Evaluation of haematological alteration and oxidative stress with reference to antioxidant potential of ascorbic acid in CPV gastroeteritis

Introduction

Canine parvovirus type 2 (CPV-2) is a highly contagious DNA virus that primarily affects dogs, with puppies and unvaccinated animals being especially susceptible. Since its first recognition in 1967, the virus has remained a significant cause of morbidity and mortality in canine populations, exhibiting nearly 100% morbidity and mortality rates of up to 91% in the absence of appropriate treatment (Nandi and Kumar, 2010;Sykes, 2014). Canine parvovirus type 2 (CPV-2) is a small, non-enveloped virus possessing a single-stranded DNA genome. Among its variants, CPV-2a and CPV-2b are the most prevalent strains worldwide and are responsible for the majority of canine infections. Viral replication is dependent on the host cell machinery, particularly within the cell nucleus. Transmission occurs primarily through the fecal-oral route or through contact with fomites contaminated by infected fecal material. CPV-2 exhibits remarkable environmental stability, remaining infectious for up to one year under adverse conditions and surviving in contaminated soil for approximately five International Journal of Advanced Biochemistry Research https://www.biochemjournal.com months. Following entry into the host, the virus preferentially infects rapidly dividing cells, especially those located in lymphoid tissues and bone marrow progenitor cells (Nandi and Kumar, 2010) [32] . Approximately 3-4 days after infection, CPV-2 spreads hematogenously to the intestinal crypts of the small intestine.

During the initial five days of viremia, the virus is widely distributed in the epithelial tissues of the oral cavity, tongue, esophagus, and small intestine, as well as in bone marrow and lymphoid organs. Infection of these rapidly proliferating cells causes cell death and impairs normal cell division, contributing to the pathogenesis of the disease (Schoeman et al., 2013) [41] .

Although canine parvovirus can affect dogs regardless of breed or sex, puppies between 6 weeks and 6 months of age are at the highest risk of infection (Goddard et al., 2008) In CPV-2 infections, oxidative stress and electrolyte disturbances are more closely related to disease severity than to primary causative factors (Ukwueze et al., 2020). Diagnosis is often challenging due to nonspecific signs such as vomiting and diarrhea, which overlap with other gastrointestinal diseases; hence, clinical suspicion is usually confirmed using rapid antigen detection tests. Vitamin C acts as an antioxidant by scavenging free radicals (Carr and Frei, 1999), while N-acetylcysteine has been shown to improve clinical recovery and reduce hospitalization time in infected dogs (Gaykwad et al., 2018). Combined use of vitamin C, Nacetylcysteine, and antibiotics has been reported to enhance recovery in CPV cases (Kataria et al., 2020). Ascorbic acid, a water-soluble vitamin, acts as a major extracellular antioxidant that protects cellular components such as lipids, proteins, and DNA from oxidative injury and helps modulate inflammation. Therefore, this study was undertaken to assess the antioxidant efficacy of ascorbic acid in the treatment of CPV-associated gastroenteritis [15, 17, 29, 18, 45, 38, 32, 44, 41, 2, 4, 19] .

Material and methods

The study commenced with the collection of fecal samples from dogs showing clinical signs of hemorrhagic diarrhea and vomiting. These samples were screened for canine parvovirus using a rapid antigen detection kit (PetX, Secure Diagnostic Private Limited, Bhopal). During the study period, a total of 613 cases of hemorrhagic gastroenteritis in dogs were recorded at the Veterinary Clinical Complex, College of Veterinary and Animal Sciences, Pantnagar. Approximately 5 mL of blood was collected from each enrolled dog via cephalic vein puncture. The blood samples were divided into vials with and without EDTA. Whole blood samples were used for hematological analysis, whereas non-anticoagulated blood was allowed to clot and centrifuged at 3000 rpm for 5 minutes to obtain serum, which was subsequently stored at -20°C until further analysis. Hematological parameters, including hemoglobin concentration, total and differential leukocyte counts, and packed cell volume (PCV), were evaluated on days 0 (pretreatment), 3, and 5 post-treatment using standard laboratory procedures. Oxidative stress parameters, including malondialdehyde (MDA) concentration as described by Draper and Hadley (1990) serum nitric oxide (NO) levels as per Sastry et al. (2002) and serum ceruloplasmin concentration following the method of Sunderman and Nomoto (1970), were estimated using spectrophotometric techniques at the Clinical Diagnostic Laboratory, ICAR-IVRI, Izatnagar [12, 40, 43] . A total of 18 dogs were randomly selected for the therapeutic study and allocated into three groups (T 1 , T 2 , and T 3 ), with six animals in each group. Group T 1 served as the healthy control. Dogs in Group T 2 received supportive and symptomatic treatment as required. The treatment protocol included ceftriaxone at a dose of 25 mg/kg body weight, administered twice daily for five days to prevent secondary bacterial infections. Intravenous fluid therapy with Improlyte was provided to correct electrolyte imbalances resulting from vomiting and diarrhea. Metoclopramide was administered intravenously at 0.2 mg/kg body weight for five days to control vomiting, while pantoprazole was given intravenously at 1.0 mg/kg body weight for five days to suppress gastric acid secretion and protect the gastrointestinal mucosa from ulceration. Metronidazole was administered at a dose of 20 mg/kg body weight twice daily for five days to manage secondary infections. A haemostatic agent was given until hemorrhage was controlled. Botropase was used as the haemostatic agent at a dose of 0.5-1.0 mL, adjusted according to the individual animal's body weight. During the recovery phase, after the resolution of vomiting and diarrhea, multivitamin supplements and a liver tonic were recommended to support hematopoietic recovery and restore energy levels as required. Group T3 received supportive and symptomatic therapy as needed, along with intravenous ascorbic acid at a dose of 15 mg/kg body weight for five consecutive days, which was administered as antioxidant therapy to mitigate oxidative stress.

Results & Discussion

Out of 613 affected dogs included in the study, 139 were randomly selected for therapeutic evaluation. Among these, 54 dogs were confirmed positive for canine parvovirus (CPV) gastroenteritis using a rapid antigen detection test, as evidenced by the presence of both the test (T) and control (C) lines. The most common clinical manifestations observed in CPV-infected dogs were depression, anorexia, vomiting, and diarrhea.

Analysis of mean hemoglobin concentration (g/dl) revealed no significant difference between treatment groups T 2 (ST) and T 3 (ST+AA) on day 0. However, prior to the initiation of treatment, both T 2 and T 3 exhibited significantly lower hemoglobin levels (p<0.05) compared with the healthy control group (T 1 ). Figures with atleast one common superscript a,b,c along rows are non-significant and different subscripts x,y,z along columns differ significantly (p<0.05). By day 5, no significant differences in mean hemoglobin concentration were observed between the therapeutic groups, and their values had approached those of the healthy control group. Within-group analysis showed that the mean hemoglobin concentration (g/dl) on day 5 was significantly higher (p<0.05) in both treatment groups, T 2 (ST) and T 3 (ST+AA), compared with their respective baseline values on day 0 (Table 1). All dogs in the treatment groups received supportive therapy consisting of balanced electrolyte fluids, an antiemetic (Metoclopramide @ 0.5 mg/kg body weight, IV, BID), a proton pump inhibitor (Pantoprazole @ 1 mg/kg body weight, IV, OD), antibiotics, and haemostatic agents as required. This therapeutic regimen may have helped minimize blood loss and contributed to the observed improvement in hemoglobin levels. The mean total leukocyte count (×10³/cumm) was compared among the study groups. Before the initiation of treatment (day 0), the mean total leukocyte count was significantly lower (p<0.05) in both therapeutic groups, T 2 (ST) and T 3 (ST+AA), compared with the healthy control group (T 1 ). However, no significant difference was observed between the therapeutic groups at this stage. A similar trend was noted on day 3, with no significant variation between T2 and T3. On day 5, although the leukocyte counts in both treatment groups remained significantly lower (p<0.05) than those of the healthy control group, no significant difference was detected between the two therapeutic groups. Within-group analysis revealed that the mean leukocyte count in group T 2 (ST) did not change significantly throughout the treatment period from day 0 to day 5. In contrast, group T 3 (ST+AA) exhibited a significant increase (p<0.05) in leukocyte count by day 5 following the initiation of treatment.

The findings of the present study were not in agreement with those reported by Salem (2014) [42, 46, 13, 49, 14, 26, 13, 6, 20, 38, 48, 7, 6, 20] . Comparison of the mean neutrophil (%) values among the study groups revealed that, on day 0 and day 3, both therapeutic groups T 2 (ST) and T 3 (ST+AA) had significantly lower (p<0.05) neutrophil percentages than the healthy control group (T 1 ). However, following treatment, no significant difference in neutrophil (%) values was observed between the therapeutic groups and the healthy control group on day 5. Within-group analysis showed that the mean neutrophil (%) values in both T 2 (ST) and T 3 (ST+AA) increased significantly (p<0.05) by day 5 compared with their respective baseline values on day 0 ( (2021) reported that lymphopenia in CPV infection may be due to the virus's affinity for lymphoid tissues and lymphocytes, leading to their depletion.

In the present study, the mean monocyte (%) values were compared both between the therapeutic groups and within each group. It was observed that there was no significant difference in monocyte percentages among the treatment groups T 2 (ST) and T 3 (ST+AA) (Table 1). Similar findings were reported by Khare et al., (2020), who also observed nonsignificant changes in monocyte counts. In contrast, Bastan et al., (2013) reported a decrease in monocyte values in dogs affected with parvoviral infection and emphasized the beneficial role of monocytes in recovery from leukopenia in canine parvovirus gastroenteritis. This improvement was attributed to bone marrow recovery and monocyte production, which typically occurs within approximately three days. Statistical analysis of basophil (%) values showed that, both between and within the groups, there was no significant difference observed at day 0, day 3, and day 5 (Table 1). Before the initiation of treatment (day 0) and on day 3 posttreatment, NO levels were significantly higher (p<0.05) in both therapeutic groups T 2 (ST) and T 3 (ST+AA) compared with the healthy control group (T 1 ). On day 5, NO levels remained significantly elevated (p<0.05) in group T 2 (ST) when compared with both group T3 and the healthy control group following symptomatic treatment. In contrast, the mean NO concentration in group T 3 (ST+AA) was closer to that of the healthy control group, indicating a more pronounced normalization effect. Within-group analysis of nitric oxide (NO) levels (nM/ml) from day 0 to day 5 following therapeutic protocols revealed that there was no significant variation in NO concentration in the healthy control group (T 1 ) over the study period. In addition, no significant difference was observed in NO levels at day 3 compared with day 0. In contrast, group T 3 (ST+AA) showed a significant reduction (p<0.05) in NO levels on day 3 posttreatment compared with day 0 (Table 1), indicating an improvement following the therapeutic intervention.

In the present study, a marked increase in nitric oxide levels was observed in dogs affected with canine parvovirus infection, which is in agreement with earlier reports by Nykky et al., (2014), Gaykwad et al., (2018) and Chethan (2020). These authors also reported pronounced oxidative stress in CPV-infected dogs, as evidenced by elevated NO levels prior to the initiation of treatment.

Comparison of mean NO concentrations among the treatment groups revealed a significant (p<0.05) reduction in group T3 (ST+AA) on day 3 when compared with group T2 (ST). These findings suggest an improvement in oxidative stress status in CPV-infected dogs following antioxidant supplementation, as also reported by Gaykwad et al., (2018). The mean ceruloplasmin (g/dl) levels were compared among the study groups, and it was observed that before the initiation of treatment, both therapeutic groups T 2 (ST) and T 3 (ST+AA) had significantly higher (p<0.05) ceruloplasmin levels compared with the healthy control group (T 1 ). However, on day 5, the mean ceruloplasmin levels showed no significant difference among all groups and were comparable to the healthy control group. Within-group analysis revealed that in group T 2 (ST), ceruloplasmin levels did not differ significantly from day 0 to day 5. In contrast, group T 3 (supportive treatment plus ascorbic acid) showed a significant reduction (p<0.05) in ceruloplasmin levels on both day 3 and day 5 following the initiation of treatment protocols (Table 1). Kocaturk et al., (2015) proposed that, in addition to inflammation, the elevation of serum ceruloplasmin in CPVinfected dogs may also reflect a compensatory response against oxidative stress, owing to its antioxidant properties.

In the present study, a significant (p<0.05) reduction in ceruloplasmin levels was observed in groups T3 (supportive treatment plus ascorbic acid) on day 3 post-treatment when compared with group T 2 (supportive treatment alone). This decrease may be attributed to the reduction in inflammatory status following therapeutic intervention, where passive viral neutralization contributes to decreased systemic inflammation and promotes clinical recovery. In line with this, Kalli et al., (2010) also reported a decline in serum ceruloplasmin levels following passive viral neutralization, suggesting that the reduction is associated with attenuation of the systemic inflammatory response. In CPV infection, pro-inflammatory cytokines and endotoxins play a key role in triggering and sustaining the systemic inflammatory response [11, 3, 34, 18, 31, 2, 30, 36, 25, 24, 10, 1, 39, 16, 22, 5, 37, 21, 35, 28, 33, 14, 9, 35, 24, 27, 33, 18] .

Summery and conclusion

During the study period, a total of 613 cases of haemorrhagic gastroenteritis in dogs were reported at the Veterinary Clinical Complex, College of Veterinary and Animal Sciences, Pantnagar. Out of these, 139 samples were randomly selected for therapeutic evaluation, of which 54 faecal samples were confirmed positive for canine parvovirus (CPV) gastroenteritis using a rapid antigen detection test kit. Analysis of oxidative stress indices revealed a significant (p<0.05) improvement, with marked reductions in malondialdehyde (MDA), nitric oxide (NO), and ceruloplasmin levels in group T 3 (supportive treatment plus ascorbic acid) compared with group T 2 (supportive treatment alone). No adverse effects associated with ascorbic acid administration were observed. The antioxidant action of ascorbic acid likely contributed to the scavenging of free radicals and mitigation of oxidative stress. Based on these findings, ascorbic acid may be considered a beneficial adjunct therapy in the management of canine parvovirus gastroenteritis.

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