV | qPCR FPV | qPCR CPV
Cats (n = 100) | 8 (8%) | 10 (10%) | 11 (11%) | 26 (26%) | 12 (12%)
Dogs (n = 100) | 2 (2%) | 70 (70%) | 28 (28%) | 1 (1%) | 85 (85%)
Total (n = 200) | 10 (5%) | 80 (40%) | 39 (19.5%) | 27 (13.5%) | 97 (48.5%)

Figure 5: Cytopathic changes in Crandell–Rees feline kidney (CRFK) cells after parvovirus inoculation. Left: Uninfected CRFK cells showing normal confluent monolayer morphology (40×, inverted microscope). Right: CRFK cells inoculated with suspected parvovirus showing mild cytopathic changes (40×, inverted microscope).

Figure 6: Polymerase chain reaction amplification of the VP2 gene from infected cell culture confirming parvoviral DNA (681 bp). Lane W: 100 bp DNA ladder (Catalog #SM0243, Applied Biosystems™, Thermo Fisher Scientific Inc.); lanes S1, S4, and S6: Negative samples; lanes S2, S3, and S5: Positive samples showing the target-specific amplicon of 681 bp; lane PC: Positive control; lane NTC: No-template control.

Molecular characterization of parvovirus isolates

Sequencing of the VP2 gene was performed on 10 representative isolates, including 3 FPV and 7 CPV samples. FPV isolates showed 99.8%–100% nucleotide identity with classical FPV strains reported globally and displayed only minor amino acid substitutions.

In contrast, CPV isolates showed greater variability and possessed characteristic amino acid signatures consistent with the CPV-2c variant. Phylogenetic analysis clustered the sequences into two distinct groups corresponding to FPV and CPV, with CPV isolates grouping closely with recent CPV-2c strains reported from Asia and Europe. Representative sequencing chromatograms illustrating nucleotide substitutions and corresponding amino acid changes in FPV and CPV isolates are presented in Figures 7–10. Detailed comparison of amino acid substitutions at selected VP2 loci is presented in Table 3, whereas sequence identity and closest GenBank matches are summarized in Table 4.

Phylogenetic analysis of partial VP2 gene sequences grouped the Gujarat isolates into two distinct clusters corresponding to FPV and CPV. The analysis revealed clustering of the study isolates with reference CPV-2c strains and classical FPV strains. Because only partial VP2 sequences were analyzed, definitive subtype assignment for all isolates should be interpreted with caution. The study isolates clustered predominantly with contemporary CPV-2c strains while maintaining genetic relatedness to previously reported CPV-2a lineages. Although partial sequences limited definitive subtype classification, the clustering pattern indicated genetic relatedness to contemporary Asian strains. The observed separation from vaccine and prototype strains suggests minor antigenic divergence among circulating parvoviruses (Figure 11). However, the use of partial VP2 gene sequences limited the ability to achieve complete subtype differentiation and full genomic characterization of circulating parvovirus strains.

Isolate details | Mutated loci of amino acid
ID | Accession No. | Subtype | Location | Year | 100 | 131 | 135 | 150 | 151 | 165 | 168 | 173 | 191 | 192 | 203 | 206
F02D4MM | PX365589 | FPV | Gujarat, India | 2025 | V | T | F | N | R | S | A | D | D | Y | E | F
F03D4MF | PX365590 | FPV | Gujarat, India | 2025 | V | T | F | N | R | S | A | D | D | Y | E | L
F04D4MF | PX365591 | FPV | Gujarat, India | 2025 | V | T | F | N | R | S | A | D | D | Y | ? | L
1986_FPV_ China | KX900570 | FPV | China | 1986 | V | T | F | N | R | S | A | D | D | Y | ? | L
2009_FPV_Japan | D88287 | FPV | Japan | 2009 | I | T | F | N | R | S | A | D | D | Y | ? | L
2010_FPV_ China | FJ231389 | FPV | China | 2010 | I | T | F | N | R | S | A | D | N | Y | ? | L
2014_FPV_ China | KP280068 | FPV | China | 2014 | V | T | F | N | R | S | A | D | D | Y | ? | L
2016_FPV_China | KX685354 | FPV | China | 2016 | V | T | F | N | R | S | A | D | D | Y | ? | L
2018_FPV_ Tamil Nadu | MH559110 | FPV | Tamil Nadu | 2018 | V | T | F | N | R | S | A | D | D | Y | ? | L
2019_ FPV_China | MT614366 | FPV | China | 2019 | V | T | F | N | R | S | A | D | D | Y | ? | L
C32D2MM | PX365592 | CPV 2c | Gujarat, India | 2025 | I | T | Y | N | R | A | G | Y | N | I | E | F
C33H11MM | PX365593 | CPV 2c new | Gujarat, India | 2025 | I | T | Y | N | R | A | G | Y | N | I | ? | L
C59H6YM | PX365594 | CPV 2c | Gujarat, India | 2025 | I | I | NS | NS | NS | NS | NS | NS | NS | NS | ? | NS
C62D1MF | PX365595 | CPV 2c | Gujarat, India | 2025 | I | T | Y | N | R | A | G | Y | N | I | ? | L
C67H14YM | PX365596 | CPV 2c new | Gujarat, India | 2025 | I | T | Y | P | T | A | G | Y | N | I | ? | L
C77D6MM | PX365597 | CPV 2c | Gujarat, India | 2025 | I | T | Y | N | R | A | G | Y | N | I | ? | L
C78D6MM | PX365598 | CPV 2c | Gujarat, India | 2025 | I | T | Y | N | R | A | G | Y | N | I | ? | L
1995_CPV_global_ | M19296.1 | CPV | Global | 1995 | I | T | F | N | R | S | A | D | N | Y | ? | L
1996_CPV 2_ New York | M38245.1 | CPV 2 | New York | 1996 | I | T | F | N | R | S | A | D | N | Y | ? | L
2019_CPV 2a _ China | MD439727.1 | CPV 2a 2b 2c | China | 2019 | I | T | F | N | R | A | G | Y | N | Y | ? | L
2016_CPV 2a _Nigeria | MH337275.1 | CPV 2 | Nigeria | 2016 | I | T | Y | N | R | A | G | Y | N | I | ? | L
2011_CPV 2a_ China | JQ268283.1 | CPV 2a | China | 2011 | I | T | F | N | R | A | G | Y | N | I | ? | L
2013_CPV 2a_ China | KF676668.1 | CPV 2a | China | 2013 | I | T | Y | N | R | A | G | Y | N | I | ? | L
2015_CPV 2a_ China | MG583676.1 | CPV 2a | China | 2015 | I | T | Y | N | R | A | G | Y | N | I | ? | L
2001_CPV 2b _ Japan | AB054221.1 | CPV 2b | Japan | 2001 | I | T | F | N | R | A | G | Y | N | Y | ? | L
2017_CPV 2b_Japan | LC270892.1 | CPV 2b | Japan | 2017 | I | T | F | N | R | A | V | Y | N | Y | ? | L
2017_CPV 2c _China | MG013488.1 | CPV 2c | China | 2017 | I | T | Y | N | R | A | G | Y | N | I | ? | L
2018_CPV 2c_China | MT010564.1 | CPV 2c | China | 2018 | I | T | Y | N | R | A | G | Y | N | I | ? | L
2018_CPV 2c_Taiwan | MN832850.1 | CPV 2c | Taiwan | 2018 | I | T | Y | N | R | A | G | Y | N | I | ? | L
2001_CPV 2c (b)_ Japan | AB054224.1 | CPV 2c(b) | Japan | 2001 | I | T | F | N | R | A | D | Y | N | Y | ? | L
2016_CPV 2a new _India | MN661243.1 | CPV 2a new | India | 2016 | I | T | Y | N | R | A | G | Y | N | I | ? | L

Statistical analysis

Descriptive statistics were used to summarize the prevalence of FPV and CPV infections among cats and dogs. No inferential statistical analysis was performed in the present study.

Sr. No. | Query | Accession No. | Coverage (%) | Identity (%) | Closest accession | Virus and strain | Country
1 | F02D4MM | PX365589 | 99 | 99.84 | OQ266795.1 | Feline panleukopenia virus | Tamil Nadu, India
2 | F03D4MF | PX365590 | 100 | 99.84 | OQ266795.1 | Feline panleukopenia virus | Tamil Nadu, India
3 | F04D4MF | PX365591 | 100 | 100 | OQ266795.1 | Feline panleukopenia virus | Tamil Nadu, India
4 | C32D2MM | PX365592 | 99 | 99.84 | OR463619.1 | Protoparvovirus carnivoran1 (CPV-2c) | Italy
5 | C33H11MM | PX365593 | 100 | 100 | MW239601.1 | Protoparvovirus carnivoran1 (CPV-2c-new) | Viet Nam: Hai Phong
6 | C59H6YM | PX365594 | 99 | 100 | MT488467.1 | CPV-2c | China
7 | C62D1MF | PX365595 | 100 | 99.84 | OR463619.1 | Protoparvovirus carnivoran1 (CPV-2c) | Italy
8 | C67H14YM | PX365596 | 100 | 99.39 | MW239601.1 | Protoparvovirus carnivoran1 (CPV-2c-new) | Viet Nam: Hai Phong
9 | C77D6MM | PX365597 | 100 | 100 | OR296263.1 | CPV-2c | India: Chennai, Tamil Nadu
10 | C78D6MM | PX365598 | 100 | 100 | OR463619.1 | Protoparvovirus carnivoran1 (CPV-2c) | Italy

Figure 7: Sequencing chromatogram of feline parvovirus isolate PX365589 showing TTT → F substitution.

Figure 8: Sequencing chromatogram of feline parvovirus isolate PX365590 showing TTA → L substitution.

Figure 9: Sequencing chromatogram of canine parvovirus isolate PX365596 showing CCT → P and ACA → T substitutions.

Figure 10: Sequencing chromatogram of canine parvovirus isolate PX365595 showing AAT → N and AGA → R substitutions.

Figure 11: Phylogenetic tree of the VP2 gene from feline and canine parvovirus isolates, with reference parvovirus sequences. The evolutionary history was inferred using the Maximum Likelihood method and the Tamura three-parameter model.