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Full Research Article

Molecular Identification and Epidemiological Factor Analysis of Canine Parvovirus-2 Infection in Diarrheic Dogs of Indore, Madhya Pradesh

V
Vinay Waskel1
R
Rakhi Gangil2,*
D
Daljeet Chhabra2
R
Rakesh Sharda2
R
Ravi Sikrodia2
J
Joycee Jogi2
K
K.K. Jadhav3
1Department of Surgeon, Veterinary and Animal Husbandry, Khargone-451 001, Madhya Pradesh, India.
2Department of Veterinary Microbiology, College of Veterinary Science and Animal Husbandry, Mhow-453 446, Madhya Pradesh, India.
3School of Wild life Forensic and Health, Nanaji Deshmukh Veterinary Science University, Jabalpur-482 001, Madhya Pradesh, India.

ABSTRACT

Background: Canine parvovirus type 2 (CPV-2) is a highly contagious and economically important viral disease in dogs. The present study was designed to detect CPV infection in diarrhoeic dogs, estimate its incidence and identify the circulating antigenic variant in the study area.

Methods: A total of 100 fecal swab samples were collected from dogs showing clinical signs suggestive of CPV-2 infection. The samples were tested by single-step PCR and nested PCR targeting the VP2 gene. Positive PCR products were sequenced commercially and phylogenetic analysis was performed using DNASTAR and MEGA software. Statistical analysis was used to assess the association between CPV infection and different risk factors.

Result: Nested PCR detected a CPV-2 incidence of 64% and was more sensitive than conventional PCR. Infection was more common in puppies below 3 months of age. Medium-sized breeds were most frequently affected and males showed higher positivity than females. The highest prevalence was observed during the rainy season, followed by winter and summer. CPV infection was also found in vaccinated dogs. Phylogenetic analysis revealed that the circulating virus belonged to the CPV-2b antigenic variant.

INTRODUCTION

Canine parvovirus (CPV) emerged in 1978 and has since become a major pathogen of domestic and wild canids, particularly affecting puppies below 6 months of age. CPV-2 is a non-enveloped virus with an icosahedral structure and is classified under the genus Protoparvovirus, subfamily Parvovirinae and family Parvoviridae. It has a single-stranded, negative-sense DNA genome of approximately 5.3 kb that encodes two non-structural proteins, NS1 and NS2 and three capsid proteins, VP1, VP2 and VP3. Among these, VP2 is the major structural protein involved in cell entry and a key site for genetic mutation (Cavalli et al., 2008). CPV primarily infects intestinal epithelial cells, where it replicates and is shed through faeces (Hoelzer et al., 2008). The infection is highly contagious and may present as hemorrhagic enteritis with bloody diarrhoea and vomiting, or as cardiac syndrome/myocarditis, especially in pups below 3 months of age (Hayes et al., 1979).
       
Numerous studies have reported CPV infections in the Indian subcontinent (Mandawat et al., 2025; Mohan et al., 2025). Recent molecular epidemiological studies show the continued evolution and global spread of CPV, with CPV-2a, CPV-2b and CPV-2c co-circulating in many regions, including Asia and India. CPV-2b and CPV-2c have been frequently associated with diarrhoeic dogs and vaccine breakthrough infections due to VP2 antigenic drift (Mittal et al., 2022; Nandi et al., 2023; Reddy et al., 2024; Chethan et al., 2025).
       
Because CPV is highly contagious and often fatal, early diagnosis is essential. Molecular methods such as nested PCR and sequencing are now vital for rapid detection, accurate typing and surveillance of circulating CPV strains (Zhang et al., 2023; Singh et al., 2024). Nested PCR (NPCR) is highly sensitive and can even detect 106 particles of CPV/g of faeces (Kumar et al., 2011), thus giving an early diagnosis of even sub-clinical infections. PCR and NPCR are used to detect the VP2 gene in CPV, which encodes the capsid protein that is important to determine its antigenicity (Phromnoi et al., 2010). Many risk factors, such as age, season, breed size and vaccination are also associated with the CPV-2 infection. CPV immunization is viewed as a core factor to prevent the infection in newborn pups. As an integral part of the canine core vaccine, vaccination failure is also not uncommon (Muzaffar et al., 2006). Till date no study has been conducted to determine the incidence of CPV infection with nested PCR and circulating variant in the Indore region. Hence, the present study was undertaken to confirm the presence of CPV-2 in dogs with acute gastroenteritis and to characterize the antigenic variants circulating in the Indore region. Further, study of associated risk factors like season, age, sex, breed of dogs and vaccination status was taken into account.

MATERIALS AND METHODS

Collection of samples
 
The study was conducted in the Department of Veterinary Microbiology, College of Veterinary Science and AH MHOW. Samples were collected from Veterinary Clinical Complex (VCC), College of Veterinary Science and Animal Husbandry, Mhow (M.P.) and from private pet clinics of Indore during 2022-2023. One hundred (100) rectal swab samples were aseptically collected from suspected dogs with a history of diarrhoea/ haemorrhagic diarrhoea, vomition, reduced appetite, dehydration with or without pyrexia and transported to the laboratory in 2 mL of phosphate-buffered saline (PBS; pH 7.2) under chilled conditions. Samples were collected with the questionnaire, viz., date of collection, age, sex, breed, vaccination status and season of infection, etc., to investigate various risk factors correlated to the occurrence of CPV in dogs in the study area. The experimental protocol (No. 04/Ethical/2021, Dated 05/02/2021) was approved by the Institutional Animal Ethics Committee (IAEC) College of Veterinary Science and AH MHOW, NDVSU Jabalpur.
 
Preparation of virus inoculum/sample processing
 
All the swabs with PBS were squeezed against the collection tube wall and transferred to a 2 mL microcentrifuge tube and centrifuged at 3000 rpm for 10 min. Supernatant was kept at -20°C till further use.
 
Detection of canine parvovirus (CPV-2) by PCR and NPCR
 
Total genomic DNA was extracted from all the supernatants of faecal samples using phenol-chloroform extraction method from all clinical swab samples and vaccine (Megavac-7 DHPPi) using standard protocol prescribed by Sambrook and Russell (2001) and stored at -20°C for further use. The screening of CPV-2 in faecal samples was carried out by the detection of VP2 gene using polymerase chain reaction (PCR) and Nested PCR.
 
Polymerase chain reaction (PCR) for the detection of canine parvovirus-2
 
For first round of PCR, forward primer CPV-2 and reverse primer CPV-4 were used for amplification of 1198 bp product of the VP2 gene (Mizak and Rzezutka, 1999). PCR amplification with a final reaction volume of 25 µL which included 2.5 µL of 10× PCR buffer with 15 mM MgCl‚ 1µL of 10 mM dNTPs, 1 µL each of forward (CPV-2) and reverse (CPV-4) primers at 20 pmol/µL, 0.5 µL of 10 mM MgCl and 0.2 µL of Taq DNA polymerase (5U/µL) and 10 µL of DNA template (400 ng). Volume was completed to 25 µL employing nuclease-free water. Amplification protocol consisted of 35 cycles with denaturation at 94°C for 1 min, annealing at 56°C for 1 min and extension at 72°C for 150 sec, followed by a final extension at 72°C for 10 min. Appropriate controls were included in each run: Vaccine-derived DNA served as the positive control, while rectal swab samples obtained from clinically healthy dogs were used as the negative control.
 
Nested PCR (NPCR) for the detection of canine parvovirus-2
 
For second round of PCR or Nested PCR (NPCR), forward primer CPV-6 and reverse primer CPV-4 were used (Mizak and Rzezutka, 1999) for amplification of 548 bp size product. Reaction mixture of 25 µl by adding 2.5 µl of 10X PCR Buffer (with 15 mM MgCl2), 1 µl of dNTPs (10 mM), 1 µl of CPV-6 (Forward) and CPV-4 (Reverse) primers (20 pmol/µl) each, 0.5 µl of MgCl2 (10 mM), 0.2 µl of Taq DNA Polymerase (5 unit/µl) and 5 µl of PCR product of first round of PCR as a DNA template and making the volume of 25 µl by adding Nuclease Free Water (NFW). Thermocyclic conditions was same as first round of PCR. PCR products were resolved on a 1.5% agarose gel and visualized using a gel documentation system.  
 
Sequencing of VP2 specific gene of CPV
 
Following the manufacturer’s instructions, nested PCR products (partial VP2 gene) have been purified employing a gel extraction kit (GenEluteTM PCR Clean-UP Kit, Sigma). The DNA’s purity was verified and it was subsequently submitted to Eurofins Genomic India Pvt Ltd for sequencing using Sanger’s method (BigDye V3.1 terminator technology). Software such as DNASTAR and MEGA7.0 have been employed for processing and analyzing the collected data. DNA sequences were aligned utilizing the ClustalW technique in the MegAlign program by DNASTAR, USA and MEGA7 with reference sequences that had already been published and obtained from GenBank NCBI (Kumar et al., 2016). Phylogenetic relationships were analyzed utilizing Maximum Likelihood method implemented in MEGA7 and evolutionary history was inferred employing Tamura-Nei nucleotide substitution model (Tamura and Nei, 1993). A bootstrap consensus tree was constructed from 1000 replicates for assessing robustness of the branches (Felsenstein, 1985). The VP2 gene sequences of Canine parvovirus type 2 were submitted to GenBank to obtain accession numbers.
 
Statistical analysis
 
The data were analyzed and summarized using descriptive statistical methods.
       
Results were expressed as percentage positivity of the detected infections.

RESULTS AND DISCUSSION

Molecular detection of CPV infection by PCR and NPCR
 
In the present study, the preliminary VP2 gene-specific PCR produced the expected amplified products of 1198 bp in both the positive control and the suspected clinical samples (Fig 1).  No bands were observed in the non-template control. Among the 100 samples, 50 tested positive using PCR, indicating a 50% positivity of CPV in this PCR. However, of these 100 samples, 64 tested positive using NPCR, amplifying a product size of 548 bp (Fig 2). This suggests a 64% incidence of CPV when using NPCR. This study demonstrates that NPCR exhibits higher sensitivity compared to PCR in detecting CPV. Hirasawa et al. (1994) and Schmitz (2009) also highlighted the increased sensitivity of nested PCR compared to conventional PCR in their studies. This heightened sensitivity may be attributed to the potential presence of inhibitory substances in samples with very few virus particles, preventing the visualization of amplified products in conventional PCR. Nested PCR (NPCR) can overcome this limitation, enabling the visualization of NPCR products in an agarose gel (Kumar et al., 2011). Similarly, Singh et al., 2021 recorded a positivity of 28% and 70% for PCR and NPCR, respectively, underscoring the superior sensitivity of NPCR over PCR. Wang et al., (2022) and Kumar et al., (2022) suggested that nested PCR and real-time PCR assays show significantly higher sensitivity than conventional PCR for detection of low viral loads in clinical and subclinical CPV infections.

Fig 1: Agarose gel electrophoresis showing amplified product (1198 bp) of VP2 gene in PCR.



Fig 2: Agarose gel electrophoresis showing amplified product (548 bp) of VP2 gene in NPCR.


 
Analysis of epidemiological factors in CPV-2 infected dogs
 
The distribution of positive cases was recorded higher in below 3 month of age group of dogs (31/39, 79.5%), compared to between 3-6 months of age (28/48, 58.3%) and more than 6 months of age (5/13, 38.5%). The increased incidence in puppies under six months may be explained by the marked tropism of Canine parvovirus type 2 for rapidly dividing cells (myocardiocytes, small intestinal crypt epithelial cells, lymphopoietic tissue and bone marrow), young animals will have a greater number of such cells than adults (Chethan et al., 2021). CPV-2 commonly affects young puppies between 6 and 20 weeks of age, but unvaccinated dogs of any age group may potentially be affected (Eregowda et al., 2020). Maternally derived antibodies protect newborn puppies from CPV-2 infection; however susceptibility increases when maternally acquired antibodies begin to wane (Mittal et al., 2022).
       
The CPV infection was found higher in male dogs (50/73, 68.4%) than in female dogs (14/27, 51.8%), which could be attributable to the fact that the number of male animals presented to the hospital was higher than female animals. There is no breed or sex susceptibility to CPV-2 infection (Chethan et al., 2021). However, Geetha and Selvaraju (2019) and Khare et al., (2019) similarly reported a higher susceptibility of male dogs to CPV- 2 infection compared with females. The majority positive cases was found to be higher in medium-sized breed (56/79, 70.9%), followed by large-sized breed (7/18, 38.9%) and small-sized breed (1/3, 33.33%). (Large breed size dogs included Dalmatian, German Shepherd and Rottweiler. Medium-sized dogs included Boxer, Siberian husky, Labrador and non-descript breeds of dogs. Small breed size dog included Pomeranian and Pug). Breed-wise analysis showed that non-descript dogs accounted for the highest number of positive cases (32/49), followed by Labrador dogs (21/27), while German Shepherds contributed 5 positives out of 13 examined. It may be because the majority of dogs presented to the hospital in the current study were non-descript dogs, which could explain the relatively high percentage of positive cases observed among non-descript breeds. Higher prevalence of CPV infection in non-descript as well as medium-sized breeds has been reported, likely due to poor vaccination coverage and higher exposure risk (Kumar et al., 2022; Singh et al., 2024).
       
Higher number of cases 53/77 (68.8%) were recorded from non-vaccinated dogs compared to vaccinated dogs 11/23 (47.8%). It was evident that the vaccine prevents infection by boosting immunity against canine Parvovirus infection. However, vaccinated dogs were also found to be positive in the present study, but the rate of incidence has been higher in unvaccinated dogs. Rafiq et al., (2019) also reported 38.7% prevalence in unvaccinated dogs and 21% in vaccinated dogs. Vaccinated dogs affected due to the unidentified CPV strain is circulating in that particular area. A significant cause of vaccine failure in puppies is interference from maternally derived antibodies (MDA), which can have a substantial impact. Mother antibodies, improper vaccine handling, as well as antigenic mismatch between vaccine strains and circulating field variants can cause vaccine failure (Mittal et al., 2022). The higher cases observed in rainy season (19/21, 90.5%) as compared to winter (34/55, 61.8%) and summer season (11/24, 45.8%). Seasonal variation has been observed in canine parvovirus infection, with higher occurrence during rainy or humid periods, probably due to prolonged environmental survival of the virus and increased contamination of soil and surroundings. Geetha and Selvaraju (2023) reported an increased incidence of CPV enteritis in the humidity and rainy seasons.
 
Phylogenetic analysis of CPV-2 variants
 
CPV-2 is constantly changing its genetic and antigenic properties because of frequent mutations in the VP2 gene. These changes have led to the emergence and worldwide spread of different antigenic variants, namely CPV-2a, CPV-2b and CPV-2c (Thomas et al., 2017). In India, CPV-2a has been reported as the most common variant, followed by CPV-2b (Thomas et al., 2017). Among the 64 NPCR positive samples, 2 samples were randomly selected for sequencing and phylogenetic analysis. Initial analysis of the VP2 gene sequences using the Blast tool at NCBI(http://blast.ncbi.nlm.nih.gov/Blast.cgi) revealed a high similarity of 99% with available sequences in GenBank. Further, when performing a multiple sequence alignment of VP2 gene sequences employing MegAlign program by DNASTAR, it was found that there was a 99.6% identity with isolates from various countries (Fig 3). The isolates from this study clustered with many Indian isolates. These isolates were closely associated with the CPV 2b strain of other Indian isolates and were distantly related to Canine Adenovirus (Out group). Therefore, it was determined that these strains belonged to the CPV-2b antigen type. Both CPV strains from Madhya Pradesh (CPV1 MP and CPV2 MP) had been submitted to NCBI GenBank and accession numbers were obtained (Accession no.- OM585418, OM585419) (Fig 4).

Fig 3: Per cent identity of VP2 gene (partial sequence) of CPV-2 MP isolates compared with published reference sequences.



Fig 4: Phylogenetic tree based on two partial VP2 gene sequences (CPV MP isolates) from the present study and published sequences including canine adenovirus (Out group).


       
Phylogenetic analysis indicated that the samples were categorized as the CPV-2b antigen type. In a previous study, Recent molecular studies from India and neighbouring countries have consistently reported CPV-2b as a predominant circulating variant, although sporadic emergence of CPV-2c has also been documented, indicating ongoing viral evolution and the need for continuous molecular surveillance (Nandi et al., 2023; Zhang et al., 2023). Nandi et al. (2010) identified CPV-2b subtypes in dogs, aligning with our findings. However, their study also reported CPV 2C subtypes. They observed nucleotide substitutions, suggesting that CPV-2c mutants might have evolved from CPV-2b mutants circulating in India. In a different study, Buonavoglia et al. (2001) reported the CPV 2b antigenic subtype in dogs with severe haemorrhagic diarrhoea. The virus’s virulence and immunogenicity are examined by the presence of particular amino acids in the VP2 gene. For determining the genotype and pathotype present, it is therefore crucial to monitor the CPV that is circulating in the field at various intervals.

CONCLUSION

This study shows that CPV 2 is highly prevalent in diarrheic dogs in Indore, Madhya Pradesh, with NPCR (64%) being more sensitive than conventional PCR (50%). The predominant circulating strain is CPV 2b and infection is strongly associated with puppies under 3 months, male dogs, medium sized breeds (especially non descript and Labrador) and the rainy season, with higher positivity in unvaccinated animals. The findings highlight the need for improved vaccination, seasonal based control and continuous molecular surveillance of CPV variants.

ACKNOWLEDGEMENT

Authors are thankful to Dean, College of Veterinary Science and A.H., Mhow for providing the necessary facilities to conduct this research in the Department of Veterinary Microbiology, College of Veterinary Science and AH MHOW, NDVSU Jabalpur.
 
Author’s contribution
 
V. Waskel: Sample collection, execution of laboratory work. R Gangil: Planning of work, Standardization of the PCR and N-PCR, drafting of manuscript. D. Chhabra: Review and editing of manuscript. R. Sharda: Analysis of results and editing of manuscript. R. Sikrodia: Collection of data of risk factors and sample collection. J. Jogi: Statical analysis of results. K. Jadhav: Bioinformatic analysis of sequences.

CONFLICT OF INTEREST

The authors declare that is there is no conflict of interest.

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    Full Research Article

    Molecular Identification and Epidemiological Factor Analysis of Canine Parvovirus-2 Infection in Diarrheic Dogs of Indore, Madhya Pradesh

    V
    Vinay Waskel1
    R
    Rakhi Gangil2,*
    D
    Daljeet Chhabra2
    R
    Rakesh Sharda2
    R
    Ravi Sikrodia2
    J
    Joycee Jogi2
    K
    K.K. Jadhav3
    1Department of Surgeon, Veterinary and Animal Husbandry, Khargone-451 001, Madhya Pradesh, India.
    2Department of Veterinary Microbiology, College of Veterinary Science and Animal Husbandry, Mhow-453 446, Madhya Pradesh, India.
    3School of Wild life Forensic and Health, Nanaji Deshmukh Veterinary Science University, Jabalpur-482 001, Madhya Pradesh, India.

    ABSTRACT

    Background: Canine parvovirus type 2 (CPV-2) is a highly contagious and economically important viral disease in dogs. The present study was designed to detect CPV infection in diarrhoeic dogs, estimate its incidence and identify the circulating antigenic variant in the study area.

    Methods: A total of 100 fecal swab samples were collected from dogs showing clinical signs suggestive of CPV-2 infection. The samples were tested by single-step PCR and nested PCR targeting the VP2 gene. Positive PCR products were sequenced commercially and phylogenetic analysis was performed using DNASTAR and MEGA software. Statistical analysis was used to assess the association between CPV infection and different risk factors.

    Result: Nested PCR detected a CPV-2 incidence of 64% and was more sensitive than conventional PCR. Infection was more common in puppies below 3 months of age. Medium-sized breeds were most frequently affected and males showed higher positivity than females. The highest prevalence was observed during the rainy season, followed by winter and summer. CPV infection was also found in vaccinated dogs. Phylogenetic analysis revealed that the circulating virus belonged to the CPV-2b antigenic variant.

    INTRODUCTION

    Canine parvovirus (CPV) emerged in 1978 and has since become a major pathogen of domestic and wild canids, particularly affecting puppies below 6 months of age. CPV-2 is a non-enveloped virus with an icosahedral structure and is classified under the genus Protoparvovirus, subfamily Parvovirinae and family Parvoviridae. It has a single-stranded, negative-sense DNA genome of approximately 5.3 kb that encodes two non-structural proteins, NS1 and NS2 and three capsid proteins, VP1, VP2 and VP3. Among these, VP2 is the major structural protein involved in cell entry and a key site for genetic mutation (Cavalli et al., 2008). CPV primarily infects intestinal epithelial cells, where it replicates and is shed through faeces (Hoelzer et al., 2008). The infection is highly contagious and may present as hemorrhagic enteritis with bloody diarrhoea and vomiting, or as cardiac syndrome/myocarditis, especially in pups below 3 months of age (Hayes et al., 1979).
           
    Numerous studies have reported CPV infections in the Indian subcontinent (Mandawat et al., 2025; Mohan et al., 2025). Recent molecular epidemiological studies show the continued evolution and global spread of CPV, with CPV-2a, CPV-2b and CPV-2c co-circulating in many regions, including Asia and India. CPV-2b and CPV-2c have been frequently associated with diarrhoeic dogs and vaccine breakthrough infections due to VP2 antigenic drift (Mittal et al., 2022; Nandi et al., 2023; Reddy et al., 2024; Chethan et al., 2025).
           
    Because CPV is highly contagious and often fatal, early diagnosis is essential. Molecular methods such as nested PCR and sequencing are now vital for rapid detection, accurate typing and surveillance of circulating CPV strains (Zhang et al., 2023; Singh et al., 2024). Nested PCR (NPCR) is highly sensitive and can even detect 106 particles of CPV/g of faeces (Kumar et al., 2011), thus giving an early diagnosis of even sub-clinical infections. PCR and NPCR are used to detect the VP2 gene in CPV, which encodes the capsid protein that is important to determine its antigenicity (Phromnoi et al., 2010). Many risk factors, such as age, season, breed size and vaccination are also associated with the CPV-2 infection. CPV immunization is viewed as a core factor to prevent the infection in newborn pups. As an integral part of the canine core vaccine, vaccination failure is also not uncommon (Muzaffar et al., 2006). Till date no study has been conducted to determine the incidence of CPV infection with nested PCR and circulating variant in the Indore region. Hence, the present study was undertaken to confirm the presence of CPV-2 in dogs with acute gastroenteritis and to characterize the antigenic variants circulating in the Indore region. Further, study of associated risk factors like season, age, sex, breed of dogs and vaccination status was taken into account.

    MATERIALS AND METHODS

    Collection of samples
     
    The study was conducted in the Department of Veterinary Microbiology, College of Veterinary Science and AH MHOW. Samples were collected from Veterinary Clinical Complex (VCC), College of Veterinary Science and Animal Husbandry, Mhow (M.P.) and from private pet clinics of Indore during 2022-2023. One hundred (100) rectal swab samples were aseptically collected from suspected dogs with a history of diarrhoea/ haemorrhagic diarrhoea, vomition, reduced appetite, dehydration with or without pyrexia and transported to the laboratory in 2 mL of phosphate-buffered saline (PBS; pH 7.2) under chilled conditions. Samples were collected with the questionnaire, viz., date of collection, age, sex, breed, vaccination status and season of infection, etc., to investigate various risk factors correlated to the occurrence of CPV in dogs in the study area. The experimental protocol (No. 04/Ethical/2021, Dated 05/02/2021) was approved by the Institutional Animal Ethics Committee (IAEC) College of Veterinary Science and AH MHOW, NDVSU Jabalpur.
     
    Preparation of virus inoculum/sample processing
     
    All the swabs with PBS were squeezed against the collection tube wall and transferred to a 2 mL microcentrifuge tube and centrifuged at 3000 rpm for 10 min. Supernatant was kept at -20°C till further use.
     
    Detection of canine parvovirus (CPV-2) by PCR and NPCR
     
    Total genomic DNA was extracted from all the supernatants of faecal samples using phenol-chloroform extraction method from all clinical swab samples and vaccine (Megavac-7 DHPPi) using standard protocol prescribed by Sambrook and Russell (2001) and stored at -20°C for further use. The screening of CPV-2 in faecal samples was carried out by the detection of VP2 gene using polymerase chain reaction (PCR) and Nested PCR.
     
    Polymerase chain reaction (PCR) for the detection of canine parvovirus-2
     
    For first round of PCR, forward primer CPV-2 and reverse primer CPV-4 were used for amplification of 1198 bp product of the VP2 gene (Mizak and Rzezutka, 1999). PCR amplification with a final reaction volume of 25 µL which included 2.5 µL of 10× PCR buffer with 15 mM MgCl‚ 1µL of 10 mM dNTPs, 1 µL each of forward (CPV-2) and reverse (CPV-4) primers at 20 pmol/µL, 0.5 µL of 10 mM MgCl and 0.2 µL of Taq DNA polymerase (5U/µL) and 10 µL of DNA template (400 ng). Volume was completed to 25 µL employing nuclease-free water. Amplification protocol consisted of 35 cycles with denaturation at 94°C for 1 min, annealing at 56°C for 1 min and extension at 72°C for 150 sec, followed by a final extension at 72°C for 10 min. Appropriate controls were included in each run: Vaccine-derived DNA served as the positive control, while rectal swab samples obtained from clinically healthy dogs were used as the negative control.
     
    Nested PCR (NPCR) for the detection of canine parvovirus-2
     
    For second round of PCR or Nested PCR (NPCR), forward primer CPV-6 and reverse primer CPV-4 were used (Mizak and Rzezutka, 1999) for amplification of 548 bp size product. Reaction mixture of 25 µl by adding 2.5 µl of 10X PCR Buffer (with 15 mM MgCl2), 1 µl of dNTPs (10 mM), 1 µl of CPV-6 (Forward) and CPV-4 (Reverse) primers (20 pmol/µl) each, 0.5 µl of MgCl2 (10 mM), 0.2 µl of Taq DNA Polymerase (5 unit/µl) and 5 µl of PCR product of first round of PCR as a DNA template and making the volume of 25 µl by adding Nuclease Free Water (NFW). Thermocyclic conditions was same as first round of PCR. PCR products were resolved on a 1.5% agarose gel and visualized using a gel documentation system.  
     
    Sequencing of VP2 specific gene of CPV
     
    Following the manufacturer’s instructions, nested PCR products (partial VP2 gene) have been purified employing a gel extraction kit (GenEluteTM PCR Clean-UP Kit, Sigma). The DNA’s purity was verified and it was subsequently submitted to Eurofins Genomic India Pvt Ltd for sequencing using Sanger’s method (BigDye V3.1 terminator technology). Software such as DNASTAR and MEGA7.0 have been employed for processing and analyzing the collected data. DNA sequences were aligned utilizing the ClustalW technique in the MegAlign program by DNASTAR, USA and MEGA7 with reference sequences that had already been published and obtained from GenBank NCBI (Kumar et al., 2016). Phylogenetic relationships were analyzed utilizing Maximum Likelihood method implemented in MEGA7 and evolutionary history was inferred employing Tamura-Nei nucleotide substitution model (Tamura and Nei, 1993). A bootstrap consensus tree was constructed from 1000 replicates for assessing robustness of the branches (Felsenstein, 1985). The VP2 gene sequences of Canine parvovirus type 2 were submitted to GenBank to obtain accession numbers.
     
    Statistical analysis
     
    The data were analyzed and summarized using descriptive statistical methods.
           
    Results were expressed as percentage positivity of the detected infections.

    RESULTS AND DISCUSSION

    Molecular detection of CPV infection by PCR and NPCR
     
    In the present study, the preliminary VP2 gene-specific PCR produced the expected amplified products of 1198 bp in both the positive control and the suspected clinical samples (Fig 1).  No bands were observed in the non-template control. Among the 100 samples, 50 tested positive using PCR, indicating a 50% positivity of CPV in this PCR. However, of these 100 samples, 64 tested positive using NPCR, amplifying a product size of 548 bp (Fig 2). This suggests a 64% incidence of CPV when using NPCR. This study demonstrates that NPCR exhibits higher sensitivity compared to PCR in detecting CPV. Hirasawa et al. (1994) and Schmitz (2009) also highlighted the increased sensitivity of nested PCR compared to conventional PCR in their studies. This heightened sensitivity may be attributed to the potential presence of inhibitory substances in samples with very few virus particles, preventing the visualization of amplified products in conventional PCR. Nested PCR (NPCR) can overcome this limitation, enabling the visualization of NPCR products in an agarose gel (Kumar et al., 2011). Similarly, Singh et al., 2021 recorded a positivity of 28% and 70% for PCR and NPCR, respectively, underscoring the superior sensitivity of NPCR over PCR. Wang et al., (2022) and Kumar et al., (2022) suggested that nested PCR and real-time PCR assays show significantly higher sensitivity than conventional PCR for detection of low viral loads in clinical and subclinical CPV infections.

    Fig 1: Agarose gel electrophoresis showing amplified product (1198 bp) of VP2 gene in PCR.



    Fig 2: Agarose gel electrophoresis showing amplified product (548 bp) of VP2 gene in NPCR.


     
    Analysis of epidemiological factors in CPV-2 infected dogs
     
    The distribution of positive cases was recorded higher in below 3 month of age group of dogs (31/39, 79.5%), compared to between 3-6 months of age (28/48, 58.3%) and more than 6 months of age (5/13, 38.5%). The increased incidence in puppies under six months may be explained by the marked tropism of Canine parvovirus type 2 for rapidly dividing cells (myocardiocytes, small intestinal crypt epithelial cells, lymphopoietic tissue and bone marrow), young animals will have a greater number of such cells than adults (Chethan et al., 2021). CPV-2 commonly affects young puppies between 6 and 20 weeks of age, but unvaccinated dogs of any age group may potentially be affected (Eregowda et al., 2020). Maternally derived antibodies protect newborn puppies from CPV-2 infection; however susceptibility increases when maternally acquired antibodies begin to wane (Mittal et al., 2022).
           
    The CPV infection was found higher in male dogs (50/73, 68.4%) than in female dogs (14/27, 51.8%), which could be attributable to the fact that the number of male animals presented to the hospital was higher than female animals. There is no breed or sex susceptibility to CPV-2 infection (Chethan et al., 2021). However, Geetha and Selvaraju (2019) and Khare et al., (2019) similarly reported a higher susceptibility of male dogs to CPV- 2 infection compared with females. The majority positive cases was found to be higher in medium-sized breed (56/79, 70.9%), followed by large-sized breed (7/18, 38.9%) and small-sized breed (1/3, 33.33%). (Large breed size dogs included Dalmatian, German Shepherd and Rottweiler. Medium-sized dogs included Boxer, Siberian husky, Labrador and non-descript breeds of dogs. Small breed size dog included Pomeranian and Pug). Breed-wise analysis showed that non-descript dogs accounted for the highest number of positive cases (32/49), followed by Labrador dogs (21/27), while German Shepherds contributed 5 positives out of 13 examined. It may be because the majority of dogs presented to the hospital in the current study were non-descript dogs, which could explain the relatively high percentage of positive cases observed among non-descript breeds. Higher prevalence of CPV infection in non-descript as well as medium-sized breeds has been reported, likely due to poor vaccination coverage and higher exposure risk (Kumar et al., 2022; Singh et al., 2024).
           
    Higher number of cases 53/77 (68.8%) were recorded from non-vaccinated dogs compared to vaccinated dogs 11/23 (47.8%). It was evident that the vaccine prevents infection by boosting immunity against canine Parvovirus infection. However, vaccinated dogs were also found to be positive in the present study, but the rate of incidence has been higher in unvaccinated dogs. Rafiq et al., (2019) also reported 38.7% prevalence in unvaccinated dogs and 21% in vaccinated dogs. Vaccinated dogs affected due to the unidentified CPV strain is circulating in that particular area. A significant cause of vaccine failure in puppies is interference from maternally derived antibodies (MDA), which can have a substantial impact. Mother antibodies, improper vaccine handling, as well as antigenic mismatch between vaccine strains and circulating field variants can cause vaccine failure (Mittal et al., 2022). The higher cases observed in rainy season (19/21, 90.5%) as compared to winter (34/55, 61.8%) and summer season (11/24, 45.8%). Seasonal variation has been observed in canine parvovirus infection, with higher occurrence during rainy or humid periods, probably due to prolonged environmental survival of the virus and increased contamination of soil and surroundings. Geetha and Selvaraju (2023) reported an increased incidence of CPV enteritis in the humidity and rainy seasons.
     
    Phylogenetic analysis of CPV-2 variants
     
    CPV-2 is constantly changing its genetic and antigenic properties because of frequent mutations in the VP2 gene. These changes have led to the emergence and worldwide spread of different antigenic variants, namely CPV-2a, CPV-2b and CPV-2c (Thomas et al., 2017). In India, CPV-2a has been reported as the most common variant, followed by CPV-2b (Thomas et al., 2017). Among the 64 NPCR positive samples, 2 samples were randomly selected for sequencing and phylogenetic analysis. Initial analysis of the VP2 gene sequences using the Blast tool at NCBI(http://blast.ncbi.nlm.nih.gov/Blast.cgi) revealed a high similarity of 99% with available sequences in GenBank. Further, when performing a multiple sequence alignment of VP2 gene sequences employing MegAlign program by DNASTAR, it was found that there was a 99.6% identity with isolates from various countries (Fig 3). The isolates from this study clustered with many Indian isolates. These isolates were closely associated with the CPV 2b strain of other Indian isolates and were distantly related to Canine Adenovirus (Out group). Therefore, it was determined that these strains belonged to the CPV-2b antigen type. Both CPV strains from Madhya Pradesh (CPV1 MP and CPV2 MP) had been submitted to NCBI GenBank and accession numbers were obtained (Accession no.- OM585418, OM585419) (Fig 4).

    Fig 3: Per cent identity of VP2 gene (partial sequence) of CPV-2 MP isolates compared with published reference sequences.



    Fig 4: Phylogenetic tree based on two partial VP2 gene sequences (CPV MP isolates) from the present study and published sequences including canine adenovirus (Out group).


           
    Phylogenetic analysis indicated that the samples were categorized as the CPV-2b antigen type. In a previous study, Recent molecular studies from India and neighbouring countries have consistently reported CPV-2b as a predominant circulating variant, although sporadic emergence of CPV-2c has also been documented, indicating ongoing viral evolution and the need for continuous molecular surveillance (Nandi et al., 2023; Zhang et al., 2023). Nandi et al. (2010) identified CPV-2b subtypes in dogs, aligning with our findings. However, their study also reported CPV 2C subtypes. They observed nucleotide substitutions, suggesting that CPV-2c mutants might have evolved from CPV-2b mutants circulating in India. In a different study, Buonavoglia et al. (2001) reported the CPV 2b antigenic subtype in dogs with severe haemorrhagic diarrhoea. The virus’s virulence and immunogenicity are examined by the presence of particular amino acids in the VP2 gene. For determining the genotype and pathotype present, it is therefore crucial to monitor the CPV that is circulating in the field at various intervals.

    CONCLUSION

    This study shows that CPV 2 is highly prevalent in diarrheic dogs in Indore, Madhya Pradesh, with NPCR (64%) being more sensitive than conventional PCR (50%). The predominant circulating strain is CPV 2b and infection is strongly associated with puppies under 3 months, male dogs, medium sized breeds (especially non descript and Labrador) and the rainy season, with higher positivity in unvaccinated animals. The findings highlight the need for improved vaccination, seasonal based control and continuous molecular surveillance of CPV variants.

    ACKNOWLEDGEMENT

    Authors are thankful to Dean, College of Veterinary Science and A.H., Mhow for providing the necessary facilities to conduct this research in the Department of Veterinary Microbiology, College of Veterinary Science and AH MHOW, NDVSU Jabalpur.
     
    Author’s contribution
     
    V. Waskel: Sample collection, execution of laboratory work. R Gangil: Planning of work, Standardization of the PCR and N-PCR, drafting of manuscript. D. Chhabra: Review and editing of manuscript. R. Sharda: Analysis of results and editing of manuscript. R. Sikrodia: Collection of data of risk factors and sample collection. J. Jogi: Statical analysis of results. K. Jadhav: Bioinformatic analysis of sequences.

    CONFLICT OF INTEREST

    The authors declare that is there is no conflict of interest.

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