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1 Available online freely at Bioscience Research Print ISSN: Online ISSN: Journal by Innovative Scientific Information & Services Network RESEARCH ARTICLE BIOSCIENCE RESEARCH, (4): OPEN ACCESS Diagnosis of hemorrhagic gastroenteritis in dogs Abeer A. Abd El-Baky 1*, S. A. Mousa 2 and W. M. Kelany 2 1 Department of Clinical Pathology, Faculty of Veterinary Medicine, Cairo University, Egypt. 2 Department of Internal Medicine and Infectious Diseases, Faculty of Veterinary Medicine, Cairo University, Egypt.. *Correspondence: abeer_abdelbaky@cu.edu.eg Accepted: 18 Nov Published online: 30 Dec Hemorrhagic gastroenteritis (HGE), especially in young dogs is a life-threatening condition due to the loss of body fluid and electrolytes along with the blood loss in vomits and stool. Canine parvovirus (CPV) is one of the causative agent of HGE in dogs. The present study aimed to diagnose dogs suspected to be infected with CPV through clinical examination, rapid detection of CPV antigen in feces along with hematological and biochemical examinations. To perform this study, 30 German shepherd dogs, aged from 1 to 3 months, admitted to the clinic of Faculty of Veterinary Medicine, Cairo University and to a private veterinary clinic in Giza governorate were used and were subjected to clinical, hematological and biochemical examinations. Dogs were divided into 2 groups; apparently healthy group (9 dogs) and HGE affected group (21 dogs, manifesting HGE). Results in comparison to the apparently healthy dogs showed that HGE affected dogs had signs of HGE including sudden onset of acute bloody vomiting, bloody diarrhoea, dehydration, anorexia, weight loss, depression, fever, congested mucous membranes and positive to rapid detection of CPV antigen in their feces (CPV infected dogs), also had the following hematological abnormalities including the presence of microcytic normochromic anemia, leukopenia, neutropenia, lymphopenia, and thrombocytopenia. Together with the following biochemical findings including hypoproteinemia, hypoalbuminemia, hypocalcemia, hypokalemia, hyponatremia, and increase in C-reactive protein (CRP) concentration. This study cleared that, diagnosis of HGE caused by CPV depending on combination of clinical, hematological and biochemical examinations together with the detection of CPV antigen in the feces. Keywords: Hemorrhagic gastroenteritis, Parvovirus enteritis, Canine parvovirus, Hematological studies, Biochemical studies, Rapid detection of parvovirus antigen. INTRODUCTION Hemorrhagic gastroenteritis (HGE) is a lifethreatening disease of dogs. HGE is an acute, severe form of diarrhoea that commonly occurs in all dog s breeds and ages, but small breeds are more likely affected than large breeds. It is characterized by sudden onset of vomiting, profuse bloody diarrhoea and depression. This diarrhoea causes large amounts of water to be lost from the body, resulting in severe dehydration. Massive amounts of blood may also be lost into the gastrointestinal tract (GIT) making HGE a serious disease that requires immediate veterinary interference to prevent animal death (Kumar et al. 2014). There are different causative agents responsible for HGE in dogs including viruses as; Parvovirus (Hoskins, 1997), Coronavirus (Toma and Moraillon, 1980) and Rotavirus (Barrios et al. 1989). Bacteria as; Salmonella spp.(chaudhary et al.1985), Escherichia coli (Prada et al. 1991) and Clostridium spp. (Turk et al. 1992). Endoparasites as; Dipyllidium caninum and Ancylostoma caninum (Kumar et al. 2001). Food allergy (Kumar et al. 2003) and irritant drugs (Waters et al. 1992) are also responsible for HGE in dogs. Canine parvovirus enteritis (PVE) is one of the leading cause of morbidity and mortality in dogs

2 (Goddard and Leisewitz, 2010; Sykes, 2014). Clinical manifestations of PVE infection are nonspecific, have clinical similarities with other causes of acute HGE (Sykes, 2014). PVE clinical signs include anorexia, weakness, depression, foul-smelling diarrhoea, which may range from mucoid to purely hemorrhagic, vomiting, dehydration, and fever (McCaw and Hoskins, 2006; Kalli et al. 2010). Canine Parvovirus (CPV) is one of the causative agent of HGE, it belongs to family Parvoviridae, genus Parvovirus. CPV is a serious, highly contagious virus that affects dogs, resulting in severe gastrointestinal symptoms and occasionally cardiac symptoms. It can be transmitted from one dog to another through contact with infected feces Judge, (2015). Canine PVE caused by three variants of CPV type 2 (CPV-2a, CPV-2b, and CPV-2c), which is the most prevalent viral cause of diarrhoea in dogs (Tefft, 2014). All these three variants are thought to have similar pathogenicity leading to indistinguishable clinical disease (Decaro and Buonavoglia, 2012; Marcovich et al. 2012; Mylonakis et al. 2016). Therefore, determining of CPV strain is not required for the clinical management of infected animals (Markovitch et al., 2012; Tefft, 2014) while, for the clinical diagnosis the combination of compatible clinical and clinicopathologic abnormalities together with the detection of the viral antigen in the feces are required (Mylonakis et al. 2016). The present study aimed to diagnose dogs having symptoms of bloody vomiting and bloody diarrhoea of unknown cause. This diagnosis based on case history, and combination of findings of both clinical and clinicopathological (hematological and biochemical) examinations. MATERIALS AND METHODS Study Design The present study was conducted on 30 dogs (German shepherd, aged from 1 to 3 months) admitted to the clinic of Faculty of Veterinary Medicine, Cairo University and to a private veterinary clinic in Giza governorate in the period from January 2016 to January Based on case history and clinical examination, dogs were divided into 2 groups; first group served as apparently healthy group, include 9 clinically healthy dogs. Second group served as HGE affected group, include 21 dogs which manifesting symptoms of HGE suggestive of parvovirus infection, fecal swab was taken from each dog of this group and tested for CPV antigen using the Rapid CPV Ag Test Kit (VetAll Laboratories, Korea). Clinical Examination All dogs were thoroughly investigated and clinically examined by abdominal palpation and tactile percussion according to the method described by Kelly (1984). At the time of examination, dogs of HGE affected group showed pain on pressing the abdomen and stomach. Respiratory rate, pulse rate and rectal temperature of all dogs were recorded. Examination of all dog s mucous membranes and superficial lymph nodes was done. Clinicopathological Examinations Blood samples were collected from cephalic vein of each dog. Obtained blood sample was divided into two parts. First part was anti coagulated by heparin and used for hemogram evaluation, mineral and electrolyte analysis. Second part was collected in clean centrifuge tube for serum separation, clear non hemolysed supernatant serum was harvested for biochemical studies. Hematological Studies Red blood cells (RBCs) count, packed cell volume (PCV%), hemoglobin (Hb) concentration, mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC), total leukocytic count (TLC) and differential leukocytic count (DLC) on Giemsa stained blood smears were performed (Feldman et al. 2000). Biochemical Studies Serum samples were prepared to assay the following biochemical studies; total proteins and albumin concentrations were measured according to Weichselbaun (1946) and Dumas and Biggs (1972), respectively. Heparinized plasma was used for estimation of calcium concentration according to Biggs and Moorhead (1974) while, potassium and sodium were determined according to Niels et al. (1984). C-reactive protein (CRP) concentration was carried according to the method described by Kindmark (1972). The before mentioned biochemical parameters were assayed using reagent kits of good quality and analytical grade. STATISTICAL ANALYSIS Analysis of the data was performed by ANOVA (Analysis Of Variance) test using the computer software Statistical Package for Social Sciences Bioscience Research, 2017 volume 14(4):

3 (SPSS) for Windows (version 10.0) according to the method described by Irwan (1996). RESULTS Clinical signs and Rapid test result Based on case history, clinical examination and results of rapid test, dogs (n=30) were divided as follow, clinically healthy dogs (n=9) (apparently healthy group) had no signs of HGE. CPV infected group (n=21), dogs manifested signs of HGE including; sudden onset of acute bloody vomiting, bloody diarrhoea, dehydration, anorexia, weight loss, depression, fever, congested mucous membranes (Table, 1), and their fecal swaps gave positive results to CPV antigen rapid test. (Fig., 1) Table (1): Clinical examination of apparently healthy and CPV infected groups (mean values ± SD) Parameters Apparently healthy group CPV infected group Respiratory rate (/min.) 21.00± ±1.18 * Pulse rate 79.60± ±2.83 * (/min.) Rectal 38.43± ±0.13 * temperature ( C) Lymph nods normal normal Mucous Rosy red congested membranes color CPV: represents canine parvovirus. *Significant difference at P value 0.05 Figure (1): Haemorrhagic gastroenteritis affected dogs a. Haemorrhagic gastroenteritis (HGE) affected dogs showed bloody diarrhoea. b. Fecal swabs from HGE affected dogs showed positive result to Rapid CPV Ag Test Kit. Hematological Results In comparison to the mean values of apparently healthy dogs, erythrogram of CPV infected dogs reflected the presence of microcytic hypochromic anemia. This anemia manifested by the significant decreases in mean values of PCV%, Hb concentration, RBCs count, MCV and MCHC. Leukogram of CPV infected dogs revealed the presence of significant leukopenia due to neutropenia and lymphopenia, in addition to platelets count of this group showed significant thrombocytopenia (Table, 2). Table (2): Hematological parameters of apparently healthy and CPV infected groups (mean values ± SD) Parameters Apparently healthy group CPV infected group PCV (%) 41.00± ±1.02 * Hb (g/dl) 13.40± ±0.29 * RBCs ( 10 6 /μl) 5.80± ±1.11 * MCV (fl) 70.67± ±0.71 * MCHC (g%) 32.68± ±0.49 * TLC ( 10 3 /μl) 8.73± ±2.17 * Neutrophil ( 10 3 /μl) 5.83± ±1.87 * Lymphocyte ( 10 3 /μl) 2.66± ±0.79 * Monocyte ( 10 3 /μl) 0.17± ±0.03 * Eosinophil ( 10 3 /μl) 0.29± ±0.02 * Platelets ( 10 3 /μl) ± ±9.10 * CPV: represents canine parvovirus. *Significant difference at P value Bioscience Research, 2017 volume 14(4):

4 Table (3): Biochemical parameters of apparently healthy and CPV infected groups (mean values ± SD) Parameters Apparently healthy group CPV infected group Total proteins (g/dl) 8.01± ±0.08 * Albumin (g/dl) 3.33± ±0.59 * Globulins (g/dl) 4.68± ±0.61 * A/G 0.71± ±0.20 * Calcium (mg/dl) 8.92± ±1.01 * Potassium (meq/l) 4.05± ±0.49 * Sodium (meq/l) ± ±11.32 * CRP (mg/dl) 0.60± ±0.04 * CPV: represents canine parvovirus. A/G: represents albumin/globulin ratio. CRP: represents C-reactive protein. *Significant difference at P value Biochemical Results Compared to the apparently healthy group, biochemical analysis of CPV infected group revealed hypoproteinemia, hypoalbuminemia, hypocalcemia, hypokalemia, hyponatremia, and significant increase in CRP concentration. (Table, 3) DISCUSSION Hemorrhagic gastroenteritis (HGE) is a common disease of multiple etiological agents seen in all dog s breed and age. Canine parvovirus (CPV-2) is one of the causative agents responsible for HGE. CPV-2 spreads rapidly between dogs either by direct transmission through the fecal-oral route or indirect transmission through the oro-nasal exposure to fomites contaminated by feces. HGE is primarily diagnosed by case history, physical examination and secondly by hematological and biochemical examinations. In the present study, CPV infected group showed signs of HGE including; bloody vomiting, bloody diarrhoea, dehydration, anorexia, weight loss and depression in addition to fever which is the characteristic finding of parvovirus infection. Parvovirus replicates in rapidly dividing cells as cells of GIT. Therefore, intestinal cells are affected by the virus, which results in the observed diarrhoea and vomiting (Judge, 2015). Most of the infected dogs showed dehydration of mild degree as a result of vomiting and diarrhoea which associated with large fluid and protein losses through the GIT, and most of these dogs had lower PCV% values than those of the apparently healthy dogs. Same results were obtained by Biswas et al. (2005). In contrast to the results of the present study, Burrows (1977) reported an elevation in PCV% secondary to fluid loss, and O'Sullivan et al. (1984) reported an initial increase in PCV% with the onset of vomiting and diarrhoea which subsequently decreased below the normal values with the disease progression. It is clinically known that, both hematological and biochemical parameters can provide useful information for diagnosis, differential diagnosis, response to treatment, and prognosis (Decaro and Buonavoglia 2012; Castro et al, 2013). Decreased PCV%, Hb concentration, RBCs count, MCV and MCHC confirmed the presence of anemia in dogs suffered from HGE (Arslan et al, 2017). In the present study, microcytic hypochromic anemia was observed in CPV infected group, it might be resulted from the losses of blood in vomits and stool or might be resulted from the direct effect of parvovirus on the bone marrow resulting in decreasing of erythropoiesis (Boosinger et al, 1982; Judge, 2015). CPV infected group revealed the presence of significant leukopenia. This leukopenia based on the characteristic pathogenetic fact of CPV infection which is CPV induced destruction of rapidly dividing cells including thymus, lymph nodes, and bone marrow precursor cells (Decaro et al, 2005; McCaw and Hoskins, 2006). Sharma et al. (2008) and Judge,(2015) reported that, replication of CPV in rapidly multiplying bone marrow and lymphatic tissue may destroy the active mitotic myeloid and lymphoid precursor cells resulting in leukopenia. The present leukopenia was due to neutropenia and/or lymphopenia, and it is the remarkable hematological abnormality associated with CPV Bioscience Research, 2017 volume 14(4):

5 infection as a result of; the destruction of hematopoietic precursor cells within the bone marrow and other lymphoproliferative tissue (Tefft, 2014; Judge, 2015), the depletion of lymphoid tissues, and the increased demands of the massively inflamed GIT (Goddard and Leisewitz 2010; Mylonakis et al, 2016). Platelets are principally involved in homeostasis, inflammation, tissue regeneration and other physiological and pathological processes (Tanju et al, 2014), and they are important in a variety of pathological conditions in dogs (Moritz et al, 2005; Arslan et al, 2017). In the present study, the observed thrombocytopenia in CPV infected dogs could be resulted from the loss of blood through vomits and feces, or from decreasing platelets production as a direct adverse effect of parvovirus on bone marrow precursor cells (Decaro et al, 2005; McCaw and Hoskins, 2006; Judge, 2015). Same hematological findings of CPV infected group including anemia, leukopenia and thrombocytopenia were recorded by Ramprabhu et al. (2002); Goddard et al. (2008); Sharma et al. (2008); Kalli et al, (2010); Sykes, (2014) and Mylonakis et al. (2016). The recorded biochemical changes including hypoproteinemia, hypoalbuminemia, hypocalcemia, hypokalemia, and hyponatremia in CPV infected dogs (Table, 3), were resulted from the observed anorexia, vomiting, and diarrhoea which lead to the losses of fluid and electrolytes (Nappert et al, 2002). Additionally, these changes might be resulted from the infection with CPV which was documented by the detection of its antigen in fecal samples. Also, the infection of dogs with CPV results in destruction of intestinal villi and disruption of gastrointestinal mucosal barrier which often develop a severe proteinlosing enteropathy which associated with the above-mentioned changes (Tefft, 2014). Same biochemical findings of CPV infected group were obtained by Kalli et al, (2010); Li and Humm (2015) and Mylonakis et al, (2016). Acute phase proteins including C-reactive protein (CRP), are considered a sensitive biomarker of inflammation. CRP is considered a major positive acute phase protein that its concentration increases with inflammation (Kocaturk et al, 2010; McClure et al, 2013; Tefft, 2014). CRP is recently used in the diagnosis of parvovirus enteritis (Kocaturk et al, 2010; Kocaturk et al, 2015). In the present study, there is a significant increase in CRP concentration in CPV infected group as a result of inflammation of the GIT. CONCLUSION Depending on the results of clinical examination, hematological abnormalities, biochemical findings and rapid detection of CPV antigen in feces, diagnosis of CPV infection might be confirmed. CONFLICT OF INTEREST The authors declared that present study was performed in absence of any conflict of interest. AUTHOR CONTRIBUTIONS AA and WM designed the experiment. AA wrote the manuscript. SA and WM performed clinical examination of animals. AA performed hematological and biochemical examinations. AA, SA and WM analysed the data. All authors interpreted the data, revised and approved the final version. Copyrights: author (s). This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author(s) and source are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. REFERENCES Arslan HH, Guzel M, Meral Y, Dalgin D, Gokalp G and Ozcan U, A new approach to blood parameters in dogs with hemorrhagic enteritis. Acta Scientiae Veterinariae 45: Barrios M, Luya ML, Reyna A, Lorenzo M and Action L, Isolation of parvovirus from a dog with haemorrhagic gastroenteritis in cuba. Revista- Cubana-Le- Ciencias- Veterinarians. 20: Biggs HG and Moorhead WR, Clin Chem, 20: Biswas S, Chakravorty D and Pradhan NR, Clinical and haemato-biochemical changes in parvovirus infection in dogs. Indian J Vet Med 25: Boosinger TR, Rebar AH, DeNicola DB and Boon GD, Bone marrow alterations associated with canine parvoviral enteritis. Vet Pathol 19: Bioscience Research, 2017 volume 14(4):

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