Evaluation of therapeutic efficacy of filgrastim against canine parvoviral gastroenteritis in dogs

Introduction

Canine parvoviral gastroenteritis is one of the most common causes of morbidity and mortality in young dogs worldwide (Mylonakis et al., 2016) [13] . Canine parvovirus (CPV) belongs to the genus Protoparvovirus, family Parvoviridae, a single-stranded DNA virus that infects rapidly dividing cells of the gastrointestinal tract, bone marrow, lymphoid tissue, and cardiac myocytes. CPV is found worldwide and signs of infection range from asymptomatic to severe gastroenteritis, leukopenia, dehydration, lethargy, and death (Goddard et al., 2010) [6] . Puppies under 6 months of age are more commonly clinically affected, however dogs of any age can succumb to disease (Decaro et al., 2012) [4] . Vaccination against CPV is routinely performed worldwide and is the most effective method to control disease (Kelman et al., 2020) [8] . Total white blood cell counts and the presence of leukoneutropenia have previously been considered to be a hallmark in patients with CPV because the virus attacks actively replicating cells of the bone marrow, thymus, and other lymphoid tissues (Castro et al., 2013) [3] . The most common method for initial screening and detection of canine parvovirus is the use of a cage-side, inclinic immunochromatographic enzyme-linked immunosorbent assay (ELISA) that has a high degree of sensitivity but intermediate to low specificity, compared with molecular methods such as PCR (Decaro et al., 2012) [4] . Fluid replacement, systemic antibiotic administration, antinausea medicines, anti-diarrhoea medicines, and a rigorous diet combined with monoclonal antibodies are the main treatment methods for CPV-2-infected dogs; however, the recovery rates vary from 27.8 to 93.5% (Qi et al., 2020) [16] . The destruction of haematopoietic progenitor cells of the various leukocyte types in the bone marrow and the loss of neutrophils through the injured gastrointestinal tract may causes leukopenia and neutropenia in parvoinfected dogs (Macartney et al., 1984) [11] . Neutrophils are most important cells required for immune system to combat with the bacterial infection. So prolonged neutropenia greatly increases the risk of death, due to sepsis. Hence strategies are required to shorten the duration of neutropenia in dogs. The use of granulocyte colony stimulating factor, a cytokine and growth factor that potently stimulates neutrophil production and release from the bone marrow, has been advocated for treatment of parvovirus induced neutropenia (Kraft et al., 1995) [9] . Filgrastim is a non-glycosylated, 175 amino acid containing protein, which is produced recombinantly by Escherichia coli. Filgrastim has a molecular weight of 18.8 kDa and it regulates the production and release of functional neutrophils from bone marrow within 24 hours of administration. Filgrastim results in increase in peripheral blood neutrophil counts with minor increase in monocytes (Areshkumar et al., 2017) [1] .

Materials And Methods

The study was conducted on 344 dogs diagnosed with gastroenteritis at the Veterinary Clinical Complex (VCC), College of Veterinary Science and Animal Husbandry, Mhow. The dogs were screened for canine parvovirus infection using the CPV Rapid Antigen Test Kit, an immunochromatographic assay-based diagnostic tool and 84 dogs were found to be positive for the CPV. Out of the 84 CPV positive cases, 24 dogs were selected and divided into four groups (each group comprised of six dogs and two groups had dogs less than 3 months of age and the other two groups had dogs between 3-6 months of age) and treatment was given as per the following schedule in Table 1. [15,19] .

Results

Among the 24 dogs included in the study, inappetence, vomiting, diarrhoea, and dehydration were observed in all cases (100%). Alterations in mucous membrane colour were also noted, with pink mucous membranes in 50% of dogs, congested mucous membranes in 25%, and pale mucous membranes in 25%. Based on the degree of dehydration, 54.17% of dogs exhibited mild dehydration, 37.50% moderate dehydration, and 8.33% severe dehydration.

The mean values of physiological parameters including heart rate and respiratory rate, were elevated on 0 th day (pretreatment) but returned to normal ranges 3 rd and 7 th day as shown in Table 2. Rectal temperature remained within the normal physiological range throughout the study period. However, no statistically significant differences were observed in physiological parameters during the course of treatment. A decreased mean total leukocyte count (TLC) was observed in all groups on 0 th day, which showed a significant increase by 3 rd and 7 th day as clinical recovery progressed as shown in Table 3. Differential leukocyte count revealed a significant reduction in neutrophil counts in all four groups on 0 th day. A significant increase in neutrophil counts was observed by 3 rd and 7 th day, particularly in Group I and Group III. Lymphocyte and monocyte counts remained within normal limits throughout the study period. Platelet counts were within the normal reference range in all groups; however, a significant decrease was noted in Group I and Group III during recovery. All biochemical parameters remained within normal reference ranges except for total protein, albumin, and globulin, which were below normal values prior to treatment as shown in Table 4. These parameters showed gradual improvement as treatment progressed. Dogs treated with filgrastim in combination with standard therapy exhibited a significantly shorter recovery period compared to those receiving standard treatment alone as shown in Table 5. Furthermore, total leukocyte and neutrophil counts showed significantly greater increases in Group I and Group III than in Group II and Group IV on both 3 rd and 7 th day post-treatment. A marked increase in total leukocyte and neutrophil counts was evident in dogs receiving granulocyte colony-stimulating factor therapy as shown in Table 3.

Discussions

A polypeptide called cachectin, produced by activated macrophages along with interleukins, has been recognized as a major contributor to the fever and anorexia seen in inflammatory diseases. Triggered by endotoxins, cachectin is believed to be a key mediator in the development of endotoxic shock (Strombeck et al., 1991) [22] . Further, decreased contractions and hypomotility has also been attributed as causes of fever and inappetence (Carman et al., 1982) [2] . The inflammatory damage inflicted on the stomach and intestinal mucosal barrier is considered the underlying cause of vomiting in cases of parvoviral gastroenteritis (Mccaw et al., 2006; Streck et al., 2009) [12,20] . Parvovirus infection of the germinal epithelium in the intestinal crypts leads to the breakdown and collapse of the epithelium, causing the villi to shorten. This disrupts the normal cell turnover in the small intestine. Additionally, conditions like disseminated intravascular coagulation (DIC), endotoxemia, and damage to the intestinal lining encourage secondary bacterial infections from Gram-negative and anaerobic bacteria. These intestinal changes caused by parvoviral infection are believed to contribute to the haemorrhagic diarrhoea seen in dogs with the disease. The hallmark features of parvoviral gastroenteritis in dogs are fluid and electrolyte loss resulting from persistent vomiting and diarrhoea. These symptoms are attributed to damage in the gastrointestinal epithelium, which leads to significant fluid and protein loss. Congested mucous membranes are indicative of endotoxemia in canine parvoviral gastroenteritis, potentially resulting from the invasion of Gram-negative organisms following epithelial damage. This congestion is attributed to endotoxemia and the resultant pooling of blood in capillaries and blood vessels (Strombeck et al., 1991) [22] . Severe vomiting and haemorrhagic diarrhoea associated with parvoviral gastroenteritis can result in blood loss, leading to reductions in RBC count, haemoglobin concentration, and packed cell volume (Otto et al., 1999) [14] . Parvovirus also targets rapidly proliferating cells in the bone marrow, inhibiting erythropoiesis and exacerbating anaemia. Initially, dehydration may cause haemoconcentration, but fluid replacement therapy often leads to a subsequent decline in PCV due to dilutional effects (Greene et al., 2012) [7] . The destruction of haematopoietic progenitor cells responsible for the production of various leukocyte types in the bone marrow, coupled with the loss of neutrophils through the damaged gastrointestinal tract, likely contributes to leukopenia and neutropenia in dogs infected with parvovirus (Macartney et al., 1984) [11] . Additionally, neutropenia may arise from an overwhelming demand for neutrophils in the gut due to ongoing inflammation and infection (Ettinger et al., 2016) [5] .

The thrombocytopenia observed in dogs with parvoviral gastroenteritis is thought to result primarily from the destruction of megakaryocyte precursors in the bone marrow, which limits platelet production. Additionally, an increased utilization of platelets within the damaged gastrointestinal system exacerbates the depletion of circulating platelets. This dual mechanism of bone marrow suppression and increased consumption contributes to the marked reduction in platelet levels seen in affected dogs (Rewerts et al., 2000) [17] . Filgrastim stimulates the production and release of neutrophils from the bone marrow, but its effects on other blood cell lineages, such as megakaryocytes (precursors to platelets), can lead to a relative decrease in platelet production. Additionally, rapid neutrophil proliferation may cause bone marrow exhaustion, limiting the availability of resources for megakaryocyte development, thereby contributing to thrombocytopenia in some cases (Wu et al., 2020) [22] . Anorexia, along with reduced food intake, induces a decrease in hepatic protein synthesis as amino acids are preferentially utilized for the production of positive acute phase proteins in response to inflammatory stimuli and tissue injury. This shift is a key component of the acute phase response, where the liver alters its metabolic function to support immune activation and repair mechanisms. As a consequence, there is a decrease in the synthesis of albumin and other essential proteins, contributing to hypoproteinaemia (Li et al., 2015) [10] . Numerous studies have shown the effectiveness of recombinant human granulocyte-colony stimulating factor (rhG-CSF) in treating leukopenia, particularly neutropenia, in dogs. This evidence provides strong support for the inclusion of filgrastim as an adjuvant therapy in the current study for treating canine parvoviral gastroenteritis in dogs with leukopenia (Kraft et al., 1995) [9] . The role of granulocyte colony-stimulating factor (G-CSF) in modulating neutrophil function is well-established in the literature. As a key cytokine, G-CSF regulates both the generation and differentiation of neutrophils within the bone marrow, promoting the release of mature neutrophils into peripheral circulation. In addition to increasing neutrophil numbers, G-CSF enhances several critical neutrophil functions, including chemotaxis, antibody-dependent cellular cytotoxicity (ADCC), immunoglobulin A (IgA)-mediated phagocytosis, and superoxide production. These enhanced functions significantly contribute to improved microbial clearance and a more robust immune response in treated subjects (Roberts, 2005) [18] .

Conclusion

The present study evaluated the clinical and haematological alterations in dogs affected with parvoviral enteritis and assessed the therapeutic efficacy of filgrastim as an adjunct to standard supportive treatment. All dogs exhibited severe clinical signs including inappetence, vomiting, diarrhoea, and dehydration, along with notable leukopenia and neutropenia at presentation. Standard supportive therapy led to gradual clinical improvement and restoration of haematological parameters; however, the addition of filgrastim resulted in a significantly faster increase in total leukocyte and neutrophil counts. This accelerated haematological recovery was associated with a markedly shorter recovery period compared to dogs receiving standard treatment alone. Biochemical parameters showed progressive improvement across all groups during the treatment period. The findings indicate that filgrastim is a safe and effective adjunctive therapy that enhances immune recovery and reduces the duration of illness in dogs with parvoviral enteritis. This study suggests that granulocyte colony-stimulating factor therapy can be beneficial in the management of canine parvoviral enteritis, particularly in cases presenting with severe neutropenia. Further studies with larger sample sizes are warranted to confirm these observations and to establish optimal treatment protocols.

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