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Table of Contents
RESEARCH ARTICLE
Year : 2020  |  Volume : 57  |  Issue : 3  |  Page : 226-233

Molecular diagnosis and clinico-hemato-biochemical alterations and oxidant–antioxidant biomarkers in Babesia-infected dogs of Mizoram, India


1 Department of Veterinary Medicine, College of Veterinary Sciences and Animal Husbandry, Central Agricultural University, Selesih, Aizawl, Mizoram, India
2 Department of Veterinary Microbiology, College of Veterinary Sciences and Animal Husbandry, Central Agricultural University, Selesih, Aizawl, Mizoram, India
3 Department of Physiology and Biochemistry, College of Veterinary Sciences and Animal Husbandry, Central Agricultural University, Selesih, Aizawl, Mizoram, India
4 Department of Veterinary Pathology, College of Veterinary Sciences and Animal Husbandry, Central Agricultural University, Selesih, Aizawl, Mizoram, India
5 Department of Animal Reproduction, College of Veterinary Sciences and Animal Husbandry, Central Agricultural University, Selesih, Aizawl, Mizoram, India
6 Department of Breeding and Genetics, College of Veterinary Sciences and Animal Husbandry, Central Agricultural University, Selesih, Aizawl, Mizoram, India

Date of Submission08-Aug-2018
Date of Acceptance24-Oct-2018
Date of Web Publication26-Aug-2021

Correspondence Address:
Dr Kalyan Sarma
Department of Veterinary Medicine, College of Veterinary Sciences and Animal Husbandry, Central Agricultural University, Selesih, Aizawl, Mizoram
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-9062.311775

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  Abstract 

Background & objectives: Babesiosis is a tick transmitted disease, infecting a wide variety of wild and domestic animals, as well as humans. This study was designed to investigate molecular diagnosis and clinic-hemato-biochemical and oxidant/antioxidant status in dogs of Mizoram, India.
Methods: A total 1200 dogs screened for babesiosis during 2017–18 and 53 dogs suspected for babesiosis by clinical signs and were confirmed by molecular diagnosis. Clinical signs were recorded; also blood samples were taken to investigate hematologic changes, serum biochemical variations and oxidative stress biomarkers.
Results: The overall incidence of babesiosis in dogs of Aizawl, Mizoram, India during the study period recorded was 1.25% (15/1200) and 28.3% cases confirmed from 53 suspected dogs (15/53). The most commonly observed clinical signs were fever, emaciation, depression and icterus and lymphadenopathy. Significant reduction in PCV, HB, RBCs, MCHC, total protein, and albumin along with significant increase in MCV, WBCs, monocytes and BUN were the most consistent hemato-biochemical changes. Oxidant/antioxidant assessment showed significant reduction in superoxide dismutase, catalase and total anti-oxidant (TAC) along with significant increase in lipid peroxidase (LPO) activities.
Interpretation & conclusion: The findings of this study demonstrated that the main causative agent of babesiosis in dogs in Mizoram Province is Babesia gibsoni which caused significant alteration of hemato-biochemical and oxidant-antioxidant status in dogs.

Keywords: Babesia spp, dog, PCR, hematological, biochemical, oxidant–antioxidant


How to cite this article:
Gonmei C, Sarma K, Roychoudhury P, Ali M A, Singh D, Prasad H, Ahmed F A, Lalmuanpuii R, Shah N, Singh NS, Choudhury JK. Molecular diagnosis and clinico-hemato-biochemical alterations and oxidant–antioxidant biomarkers in Babesia-infected dogs of Mizoram, India. J Vector Borne Dis 2020;57:226-33

How to cite this URL:
Gonmei C, Sarma K, Roychoudhury P, Ali M A, Singh D, Prasad H, Ahmed F A, Lalmuanpuii R, Shah N, Singh NS, Choudhury JK. Molecular diagnosis and clinico-hemato-biochemical alterations and oxidant–antioxidant biomarkers in Babesia-infected dogs of Mizoram, India. J Vector Borne Dis [serial online] 2020 [cited 2021 Oct 18];57:226-33. Available from: https://www.jvbd.org/text.asp?2020/57/3/226/311775


  Introduction Top


Canine babesiosis is a clinically important and well-known hemoprotozoan parasitic disease of dogs. Newly recognized Babesia with zoonotic potential continues to emerge around the world and causes substantial economic impact on livestock and companion animals especially in the tropics and subtropics[1],[2]. Traditionally, identification of species has been based on host specificity and morphology of the intra-erythrocytic piroplasms. Based on these, canine piroplasms have been originally recognized to belong to two distinct species, Babesia canis and Babesia gibsoni[3]. These parasites infect the erythrocytes of dogs, leading to hemolytic anemia. Infection with B. gibsoni is known to cause more severe clinical signs than infection with large Babesia spp. and may result in multiple organ dysfunction syndromes[4]. Petra et al[5] reviewed the patho-physiology of canine babesiosis and found that molecular mediators of multiple organ dysfunction including cytokine, nitric oxide and free oxygen radicals, are generated by the host itself in response to parasite. There is strong evidence of the role played by erythrocytic peroxidation in pathogenesis of several hemoparastic infections[6]. Infection with B. gibsoni can result in severe clinical manifestations in some dogs, while others only exhibit mild clinical signs and minimal hematologic or biochemical abnormalities[7].

Accurate and correct diagnosis of Babesia infections plays an important role in monitoring, management and control[8]. Today, polymerase chain reaction (PCR) is being utilized to diagnose and distinguish between the different infections caused by various Babesia subspecies[9],[10],[11].

The North Eastern Region (NER) of India comprising of eight states viz. Arunachal Pradesh, Assam, Manipur, Meghalaya, Mizoram, Nagaland, Tripura and Sikkim falls under the high rainfall zone and the climate ranges from subtropical to alpine. India’s dog population is estimated to be over 25 million and 80% of this population is either partially restricted or community (stray), or feral (unrestricted) dogs[12]. The NER is bordered by three countries viz. China, Myanmar and Bangladesh. A porous international border make this area a high-risk area for outbreaks of emerging and new diseases[13]. Therefore, there is every possibility of disease transmission from these countries. Dog, a multipurpose highly demanding companion animal in the present day society suffers from several vector borne diseases caused by bacterial, viral and parasitic pathogens. Majority of these infections in the changing environment have attained emerging/ re-emerging and zoonotic status[14] due to rapid extension of their geographic distribution through travelling pets, working dogs owned by defense organizations and unabated dog trading at national and international levels[15],[16]. This has warranted monitoring and surveillance of diseases for better information to veterinarians and pet owners about their prevalence, risk of spread and public health importance. Despite the economic importance of these diseases, information on their epidemiology in many countries, including India, is either inadequate or completely absent. In North Eastern Region, in particular the state of Mizoram, no such base line is available. A PCR-based study will provide detail information on the prevalence and incidence of babesiosis in dogs reared in Mizoram state of NER. Such information is a necessary prerequisite for designing appropriate strategies for control of the disease. So, the present study was taken up for molecular diagnosis of babesiosis in dogs of Mizoram.


  Material and Methods Top


The study was conducted at Teaching Veterinary Clinical Complex (T.V.C.C), College of Veterinary Sciences and Animal Husbandry, Central Agricultural University, Selesih, Aizawl, Mizoram, India and State Veterinary Dispensary, Khatla, Aizawl, Mizoram, India. A total of 1200 dogs were examined and age, sex, breed and history were recorded over a period of 12 months from June 2017 to May 2018. The selection was done on the basis of medical history and co-relating clinical findings, which included lethargy, depression, dehydration, anorexia, weight loss, fever, lameness, hemorrhages, pale mucous membrane, lymphadenomegaly, gastrointestinal alterations, jaundice, dermatological or ocular abnormalities, anemia and history of tick infestation.

Ethical statement

The study does not involve ethical issues.

Clinical Examinations

Detail history of the case was recorded including age, sex, breed and duration of illness. Each dog was subjected to detailed clinical examination.

Collection of samples

5 ml blood was collected from cephalic vein aseptically, from which 2 ml of blood was transferred in vial with anticoagulant (K3 EDTA- 1ml/10ml blood) for hematological and molecular studies and 3 ml blood was transferred in sterile Clot Activator vial for separation of the serum for biochemical and oxidative stress studies.

Preparation of smears for microscopic examination

For incidence study, blood smears were prepared on clean grease free glass slides. The smears were stained with Giemsa stain as per standard procedure and examined under oil immersion objective and the results obtained were compared with PCR assay.

Genomic DNA extraction, amplification and sequencing DNA extraction

The DNA from each blood sample was extracted from 200 μl of anticoagulated whole blood using the commercially available DNA extraction Kit (DNeasy Blood and Tissue kits, Qiagen™ Germany), according to the manufacturer’s instructions with minor modifications. The DNA concentrations for all the samples (ranged between 230–540 ng/μl) and purity (A 260 nm/ A 280 nm) were determined by spectrophotometry (NanoDrop® ND- 2000 UV/Vis Spectrophotometer, Thermo Fisher Scientific Inc., Wilmington, Delaware, USA). The extracted DNA was eluted in 100 μL elution buffer and stored at -20°C until further analysis.

DNA amplification and sequencing

Molecular detection of Babesia species was performed by a conventional PCR assay with the primer, Piroplasmid-F(5′-CCAGCAGCCGCGGTAATTC CTTTC-3′) and Piroplasmid-R (5′-CTTTCGCAG TAGTTYGT-CTTTAACAAATCT-3′) to amplify an approximately 400 bp region of 18S rRNA gene, described by Tabar et al.[17]. DNA extracted from a dog infected with B. gibsoni confirmed by PCR and sequencing was used as positive control.

The PCR master mix (Thermo Scientific™, USA, Catalog number: K0171) consisted of 10 X PCR Buffer (μl), 10mM dNTP (1 μl), forward primer and reverse primer @20pmol concentration (0.5 μl) each, Taq DNA Polymerase (Promega, Madison, WI, USA) (5U/μl) (0.2 μl, DNA template (500 ng) and the final volume was made up to 25 μl by nuclease free water (NFW). The cycling conditions were as follows: an initial denaturation step at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 30 sec, annealing at 51.6°C for 30 sec, and extension at 72°C for 30 sec, and a final extension was done at 72 °C for 5 min followed by a hold step at 4°C. PCR products were electrophoresed on 1.5% agarose gels stained with ethidium bromide and evaluated under UV light for the size of amplified fragments by comparison to a 100 bp DNA molecular weight marker.

Sequencing

PCR positive amplicons, obtained from dogs naturally infected with Babesia spp. were purified with a PCR purification kit (Qiagen, Hilden, Germany) and sequenced at DNA sequencing facility, Department of Biochemistry, University of Delhi, New Delhi, India.

Evaluation of hematological parameters

Hematological parameters including total erythrocyte count (TEC) hemoglobin (Hb), packed cell volume (PCV), total leucocyte count (TLC), differential leucocyte count (DLC), erythrocytic Indices viz. MCV, MCH, MCHC and platelet count (× 103μl), were evaluated by semi-automated blood analyzer (Melet Schloesing4e, France).

Evaluation of serum biochemical parameters

Biochemical parameters viz. blood urea nitrogen, creatinine, total protein, albumin, total bilirubin, direct bilirubin, indirect bilirubin, globulin, alanine amino transferase, alkaline phosphatase were determined by automated biochemical analyzer (automated Fuji Drichem 4000i biochemistry analyzer, Japan).

Evaluation of oxidant-antioxidant indices

Oxidant-antioxidant indices including lipid peroxidase (LPO), superoxide dismutase (SOD), Total antioxidant (TAO) and Glutathione (GSH) were analyzed by using commercial kit (Cayman Chemical, USA, cat. No. 705002, 706002, 709001 and 703002 respectively) as per manufacturer’s instruction.

Statistical analysis

All the data were analyzed by using Independent t-test by Statistical Package SPSS 16 (SPSS, Science, Chicago, USA).


  Results Top


The overall incidence of babesiosis in dogs of Aizawl, Mizoram during the study period recorded was 1.25% (15/1200) and 28.3% cases confirmed from 53 suspected dogs (15/53). Cytological studies revealed that 15.09 % cases (8/53) were Babesia sp which were identified by the morphology of merozoites such as pear-shaped, round, oval, pleomorphic or signet ring-shaped under oil immersion objective (magnification 100×) whereas 15 numbers of dogs infected with babesiosis out of 53 suspected dogs were confirmed by DNA extraction with PCR [Table 1] and [Figure 1] which were further confirmed by sequencing. DNA sequences were compared for similarity with sequences available in GenBank®, using the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/). The species identities found were determined according to the closest BLAST match with an identity of 97–100 % to an existing GenBank® accession. By BLAST analysis, the sequences obtained from the Babesia-PCR showed that 11 samples out of 15 were positive for Babesia gibsoni, which were 100% identical with available sequences of B. gibsonii (AB478321.1) and other four samples were 99% identical with B. gibsoni (KY021187.1; MF140997.1; KY433318.1 and KP666168.1). The sequence of the PCR product of positive samples of study area were annotated and submitted to NCBI Gen Bank and MH620356 accession number was obtained.
Figure 1: Gel electrophoresis showing amplification of Babesia spp. A. Lane-1: GeneRuler 100 bp Ladder, Lane-2: negative control, Lane-3: positive control, Lanes- 4 & 9: positive samples. B. Lane-7: GeneRuler 100 bp Ladder, Lane-6: positive control, Lane-5: negative control, Lanes-1 to 4 and Lanes-8 to 12 showing positive amplification of Babesia spp.

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Table 1: Screening of dogs for Canine babesiosis by blood smear examination and PCR

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In the present study physical examination of the dogs suffering from babesiosis revealed an increased rectal temperature (>102.4°F) and respiration rate (>35/min) in 73.33 % (11/15) of the cases, while 26.67 % (4/15) of the dogs were within normal range of temperature (99.5–102.4°F) and respiration rates (10-35/min). Clinical manifestation of naturally occurring cases of canine babesiosis was recorded [Figure 1] as high rise of temperature (11/15, 73.33 %), respiratory signs (11/15, 73.33 %), anorexia (8/15, 53.33 %), dehydration (8/15, 53.33 %), lymphadenopathy (8/15, 53.33 %), pale mucous membrane/ anemia (7/15, 46.67 %), nervous signs/staggering gait (4/15, 26.67 %), ocular signs and ascites/edema (3/15, 20 %), vomiting and diarrhea, tick infestation and epistaxis (2/15, 13.33 %) and melena and haemorrhage (1/15, 6.67 %) with an overall mean clinical score was 4.53± 0.84 with an individual score varying from 1 to 11. USG revealed 13.33% (2/15) hypo echogenicity of liver and 6.66% (1/15) each case showed splenomegaly, hepatosplenomegaly and ascites [Figure 3].
Figure 2: Clinical manifestation in babesiosis of dogs of Mizoram.

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Figure 3: Results of ultrasonographic examination A. Hyperechoic liver parenchyma with irregular edges indicating hepatomegaly B. Rounding of the spleen margin & non uniform echogenec-ity indicating splenomegaly C. Abdominal organs are floating freely in the ascitic fluid.

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The values of hemoglobin concentration (8.44±0.31 g/dl), total erythrocyte count (3.60±0.05 × 106/μl) and PCV value (26.17±0.86 %) were significantly (p<0.01) decreased in Babesia infected dogs as compared to the healthy group. Similarly, the Thrombocyte count was significantly (p<0.01) decreased in Babesia infected dogs (158.47±9.96 × 103/μl) as compared to the healthy dogs (323.50±31.88 × 103/μl) [Table 2]. The MCHC values of Babesia infected dogs (32.00±0.37 g/dl) was significantly (p<0.01) decreased than healthy dogs (34.95±0.98 g/dl). The percent of lymphocyte and monocyte count were significantly (p<0.05) decreased in Babesia infected dogs (15.38±1.21 % & 2.86±0.33 %) as compared to healthy dogs (22.17±2.40 % & 6.17±1.76 %.) whereas the granu-locyte count was significantly (p<0.01) increase in Babesia infected dogs (82.31±1.32%) as compared to healthy dogs (70.17±2.55 %) in the present study.
Table 2: Hematological status in babesiosis infected dogs

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The present study, total protein, albumin and globulin (5.05±0.05 g/dl, 1.81±0.05 g/dl and 3.25±0.03 g/dl respectively) were significantly decreased in infected dogs as compared to healthy dogs [Table 3]. However, there was a significant (p<0.01) increase in enzymes ALT and ALP in Babesia infected dogs (62.60±1.93 U/L and 122.40±3.95 U/L respectively) as compared to healthy dogs. On the other hand, total bilirubin, direct bilirubin and indirect bilirubin of infected dogs did not differ significantly from the healthy dogs [Table 3]. There was no significant difference of serum blood urea nitrogen (11.35±1.34 mg/dl) and creatinine (1.37±0.09 mg/dl) level of babesiosis infected dogs and healthy dogs (8.20±2.47 mg/dl and 1.22±0.05 mg/dl, respectively).
Table 3: Biochemical status in babesiosis infected dogs

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The Mean±SE value of total antioxidant status (TAS), SOD and GSH ((0.92±0.02 mM, 0.39±0.01 U/ml and 0.48±0.01 μM, respectively) was significantly (p<0.01) decreased in Babesia infected dogs whereas the level of LPO (0.08±0.01 nmol) was significantly (p<0.01) increased as compared to healthy dogs [Figure 4]
Figure 4: Oxidant-antioxidant status in Babesia infected dogs in comparison to healthy dogs.

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  Discussion Top


In India, a variable prevalence of canine babesiosis has been reported in different studies. In the present study the overall incidence of babesiosis in and around Aizawl, Mizoram was observed as 1.25%. Similar findings were also reported by Vatsya et al[18] and Wadhwa et al[19] who reported 1.15% incidence of babesiosis in various part of northern India viz. Uttar Pradesh, Pantnagar, Uttarakhand and Kangra Valley of Himachal Pradesh, respectively. A relatively higher prevalence of 84.9% was reported in southern India[20], 8% in Nagpur[21], and 48.57% was reported at Guwahati[22]. The variation in the prevalence reported by various workers in different parts of India may be attributed to sample size, geographical area, climatic conditions which directly influence the tick population and time of sample collection. To our knowledge, the current study is the first report of molecular detection of B. gibsoni, infection in dogs in Mizoram, India. On the basis of present findings PCR-based assay was able to detect fifteen cases for B. gibsoni. Higher detection of canine babesiosis by PCR-based assays as compared to microscopy as observed in the present study has also been reported by several authors worldwide indicating the higher sensitivity levels of PCR[23],[24]. As far as the detection of B. gibsoni with PCR-based assays is concerned, many studies have been carried out worldwide and the prevalence has been recorded to be ranging from 3.3 to 55%[24],[25]. In the present study, diagnosis of Babesia gibsoni in 11 samples was 100 % identical with B.gibsoni from Japan (AB478321.1) and other 4 samples were 99% identical with B.gibsoni from Czech Republic (KY021187.1), Qatar (MF140997.1), Romania (KY433318.1) and China (KP666168.1). It was indicated that the occurrence and distribution of these pathogens and their respective diseases in animals can be correlated with the geographical dispersion of ticks and other arthropod vectors[26].

Clinical manifestations of naturally occurring cases of canine babesiosis recorded during the study period were also observed by Nalubamba et al[27]. Anorexia and depression were frequently found in dogs with babesiosis and it might be due to rise of temperature. The pathogenesis of neurological symptom in babesiosis is related to parasitized erythrocytes that become sequestrated in the central nervous system microvasculature and to the release of inflammatory mediators and tissue hypoxia, which can lead to neurological signs[28]. Jaundice is one of the most commonly reported complications of canine babesiosis[29]. Ascites in babesiosis might be due to lower plasma colloid osmotic pressure which leads to the formation of edema including ascites; pleural effusion and pitting edema[30]. The large variation in clinical signs seems to be due to a number of factors including differences in the pathogenicity between strains of Babesia, breeds of the dog, concurrent infection with other diseases, and nutritional and immune status of the dog.

The study revealed that the hematological parameters viz. Hb, PCV, TEC, thrombocyte, MCHC, lymphocyte and monocyte were significantly (P<0.01) decreased in comparison to healthy dogs which was in agreement with other reports[27]. Decreased Hb and TEC levels could be due to epistaxis, petechial haemorrhages and intra-vascular haemolysis or due to severe anaemia. The throm-bocytopenia during the infection might be due to platelet sequestration in the spleen or immune mediated platelet destruction and development of disseminated intravascular coagulation[31]. Severe microcytic-hypochromic anemia may have been initiated by antibody-mediated cytotoxic destruction of erythrocytes and/or by auto-antibody directed against components of the membranes of infected and uninfected erythrocytes which has also been reported previously in B. gibsoni infection[32]. Lymphocytopenia associated to thrombocytopenia reveals the severe action of Babesia spp. on the body, the stress factors and the development of hyporegenerative anemia confirming the lymphocytic response of the body[33]. On the other hand, the levels of neutrophils increased significantly (P<0.01) in dogs with babesiosis than healthy dogs. This finding was in agreement with other work[34],[35]. The reason might be due to co-infection.

In the present study, it was observed that there was a significant (p<0.01) increase in enzymes ALT and ALP in Babesia infected dogs as compared to healthy dogs. This finding was in agreement with Reddy et al[36]. Increase in level of ALP was may be due to damage or abnormal function of biliary system[37]. Increased activities of AST and ALT were might be due to escape of these enzymes from the damaged hepatic parenchymal cells with necrosis or altered membrane permeability indicating hepatic dysfunction[19]. Levels of protein and albumin decreased significantly (p<0.01) in dogs with babesiosis than healthy dogs. These results were in accordance with Yadav et al[38]. This might be the consequence of peripheral loss of albumin to edematous inflammatory fluids as a result of increased vascular permeability, blood loss, or decreased protein production due to concurrent mild liver disease. Babesia can cause disruption in liver function that leads to decrease albumin synthesis and consequently affect total protein levels.

In recent years, many studies have focused on the assessment of the potential role of ROS in the pathogenesis of various parasitic infections. They often indicate that infections caused by various parasites are associated with a significant increase in lipid peroxidation[38]. Malondialdehyde is one of the end products of lipid peroxidation and is excreted in the urine, blood, and other body fluids, so it serves as a marker of lipid peroxidation and the presence of oxidative stress[39]. In the present study, LPO concentrations were significantly increased in dogs with babesiosis in comparison to the healthy group. Similarly, increases in LPO have also been reported in research conducted by other groups on dogs suffering from B. canis[37] and B. gibsoni[40]. This increase in LPO concentrations might be an indicator that oxidative stress plays a role in the pathogenesis of babesiosis in dog. SOD plays an integral part in RBCs protection system against oxidative damage[41]. In this study SOD activity showed a significant decrease compared to control data. Despite several studies in agreement with our results of a negative correlation of antioxidant enzyme activities of RBCs with the level of parasitemia and also a significantly higher activity of those enzymes in uninfected animals[42]. Chaudhuri et al[40] reported that SOD and catalase activities exhibit a significant rise in dogs naturally infected with B. gibsoni. They concluded that the increased level of SOD during parasitemia could be due to the high percentage of reticulocytes in the infected dogs, since the activity of enzyme is higher in reticulocytes than in mature erythrocytes[43]. In contrast, in this study, the significantly decreased level of catalase activity in the infected group compared to controls, its substantial positive correlation with the activity of enzymes SOD, and also, the decrease in the activity of these enzymes with an increase in the level of parasitemia indicated that all of these antioxidant agents could act as determinative factors. Reduction in the level of TAS in the infected sheep may probably be ascribed to the consumption of antioxidant enzymes as free radical scavengers during the oxidative process in natural B. gibsoni infection in dogs.


  Conclusion Top


In the present investigation, oxidant/antioxidant imbalance along with the synchronized alterations in hema-to-biochemical was detected in Babesia gibsoni infected dogs. It can be concluded that oxidative shocks to RBCs may play an imperative role in the pathogenesis of anemia in canine babesiosis caused by B. gibsoni.

Conflict of interest: None


  Acknowledgments Top


The authors are thankful to Dean of College of Veterinary Sciences and Animal Husbandry, Central Agricultural University, Selesih, Aizawl, Mizoram, India for providing necessary support required during study.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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Abstract
Introduction
Material and Methods
Results
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