Indian Journal of Animal Research

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Haemato-biochemical Alterations in Canine Rotaviral Enteritis of Dogs and its Management

Chayanika Mazumder1,*, Gauranga Mahato1, Dullav Prasad Bora2, Deepa Lahkar1, Sayed Abdul Arif3, Bhaben Chandra Baishya3
1Department of Veterinary Epidemiology and Preventive Medicine, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati-781 022, Assam, India.
2Department of Veterinary Microbiology, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati-781 022, Assam, India.
3Department of Veterinary Clinical Medicine Ethics and Jurisprudence, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati-781 022, Assam, India.

ABSTRACT

Background: Rotaviruses cause acute diarrhoea, mostly in young puppies. Rotavirus associated gastroenteritis in dog is of great concern not only because of their high morbidity and mortality, but became of the reason that, they act as potential source of zoonotic infection and inter-species transmission. There is no report on rotavirus infection in dog from Assam. Therefore, there is a need to study the haemato-biochemical alterations in rotavirus infected dogs while treating such type of clinical cases. 

Methods: A total of 157 faecal and blood samples were collected from the dogs affected with gastroenteritis, registered at Veterinary Clinical Complex, Khanapara, Assam during November, 2020 to April, 2021. Diagnosis of rotavirus was done by RNA-PAGE and RT-PCR. Haematological study of the canine rotavirus (CRV) infected dogs were carried out with automated haematological cell counter and biochemical analysis was done with semi-automated biochemistry analyser using commercial kit. 

Result: Out of 157 samples, 8 samples were found positive for canine rotavirus by RNA-PAGE and 17 samples by RT-PCR. Haemato-biochemical alterations in canine rotavirus infected dogs revealed significant increase in Hb, PCV, TEC, monocyte, lymphocyte, AST, ALT, BUN and creatinine while significant decrease in TLC, neutrophil, total protein, serum sodium, potassium and chloride.

INTRODUCTION

Gastroenteritis is a common clinical problem in puppies and etiological agents of gastroenteritis are often multi-factorial (Nadia et al., 2019). Among the various infectious agents, rotavirus (RV) is considered as an important enteric pathogen of various animal species and human (Martella et al., 2010). Rotaviruses (RVs) are classified under the family Reoviridae and genus Rotavirus (Broor et al., 2003). It is a non-enveloped virus and consists of 11 segments of double-stranded RNA (dsRNA) genome. Most commonly RV is transmitted by fecal-oral route but airborne or droplet transmission has also been recorded (Ansari et al., 1991). Rotaviruses usually multiplies in the intestinal epithelium where it damages the epithelial cells on the tips of the small intestinal villi resulting from mild to moderate villus atrophy (Mosallanejad et al., 2015). These viruses induce diarrhoea through destruction of enterocyte by stimulating intestinal secretion and activation of enteric nervous system (Crawford et al., 2017). Canine rotaviral enteritis (CRV) usually occurs in sub-clinical to mild forms. Anorexia, abdominal pain, lethargy, hyperthermia, diarrhoea (mucoid, haemorrhagic or non-haemorrhagic) with vomition are the common clinical signs. Rotaviral diarrhoea usually self limiting in nature and infected dog may recover spontaneously within 8-14 days. However, severe enteritis along with secondary bacterial infection may cause death in pups (Fulton et al., 1981). The clinical sign of rotavirus infection is not specific; therefore laboratory diagnosis is essential for immediate treatment and control of the disease. There is no specific treatment for the disease, symptomatic and supportive therapy helps in early recovery from gastroenteritis and dehydration. It is necessary to use antibiotics to controls secondary bacterial infection. Rotavirus is species specific but several reports of zoonotic and interspecies transmission of RV under natural conditions have been recorded (Martella et al., 2010). It is of utmost importance to isolate the canine rotaviral strains circulating in Assam to adopt preventive and control measures. As detail studies on the haemato-biochemical alterations in rotavirus enteritis in dogs have not been reported, hence it was felt necessary to study the changes for the therapeutic management. Supportive and symptomatic treatment is rendered to the infected pups.

MATERIALS AND METHODS

A total of 157 faecal and blood samples were collected from dogs with complaints of gastroenteritis or related symptoms, registered at Veterinary Clinical Complex (VCC), College of Veterinary Science, Assam Agricultural University, Khanapara, during the period November, 2020 to April, 2021. Approximately 2 g of faecal samples were collected aseptically in sterile vials or rectal swabs and stored at -20°C till diagnosis. Canine rotavirus (CRV) suspected faecal samples were screened by RNA-polyacrylamide gel electrophoresis (RNA-PAGE) as per the method of Herring et al., (1982) and by reverse transcription-polymerase chain reaction (RT-PCR) targeting VP6 gene of CRV as per the method mentioned by Ortega et al., (2017). Haematological and biochemical study of the rotavirus infected dogs were carried out on 0 day and 7th days of treatment. About 5 ml of whole blood was collected, out of which, 2 ml was transferred into properly labelled EDTA vacutainer for haematological study and 3 ml in serum separator tube with clot activator for biochemical analysis. Haematological parameters viz. Hb, PCV, TEC, TLC and DLC were analysed by an automated  haematological cell counter (MS 4e, France). For biochemical studies, serum was separated from blood by centrifugation at 3000 rpm for 15 min and was used for estimation of AST, ALT, BUN, creatinine, total protein, serum sodium, potassium and chloride using commercial kit as per manufacturer protocol with the help of semi-automated biochemistry analyzer by spectrophotometric method. Six apparently healthy dogs registered for vaccination were selected as healthy control group to generate normal data for comparison of parameters under study.

Two groups of pups viz. Group A (unaffected healthy control with six animals) and Group B (Nine infected pups were selected). In group B, infected dogs were treated with conventional therapy comprising fluid therapy (NS/RL/DNS) @ 10-20 ml/kg body weight), antiemetic (Ondansetron @ 0.1-0.2 mg/kg body weight), antibiotic (Ceftriaxone @ 25 mg/kg body weight daily for 5 days), proton pump inhibitor (Pantoprazole @ 1 mg/kg body weight), multivitamin/vitamin B complex (Tribivet @ 1.0 to 1.5 ml) and haemostatic (Botropase @ 10 mg/kg body weight) through IV route.
 
Statistical analysis
 
Statistical analysis was performed using SPSS software. Haematological and biochemical parameters were analysed by one-way analysis of variance (ANOVA).

RESULTS AND DISCUSSION

Out of 157 samples tested, 8 samples were found positive for canine rotavirus (CRV) by RNA-PAGE revealed migration pattern of 4:2:3:2 (Fig 1) indicating group A rotavirus and a total of 17 samples were found positive for CRV infection by RT-PCR. In RT-PCR amplified product targeting VP6 gene produced band of 379 bp (Fig 2).

Fig 1: RNA electrophoresis of canine rotavirus RNA (Group A). L1, L2, L3 and L4: Positive faecal samples of dog, L5: Positive control (Human RV), L6: Negative control.



Fig 2: Agarose-gel electrophoresis of RT-PCR product of VP6 region of canine rotavirus. L1: 100 bp ladder, L2: Positive control (Human RV), L3, L4, L5, L6 and L7: Positive faecal samples of dog, L8: Negative control.



Alterations (mean±SE) in haematological parameters of canine rotaviral enteritis (n=9) dogs in pre-treatment and post-treatment group are represented in (Table 1).

Table 1: Haematological alterations (mean±SE) of rotavirus infected dog in pre-treatment and post-treatment group.



The mean value of Hb (P<0.05), TEC (P<0.05) and PCV (P<0.01) were significantly higher in pre-treatment group in comparison to healthy control group. This corroborates the findings of Malik et al., (2014) in piglets with rotavirus diarrhoea; Bhat et al., (2015) in dog with gastroenteritis; Chethan et al., (2017) in rotavirus infected piglets. Increased level of Hb, TEC and PCV values might be due to haemoconcentration or fluid loss associated with severe diarrhoea and vomition episodes (Biswas et al., 2005). Different mechanisms that attributed to the fluid loss in rotaviral diarrhoea are malabsorption secondary to enterocyte destruction, direct inhibition of glucose-mediated sodium absorption by virus-encoded toxin (NSP4), stimulation of the enteric nervous system (ENS) and villus ischaemia (Raming, 2004; Yin et al., 2017). In CRV positive dogs, mean value of TLC and neutrophil were significantly (P<0.01) lower in pre-treatment group than healthy control group. A marked leukopenia along with neutropenia in the study is in agreement with the finding of Green et al., (2003), who has reported neutropenia in rotavirus positive children; Chethan et al., (2017) reported decrease in TLC values along with marked neutropenia in rotavirus infected piglets; Arora et al., (2018) in dog with gastroenteritis; Barua et al., (2018) in rotavirus infected calves. This might be due to result of non bacterial systemic infection caused by rotavirus (Dalgic et al., 2010). Generally, the demand for WBC (particularly neutrophil) in inflamed gastrointestinal tract is more but during infection due to destruction of haemopoietic progenitor cells of various leucocytes types in bone marrow and other lymph proliferative organs and also due to loss of neutrophils through the damaged gastrointestinal tract, causes inadequate supply of leukocytes (specifically neutrophils) in the inflamed gastrointestinal tract (Goddard et al., 2008). The mean value of lymphocyte (P<0.01) and monocyte (P<0.05) were significantly higher in CRV positive dogs in pre-treatment group in comparison to healthy control group. In the study, lymphocytosis was recorded in rotavirus infected dogs and the finding reframes with that of Chethan et al., (2017); Arora et al., (2018); Barua et al., (2018). Lymphocytosis might be due to viral etiology of the disease. Increased level of monocyte in the study was in accordance with the finding of Chethan et al., (2017). The increased level of monocytes might be due to general immune reaction to infection.

Alterations (mean±SE) in biochemical parameters of CRV infected dog in pre-treatment and post-treatment group are presented in (Table 2).

Table 2: Biochemical alterations (mean±SE) of rotavirus infected dog in pre-treatment and post-treatment group.



The mean values of AST, ALT, BUN and creatinine were significantly (P<0.01) higher in pre-treatment group when compared to healthy control group. The increased level of AST, ALT, BUN and creatinine in the study might be due to extra-intestinal spread of the rotaviruses. In previous study, RV has been demonstrated in other organs like lamina propria, Peyer’s patches, mesenteric lymph nodes, liver, lung, kidney and bile duct of children, animals and laboratory animals, which indicated extra-intestinal spread of the virus (Mossel and Ramig, 2003; Alfajaro and Cho, 2014). Increase level of AST was noted in the study which corroborates the findings of Khatib and Khan, (2012) in children with rotavirus diarrhoea; Chethan et al., (2017) in piglets with rotavirus diarrhoea; Arora et al., (2018) in dogs with gastroenteritis. Increase in AST level might be due to involvement of liver (Grigonis et al., 2002a). Hepatic damage caused by viral infections lead to increased levels of enzyme activity in serum. Increase level of ALT is consistent with the previous reports of Chethan et al., (2017), which might be due to hepatic hypoxia secondary to severe hypovolemia or the absorption of toxic substances due to loss of the gut barrier (Shah et al., 2013). Increased level of BUN and creatinine in the study were similar with the findings of Chethan et al., (2017). The increased level of BUN and creatinine are suggestive of pre-renal uremia which might be due to reduced glomerular filtration rate (GFR) because of haemoconcentration, dehydration, decrease tissue perfusion and also due to catabolic breakdown of tissues as a result of fever (Biswas et al., 2005; Bhat et al., 2015). CRV infected dogs showed, significant (P<0.01) lower values of TP in pre-treatment group in comparison to healthy control group. Decrease level of TP in the study was in agreement with the findings of Biswas et al., (2005); Khatib and Khan, (2012); Bhat et al., (2015) and Chethan et al., (2017). Hypoprotenemia might be due to anorexia, leakage of serum protein through damaged capillaries of the villi of intestines and also due to malabsorption through villi. The mean values of sodium (P<0.01), potassium (P<0.01) and chloride (P<0.05) were significantly lower in pre- treatment group comparison to healthy control group. Hyponatremia, hypochloremia and hypokalaemia in the study corroborates with the findings of Bhat et al., (2015) in dog with gastroenteritis; Khatib and Khan, (2012) who has recorded significant decrease of serum sodium and potassium in rotavirus infected children. Whereas, Barua et al., (2018) revealed lower value of serum sodium; higher level of potassium in diarrhoeic calves and significantly higher level of serum chloride in rotavirus positive cases. Hyponatremia might be due to loss of sodium ions through vomition and diarrhoea (Joshi et al., 2012). Hypochloremia might be due to the loss of chloride ions through vomition and loss in the secretion of intestinal fluid during diarrhoea, resulting in intestinal villous atrophy (Burchell et al., 2014). Hypokalaemia might be due to the loss of potassium in the diarrhoeic fluid along with sodium and bicarbonate (Agnihotri et al., 2017).

The dogs after treatment were active, alert and there was improvement in appetite. Haemato-biochemical parameters revealed significant increase in TLC (P<0.01), neutrophil (P<0.01), serum sodium (P<0.05) and potassium (P<0.05) with non-significant (P>0.05) increase in TP and serum chloride in post-treatment group in comparison to pre-treatment group. While, significant decrease in lymphocyte (P<0.01), BUN (P<0.01) and creatinine (P<0.05) with non-significant (P>0.05) decrease in Hb, PCV, TEC, monocyte, eosinophil, AST and ALT values were recorded in post-treatment group in comparison to pre-treatment group. Haemato-biochemical profiles also have reached nearer to the blood profile of healthy control group post 7th day of treatment. Fluid therapy helped in improvement of electrolyte and acid-base imbalances. Balanced isotonic crystalloid solution (lactated Ringer’s) was the fluid of choice for initial restoration of intravascular fluid and rehydration. Lactated Ringer’s solution approximately related to the composition of extracellular fluid which is lost in diarrhoea and vomition episodes (Waterman, 1991). Restoration of Hb, PCV, TEC and TP in post-treatment group was because of hydration improvement with fluid therapy. The values of AST and ALT were restored to normal in post-treatment group. The level of BUN and creatinine decreased to normal after 7th days of treatment indicating increased glomerular filtration rate. In the study, ceftriaxone (third generation cephalosporin) has been used which has broad spectrum activity. Bhat et al., (2015) has mentioned in his study that in vitro sensitivity of associated bacteria responsible for secondary infection was 91.66% (highly sensitive) for ceftriaxone.

CONCLUSION

This study was conducted on dogs infected with rota viral enteritis. CRV positive dogs showed significant increase in Hb, PCV, TEC, monocyte, lymphocyte, AST, ALT, BUN and creatinine with significant decrease in TLC, neutrophil, total protein, serum sodium, potassium and chloride. All the infected dogs recovered and the haemato-biochemical values became almost similar to healthy control group post 7th day treatment. Being the first report of RV detection in dog from Assam, this data will serve as a baseline to conduct further detailed study of RV infection in dogs. The evaluation of haemato-biochemical profiles might be helpful to clinicians while treating such type of clinical cases.

ACKNOWLEDGEMENT

The article is a part of MVSc research work of the first author. The authors gratefully acknowledged all the necessary facilities and helps received from the VCC Khanapara, Assam Agricultural University, Department of Veterinary Epidemiology and Preventive Medicine and Department of Microbiology for providing necessary facilities and support required during research period.     

Conflict of interest

None

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