DISCUSSION

Sample collection and epidemiological characteristics

The present study provides a comprehensive epidemiological assessment of parvoviral infections in companion animals in Gujarat, India, based on samples collected from diverse sources, including veterinary hospitals, private clinics, rescue organizations, shelters, and apparently healthy animals. Such heterogeneous sampling strengthens the reliability of the findings and provides a broader understanding of viral circulation within the companion animal population. The inclusion of both clinically affected and apparently healthy animals enabled the detection of subclinical infections, which are recognized as important contributors to viral maintenance and transmission within susceptible populations [14, 15].

A higher proportion of animals in this study exhibited clinical signs consistent with gastroenteritis (58.5%), including diarrhea, vomiting, dehydration, and anorexia, which are classical clinical manifestations of FPV and CPV infections [1, 16,17]. However, the detection of infection in apparently healthy animals demonstrates the presence of asymptomatic or subclinical carriers capable of shedding virus into the environment. Such animals may play an important epidemiological role by maintaining viral circulation and facilitating indirect transmission, particularly in high-density environments such as shelters, breeding facilities, and multi-pet households [14,18]. These findings emphasize that clinically healthy animals should not be overlooked in surveillance programs, as they may serve as hidden reservoirs of infection.

Most positive animals were in the 3–6-month age group, consistent with previous reports that young animals are particularly susceptible because maternally derived antibodies gradually decline while the immune system remains immature [2, 16]. During this transitional period, maternal antibodies may decrease below protective concentrations yet remain sufficiently high to interfere with vaccine-induced immunity, thereby creating a period of increased susceptibility to infection [2]. Furthermore, the rapid proliferation of intestinal crypt epithelial cells in young animals provides an ideal environment for parvoviral replication, contributing to severe intestinal damage and clinical disease [1]. These findings reinforce the importance of appropriate vaccination schedules and timely booster immunization in young companion animals.

No apparent association was observed between sex and infection status, suggesting that susceptibility to FPV and CPV is independent of sex. Similar observations have been reported previously, where no consistent sex predisposition was identified among naturally infected companion animals [3, 19]. Breed-related observations indicated relatively higher representation of infections among Labrador Retrievers and German Shepherds. Previous investigators have suggested that this trend may reflect genetic susceptibility, breed-associated immune variability, or management-related factors rather than true breed-specific susceptibility [3]. In contrast, Persian and nondescript cats exhibited comparable infection rates, indicating relatively uniform susceptibility across feline breeds.

Vaccination status appeared to influence infection dynamics. Fully vaccinated cats showed lower FPV detection rates, supporting the protective effectiveness of current vaccination programs [2]. Nevertheless, CPV infection was detected in vaccinated dogs, suggesting possible vaccine failure, incomplete vaccination schedules, interference from maternally derived antibodies, improper vaccine handling, or the circulation of antigenically divergent viral variants that partially escape vaccine-induced immunity [4, 11]. Similar breakthrough infections associated with CPV-2c have been documented in recent studies, emphasizing the need for continuous monitoring of circulating field strains and periodic evaluation of vaccine efficacy [20].

Seasonal variation, characterized by increased positivity during summer and winter, may reflect the exceptional environmental stability of parvoviruses. These viruses can remain infectious for prolonged periods under favorable environmental conditions, thereby facilitating indirect transmission via contaminated fomites and environmental surfaces [14]. Seasonal fluctuations in temperature, humidity, animal movement, and management practices may further influence viral persistence and transmission dynamics [18]. The broad geographical distribution of samples collected from veterinary hospitals, private clinics, and shelters across the study area further demonstrates the endemic nature of parvoviral infections within Gujarat and highlights the widespread circulation of these viruses under different management systems.

Overall, the epidemiological findings of the present study are consistent with previous reports demonstrating that young, unvaccinated, and densely housed companion animals are at greater risk of parvoviral infection [2, 3, 14]. The identification of infection in both clinically affected and apparently healthy animals further emphasizes the importance of routine molecular surveillance, improved vaccination coverage, enhanced biosecurity practices, and early diagnosis to reduce viral transmission and improve disease control in companion animal populations.

RAT and conventional PCR

RATs are widely used as point-of-care diagnostic tools for parvoviral infections because they are rapid, inexpensive, and suitable for field use. In the present study, however, RAT detected parvoviral antigen in only 5% of samples, which was considerably lower than the detection rates obtained by molecular methods. This finding demonstrates the limited sensitivity of antigen-based assays, particularly when viral loads are low or samples are collected during the early or late stages of infection, when antigen concentrations may fall below the assay detection threshold [10, 15]. Similar observations have been reported previously, in which a substantial proportion of RAT-negative samples were subsequently confirmed positive by PCR or qPCR [11, 18].

The comparatively higher detection rate observed in cats than in dogs using RAT may be related to differences in viral shedding patterns or the stage of infection at sampling. An additional observation was the apparent dual positivity of two canine samples with both FPV and CPV antigen kits, suggesting antigenic cross-reactivity. Because FPV and CPV share more than 98% nucleotide identity within the VP2 gene, the viruses possess highly similar antigenic epitopes that may be recognized by monoclonal antibodies incorporated into lateral-flow immunoassays [6, 7]. Such cross-reactivity has been documented previously and may complicate interpretation of antigen-based assays when closely related parvoviruses are present [15, 21].

The discrepancy between RAT and molecular assays observed in the present study may also be explained by the relatively high antigen concentration required for visual detection in lateral-flow assays. Samples containing low viral titers, immune-complexed viral antigen, or partially degraded viral proteins may yield false-negative results [10]. Conversely, nonspecific binding or faint reaction lines may occasionally produce false-positive interpretations, thereby reducing diagnostic reliability [18]. These findings indicate that RAT should primarily be regarded as a rapid screening tool rather than a definitive diagnostic assay.

Conventional PCR targeting the VP2 gene demonstrated a substantially higher detection rate (19.5%) than RAT, confirming its superior analytical sensitivity for detecting parvoviral DNA. Amplification of the expected 681 bp fragment from both feline and canine samples confirmed active circulation of parvoviruses within the study region. These findings are consistent with previous studies demonstrating that PCR reliably detects parvoviral DNA directly from fecal samples, including specimens containing antigen concentrations below the detection limit of rapid tests [5, 10,22].

Nevertheless, the PCR detection rate observed in this study was lower than those reported in several previous investigations from India, where positivity ranged from 28% to 77% among clinically suspected animals [11]. This variation is likely attributable to differences in study design, particularly the inclusion of both clinically healthy and symptomatic animals in the present investigation, as well as differences in sample type, disease stage, viral load, and DNA extraction methodology. Such methodological differences may substantially influence amplification efficiency and diagnostic sensitivity.

A major limitation of conventional PCR is its inability to distinguish FPV from CPV because the primers target highly conserved regions of the VP2 gene. Similar limitations have been reported previously, where PCR successfully confirmed parvoviral infection but required additional molecular assays for virus typing and differentiation [15, 20]. Detection of parvoviral DNA in both feline and canine samples also supports the co-circulation of FPV and CPV within the study region and raises the possibility of cross-species transmission, which has previously been documented under both natural and experimental conditions [6, 8].

Compared with qPCR, conventional PCR exhibits lower analytical sensitivity. Previous investigations have shown that qPCR can detect as few as 10–100 copies of viral DNA, making it approximately 10–100 times more sensitive than conventional PCR [20]. Consequently, the lower PCR detection rate observed in the present study may reflect reduced analytical sensitivity, particularly in samples containing low viral loads or fecal-derived PCR inhibitors [15]. Despite these limitations, conventional PCR remains a valuable diagnostic tool because of its relatively low cost, accessibility, and ability to confirm parvoviral DNA. However, precise differentiation of FPV and CPV and detailed molecular epidemiological investigations require more sensitive and discriminatory techniques such as probe-based qPCR. Overall, the present findings indicate that although RAT provides convenient field screening and conventional PCR improves diagnostic sensitivity, probe-based qPCR remains essential for accurate diagnosis, molecular surveillance, and comprehensive epidemiological investigations of companion animal parvoviruses.

Molecular detection and differentiation of FPV and CPV using qPCR

In the present study, probe-based multiplex qPCR targeting virus-specific regions of the VP2 gene demonstrated a substantially higher detection rate (62%) than conventional PCR (19.5%) and RAT (5%), confirming its superior analytical sensitivity and specificity. Detection of parvoviral infection in 124 of the 200 samples demonstrates active circulation of these viruses among companion animals in Gujarat. CPV (48.5%) was detected considerably more frequently than FPV (13.5%), indicating the predominance of CPV within the study population. Similar observations have been reported previously, where CPV variants exhibited widespread distribution, efficient transmission, and remarkable environmental persistence, facilitating continued circulation among both canine and feline hosts [15, 18, 23]. Recent reports describing the continuing global evolution and dissemination of CPV variants further support these findings [24, 25, 26].

The comparatively lower detection of FPV in cats may reflect improved vaccination coverage and increasing herd immunity within feline populations [2, 17]. Moreover, FPV infections are typically acute and associated with relatively short periods of viral shedding, reducing the probability of detection during cross-sectional investigations [14]. In contrast, CPV exhibits prolonged environmental survival and greater host adaptability, characteristics that likely contribute to its higher prevalence within the present study [1, 7].

An important finding of this investigation was the detection of CPV in feline samples and occasional detection of FPV in canine samples, suggesting possible cross-species transmission. This observation is consistent with previous reports showing that CPV variants readily infect cats, whereas FPV replicates only to a limited extent in dogs [5, 8, 27]. Such host adaptation is largely governed by amino acid substitutions within the VP2 capsid protein that influence receptor binding and host tropism [6, 7]. Detection of CPV in cats further supports previous reports describing asymptomatic or subclinical CPV infection in feline populations [21].

The superior sensitivity of qPCR compared with conventional PCR observed in this study is attributable to real-time amplification monitoring and fluorescent hydrolysis probes, which permit detection during the exponential phase of amplification. Previous studies have demonstrated that qPCR detects very low viral copy numbers and is substantially more sensitive than conventional PCR [20, 24, 28, 29]. Consequently, additional positive samples with low viral titers were identified by qPCR that would have remained undetected by conventional PCR.

The discrepancies observed between PCR and qPCR in a limited number of samples may be attributed to DNA degradation, amplification inhibitors commonly present in fecal specimens, and variation in primer or probe binding efficiency. Fecal samples frequently contain substances that can inhibit DNA amplification, thereby reducing diagnostic sensitivity and contributing to false-negative PCR results [30]. In addition, differences in sample handling, storage conditions, and nucleic acid extraction procedures may influence assay performance. The incorporation of an internal amplification control into the qPCR assay further enhanced assay reliability by minimizing false-negative results caused by amplification failure or inhibition, consistent with current recommendations for molecular diagnostics [10, 15].

Overall, the present findings demonstrate that probe-based qPCR is a highly sensitive, specific, and reliable method for detecting and differentiating FPV and CPV infections. The predominance of CPV, together with evidence of cross-species transmission, illustrates the dynamic epidemiology of companion animal parvoviruses in Gujarat. These findings emphasize the importance of continuous molecular surveillance, implementation of highly sensitive molecular diagnostic techniques, and regular monitoring of circulating viral variants to support effective disease surveillance, vaccination strategies, and long-term control of parvoviral infections.

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Virus isolation

Virus isolation remains an important confirmatory method for demonstrating the presence of infectious viral particles and investigating viral replication characteristics. In the present study, successful virus isolation was achieved in four of nine molecularly confirmed parvovirus-positive samples (44.44%) using CRFK cells, as evidenced by characteristic CPE and subsequent molecular confirmation. The observed CPE, including cell rounding, aggregation, granulation, and detachment, are consistent with the well recognized cytopathogenic properties of parvoviruses in susceptible cell cultures [1, 14].

The appearance of CPE between the third and fourth passages indicates that field isolates required an initial adaptation period before efficient replication under in vitro conditions. Similar delayed development of CPE has been reported previously, where parvoviral field isolates produced only mild to moderate cytopathic changes following serial passages in susceptible cell lines [31, 32]. This delayed adaptation may result from low viral titers in clinical specimens, partial degradation of viral particles, or the requirement for adaptation to artificial cell culture conditions.

The relatively low virus isolation rate observed in this study may be explained by several factors, including sample quality, viral load, stage of infection, and the presence of inhibitory substances or neutralizing antibodies within fecal material. Successful virus isolation depends on both the integrity of infectious viral particles and the susceptibility of the host cell line used [33]. Moreover, inclusion of both clinically healthy and clinically affected animals likely introduced variation in viral loads among specimens, thereby influencing isolation efficiency.

Confirmation of parvoviral DNA in CPE-positive cultures by PCR during the early passages verified active viral replication in CRFK cells. However, the absence of detectable amplification in later passages, despite initial PCR positivity, suggests reduced viral replication or progressive loss of detectable viral nucleic acid during serial passage. This phenomenon may reflect reduced viral fitness following repeated passaging, degradation of viral nucleic acids due to repeated freeze-thaw cycles, or suboptimal culture conditions that affect viral propagation [30]. Additionally, accumulation of defective or noninfectious viral particles during serial passage may further contribute to declining viral loads and subsequent PCR negativity.

Another possible explanation for the reduction in detectable viral DNA during later passages is activation of cellular antiviral defense mechanisms that suppress viral replication under in vitro conditions. Continuous cell culture may also exert selective pressure on field strains, resulting in viral attenuation or progressive loss of infectivity over time [7]. These findings emphasize the importance of confirming viral replication during the early passages when virus recovery is most successful.

The successful propagation of parvoviruses in CRFK cells observed in the present study supports previous reports that both FPV and CPV replicate efficiently in feline-derived cell lines. The broad host range of carnivore parvoviruses is largely determined by amino acid substitutions within the VP2 capsid protein that influence receptor binding and host tropism [6, 7]. Successful isolation of viruses from both feline and canine specimens using CRFK cells therefore further supports the concept of shared cellular tropism and receptor utilization between FPV and CPV strains.

Although alternative cell lines, including A-72 and Madin-Darby canine kidney (MDCK) cells, have been reported to exhibit greater sensitivity for parvovirus isolation, CRFK cells remain a reliable and widely accepted system for propagation of both FPV and CPV [33]. The findings of the present study reinforce the usefulness of CRFK cells while highlighting the challenges of recovering field isolates, particularly those with low viral loads or reduced viral viability.

Overall, the present study demonstrates that although virus isolation provides valuable information regarding viral infectivity and biological behavior, it is considerably less sensitive than molecular diagnostic methods such as PCR and qPCR. Combining cell culture with molecular confirmation remains essential for comprehensive characterization of circulating parvoviruses. The relatively low isolation efficiency observed in this investigation further emphasizes the importance of optimized sample handling, early passage monitoring, and integration of sensitive molecular assays to maximize virus recovery and diagnostic accuracy. These findings confirm that virus isolation serves primarily as a confirmatory method and is highly dependent on sample quality and viral viability compared with molecular diagnostic approaches.

Molecular characterization and phylogenetic analysis of FPV and CPV

Molecular characterization based on the VP2 gene provides valuable insights into parvovirus evolution, host adaptation, and antigenic diversity. In the present study, sequencing of the partial VP2 gene (approximately 681 bp) from 10 representative isolates confirmed the co-circulation of FPV and CPV among companion animals in Gujarat, India. Successful amplification and sequencing of all selected isolates further demonstrate that the VP2 gene remains a robust molecular marker for epidemiological investigations and evolutionary studies of carnivore parvoviruses [12, 20].

The FPV isolates identified in this study exhibited a high degree of nucleotide conservation with only minor amino acid substitutions compared with reference strains. Conserved amino acid residues at positions 131, 135, 150, 151, and 165 indicate considerable genetic stability among circulating FPV strains. These findings agree with previous reports indicating that FPV evolves more slowly than CPV and maintains remarkable genomic conservation across geographically distinct populations [1, 14,34,26]. Minor substitutions observed at positions 203 and 206 likely represent localized genetic drift and are unlikely to substantially influence viral antigenicity or host range.

In contrast, CPV isolates exhibited greater genetic variability, with multiple amino acid substitutions at key antigenic sites in the VP2 protein. Characteristic substitutions at positions 135 (F→Y), 165 (S→A), and 168 (A→G) are consistent with CPV-2c variant lineages. These mutations have previously been associated with alterations in viral antigenicity, receptor binding, host adaptation, and viral fitness, contributing to the successful global dissemination of CPV variants [4, 20, 35]. Most isolates displayed amino acid profiles comparable to those reported previously from India and neighboring Asian countries, indicating continued regional circulation of established CPV lineages.

One isolate possessed unique substitutions at positions 150 and 151, suggesting possible regional adaptation or emergence of a novel viral variant. Amino acid substitutions within the VP2 protein are of particular biological importance because even a single substitution may influence viral fitness, antigenicity, host range, and vaccine efficacy [7]. Consequently, these observations support the occurrence of ongoing microevolution of CPV under field conditions and further emphasize the importance of continuous molecular surveillance.

BLASTn analysis demonstrated high nucleotide identity (99%–100%) between the FPV isolates and previously reported strains from India and other Asian countries. Similarly, CPV isolates exhibited high sequence similarity with strains reported from Europe and Southeast Asia [26, 36]. These findings suggest that circulating field strains share a common evolutionary ancestry and that international movement of companion animals, together with the exceptional environmental stability of parvoviruses, may facilitate global dissemination of CPV variants [18].

Phylogenetic analysis based on partial VP2 gene sequences clearly separated the study isolates into two principal clusters corresponding to FPV and CPV, confirming their distinct evolutionary relationships. Within the CPV cluster, study isolates grouped closely with CPV-2a and CPV-2c variants while remaining distinct from prototype and vaccine strains. This clustering pattern indicates that circulating CPV strains in Gujarat are genetically related to contemporary Asian variants while continuing to undergo gradual evolutionary divergence. Similar phylogenetic relationships have recently been reported from several countries, where CPV variants continue to evolve while maintaining conserved genetic characteristics [20, 29].

The observed phylogenetic separation of field isolates from vaccine and prototype strains suggests minor antigenic divergence that may affect vaccine effectiveness under field conditions, as reported previously [26, 29]. Recent investigations have further demonstrated the increasing predominance of CPV-2c over earlier variants such as CPV-2a in several countries, including India [29]. In addition, accumulating evidence suggests that antigenic variation among circulating CPV strains may contribute to vaccine breakthrough infections, particularly in puppies with incomplete or waning immunity [37].

The inability to evaluate several important antigenic residues, including positions 297 and 426, due to the availability of only partial VP2 sequences represents a limitation of the present study. Nevertheless, the overall amino acid substitution pattern together with phylogenetic clustering strongly supports the circulation of CPV-2c-like variants in Gujarat. The observed genetic distance between field isolates and vaccine strains further indicates ongoing antigenic divergence, warranting continued molecular monitoring and evaluation of vaccine performance.

Detection of both FPV and CPV in the present investigation, together with their distinct phylogenetic clustering, reinforces the concept of co-circulation and possible cross-species transmission among companion animals [28]. CPV variants are well recognized for their ability to infect feline hosts, whereas FPV demonstrates only limited adaptation to dogs, highlighting the critical role of VP2 mutations in determining host specificity [6, 8]. Detection of CPV in feline samples within the present study further supports the expanding host range of contemporary CPV variants.

Overall, the molecular characterization performed in this study demonstrates that FPV remains genetically stable, whereas CPV continues to undergo adaptive evolution through the emergence of variant strains. The coexistence of conserved and variable regions within the VP2 gene illustrates the balance between structural conservation and evolutionary flexibility. These findings emphasize the importance of continuous genomic surveillance to identify emerging mutations, monitor viral evolution, and evaluate their potential influence on diagnostics and vaccine efficacy.

The present study has several limitations. Partial sequencing of the VP2 gene restricted comprehensive subtype characterization of all isolates. Furthermore, the absence of inferential statistical analyses prevented detailed evaluation of epidemiological risk factors associated with infection. Future investigations incorporating whole-genome sequencing, larger sample sizes, and comprehensive epidemiological datasets will provide a more complete understanding of viral evolution, transmission dynamics, and host adaptation.

The present investigation provides important baseline molecular and epidemiological information regarding the circulation of FPV and CPV in Gujarat, India. The findings demonstrate the co-circulation of genetically distinct yet closely related parvoviruses, evidence of cross-species transmission, and the continued evolution of CPV variants. Collectively, these observations highlight the need for sustained molecular surveillance, optimization of vaccination strategies, and implementation of highly sensitive diagnostic methods such as qPCR to strengthen disease surveillance and improve long-term control of parvoviral infections, particularly in regions with large populations of stray and unvaccinated companion animals.