Molecular Characterization and Assessment of Hematobiochemical and Oxidative Indices in Dog Naturally Infected with Babesia canis vogeli

Amongst the common tick-borne diseases of canidae, babesiosis is one of the major concerns in tropical countries including India. It is caused by intraerythrocytic apicomplexan protozoa of Babesia genus. Babesia canis occurs as a single pear shaped piroplasm and B. gibsoni often exists in individual ring forms. The large form is genetically characterized into B. canis canis, B. canis vogeli and B. canis rossi (Carret et al., 1999).
       
The destruction of parasitized or non-parasitized erythrocytes causes mild to severe form of anemia (Köster et al., 2015). Along with abnormal hematological parameters, there can be hypoglycaemia, increased serum activity of alanine aminotransferase (ALT), aspartate aminotransferase (AST), serum creatinine and serum urea (Brahma et al., 2019). Oxidative damage to the erythrocytes may contribute to the pathogenesis of anemia in babesiosis (Crnogaj et al., 2010). Malondialdehyde (MDA) is an end product of oxygenation of polyunsaturated fatty acids, thus commonly used as oxidant biomarker for assessing oxidative stress in the cells or tissues. There are several enzyme systems that catalyze reactions to neutralize the reactive oxygen species in the cells. Few of them are superoxide dismutase (SOD) and glutathione reductase (GR)/ glutathione disulfide reductase (GSR), which can be used as an antioxidant biomarker to evaluate the oxidative stress in canine babesiosis (Crnogaj et al., 2017). The constant stress in the Babesia infected dog causes elevated levels of basal and post-adrenocorticotropic hormone stimulated serum cortisol concentrations (Schoeman and Herrtage, 2008).
       
Nevertheless, it warrants a rapid and accurate diagnosis for instituting appropriate therapy and control in dogs in the endemic areas. Nucleic acid-based detection assays like PCR and its variants are the most promising diagnostic tools with high level of sensitivity and specificity (Kushwaha et al., 2018). One of the important gene targets for Babesia detection by PCR is 18S rRNA gene (Passos et al., 2005). It has highly conserved flanking regions, allowing the use of universal primers. Its repetitive arrangement within the genome provides excessive amounts of DNA for PCR (Hillis and Dixon, 1991).
       
Taking all the above facts into consideration, present study was aimed to identify the causative agent of canine babesiosis in South Gujarat, India using PCR and analyzing the phylogeny based on 18S rRNA gene. The level of hematobiochemical parameters, MDA (oxidant biomarker), SOD and GR/GSR (antioxidant biomarker) and plasma cortisol were also evaluated in the dogs on the day of presentation.

Animal/area under study
 
Study was conducted on dogs presented before clinician for the diseases diagnosis and treatment especially with pyrexia, anaemia and tick on body in South Gujarat, India. The work was done at Veterinary College, NAU, Navsari during 2018-2019. Fifteen PCR positive and negative dogs with canine babesiosis were selected as infected and uninfected group, respectively. 
 
Blood collection
 
Three ml blood was collected per dog in aseptic condition, 2 ml transferred in the sterile vial with K3 EDTA and 1 ml was transferred in a plain tube for plasma/serum separation.
 
Cytological examination
 
Ear tip of the dog was pricked to prepare a peripheral blood smear. Giemsa-stained smear was examined for the piroplasm of Babesia under oil immersion lens.
 
PCR examination
 
Genomic DNA was isolated from 100 ìl of the whole blood using DNEasy Blood and Tissue kit (Qiagen, Germany) as per manufacturer protocol. The eluted DNA of OD of 260 nm: 280 nm of 1.7-1.9 was stored at -20^oC. The PCR was performed to amplify the 340 bp of 18S rRNA gene using Babesia genus specific primers pair of 5'-gtcttgtaattggaatgatggtgac-3' and 5'-atgcccccaaccgttcctatta-3' (Birkenheuer et al., 2003). Reaction mixture of 25 μl contain 0.5-1  μg of template DNA, 1  μl (10-15 pmol) of each primer, 12.5 μl of 2x PCR master mix (Qiagen, Germany) and nuclease free water. Amplification condition was initial denaturation 94^oC for 5 min, denaturation at 94^oC for 1 min, annealing at 56^oC for 1 min, extension at 72^oC for 1 min, 40 cycles and final extension at 72^oC for 10 min. Each time the reaction was run with positive/negative/reagent control. All PCR products were electrophoresed through 2% agarose gel containing ethidium bromide and visualized under UV fluorescence.
 
Gene sequencing and phylogenetic analysis
 
The DNA was eluted from the agarose gel using a gel extraction kit (Qiagen, Germany) and nucleotide sequence was determined at the Sequencing Department, Eurofins Genomics, Bengaluru. Sequence data was analysed in GeneTool 1.0 Lite. The 5' and 3' ends of the 18S rRNA gene sequence were initially determined by comparing them with previously published sequences. Sequences were put to further analysis including similarity search in the BLASTN program of NCBI (http://www.ncbi.nlm.nih.gov/BLAST/). Nucleotide sequence of 100% homology was submitted into the NCBI gene bank. Phylogenetic tree was constructed in Neighbor-Joining (NJ) and Maximum Likelihood (ML) method in the MEGA 10 of version 10.1.7. Bootstrap consensus was inferred from 1000 replicates. Nucleotide sequences were of B. gibsoni, B. canis vogeli, B. canis canis, B. canis rossi and B.  microti (as out group).
 
Hemogram and hemato-biochemistry
 
One ml of fresh whole blood was analyzed in blood auto-analyzer (Nihon Kohden, Japan) to ascertain the blood parameters. The serum concentration of glucose, total protein, hepatic enzymes, urea and creatinine were estimated using commercial kits (Randox Laboratory, UK).
 
Oxidant-antioxidant and plasma cortisol estimation
 
The RBC pellet was prepared from fresh blood. It is diluted in ice cold distilled water in 1:10 ratio to make 10% hemolysate. Hemolysate was used in Sigma Aldrich assay kit to measure MDA, SOD and GR. Cortisol level was estimated using Immunotag™ Cortisol ELISA Kit (G-Biosciences, USA).
 
Statistical analysis
 
Statistical analyses were performed using R software, comparing means using one-way ANOVA with Duncan’s multiple range test. P value <0.05 or <0.01 was considered significant.

Giemsa-stained thin blood smear showed Babesia canis in blood plasma (Fig 1a) and inside RBC (Fig 1b). Intra-erythrocytic form many a time had multiplied infection of the Babesia canis parasites (Fig 1b). Out of 239 screened dogs 9 (3.76%) was infected with babesiosis in cytological examination. The PCR had produced a band of 18S rRNA gene of 340 bp (Fig 2). Current nucleotide sequences (South Gujarat isolate) (MW093397 and MW093398) showed 100% homology and 534-538 total score with published 18S rRNA DNA sequences of B. canis vogeli of Asia (KU321851.1) and also with the isolates of Europe, Africa and South America. The PCR had detected 10 more cases (=19 dogs) (7.95%) of canine babesiosis than the cytological examination (P<0.05). Sensitivity and specificity of the PCR was 100% as compared to blood smear examinations in detecting the infection. This finding was in accordance with the finding of researchers in South India (Jain et al., 2018) and North India (Laha et al., 2014). Malaysian scientist, Prakash et al., (2018) used the same gene in the PCR and successfully detected B. canis vogeli in ticks (1.4%) and dogs (2.1%).character-based methods were almost similar with a small difference in bootstrap values (Fig 3). The current sequence had formed a tight cluster with the B. canis vogeli of from different locations (Fig 3). Monophyletic lineage relationships were observed with high bootstrap proportion (98% in NJ and 99% in ML) for current and most of the published nucleotide sequences of B. canis vogeli. South Gujarat isolate revealed the closest similarity with Kerala, India isolates and also with the isolates reported from other countries of Asia, Australia and South America (Fig 3). Furthermore, the phylogram revealed separate clades of B. canis vogeli, B. canis canis, B. canis rossi and B. gibsoni (Fig 3). The same gene was used by Passos et al., (2005) and Solano-Gallego et al., (2008) to confirm the incidence of B. canis vogeli in Brazilian and Italian dogs, respectively.
 

 

 

       
Hemogram showed mild to moderate anemia and erythropenia in most of the infected dogs (Table 1). Infected dog noted decreased HCT value as compared to Babesia free dogs (P<0.05). A low level of RBC count, hematocrit and hemoglobin are an indication of anemia in the infected dogs (Varshney et al., 2003). Anemia might be due to direct mechanical disruption caused by the parasite, along with extra/intra vascular hemolysis and immune/non-immune mediated destruction of RBCs (Salem and Farag, 2014). There was non-significant difference in MCV and MCH value between the two group (normocytic normochromic anemia), but MCHC observed significant decrease in the infected animals (hypochromic microcytic anemia) (Table 1) while Salem and Farag (2014) observed non-significant difference in these parameters. Mean WBC count in infected dogs observed marked (P=0.004) leukocytosis with lymphocytosis and monocytosis. However, there was non-significant (P>0.05) increase in the granulocyte count in the infected dogs. Eosinophilia (P=0.000) and thrombocytosis (P<0.01) were also evident in the infected dogs. There was a corresponding increase in the MPV (P<0.01) and PDW (P>0.05) in the infected than the uninfected dogs. Increased value of RDW% in Babesia infected dogs was due to the formation of large size reticulocytes and young RBCs than mature erythrocytes (regenerative anemia) (Fabisiak et al., 2010).
 

       
Plasma biochemical profile of infected dogs revealed significantly (P<0.01) decreased values of blood glucose, and it was in agreement with Keller et al., (2004) (Table 1). Hypoglycemia can be due to anorexia and impaired hepatic function. Increased level of total protein has (P<0.01) been a most frequently reported feature of canine babesiosis (Hossain et al., 2003). Elevated level of ALT and AST (P<0.01) in the infected dogs was in agreement with previous observation of Furlanello et al., (2005) and it is an indication of hepatic tissue damage. Creatinine level in the infected dogs was significantly higher than the uninfected group. A similar observation was noted by Gonde et al., (2017) but Konto et al., (2014) observed lower levels of creatinine in Babesia infected dogs. Increased level of BUN andcreatinine were the indication of renal degenerative changes during canine babesiosis (Brahma et al., 2019). Consequently, an increase (p<0.05) in the level of serum urea was observed in the infected as compared with the uninfected dogs in the current (Table 1) and Lobetti et al., (2012) study.

In the infected dogs, the activity of MDA and SOD was significantly (P<0.01) increased and decreased, respectively (Table 1). Significantly an increased level of MDA was noted in dogs infected with B. canis canis (Crnogaj et al., 2010, 2017). A significantly reduced activity of SOD (P<0.01) inthe B. canis vogeli infected dogs was noted, this result agrees with the finding of Crnogaj et al., (2017) in B. canis canis infected dogs. A non-significant reduction in the amount of GR was observed in B. canis vogeli infected dogs. Overall, a low amount of antioxidant biomarkers in B. canis vogeli infected dogs, can be due to the consumption of these biomolecules in neutralizing the free radicals produced during infection. Significantly (P<0.01) higher mean cortisol concentration was noted in the B. canis vogeli infected dogs of current study which agree with the finding of Schoeman et al., (2007) in B. canis rossi infected dogs.

Babesia canis vogeli was found to be the dominant organism for canine babesiosis in South Gujarat, India, as evident in 18S rRNA gene-PCR and NCBI-BLAST study. Phylogram of 18S rRNA gene formed a tight cluster of B. canis vogeli originated from India and other parts of the world. Infected dogs measured significantly decreased value of mean hemoglobin concentration, RBC count, blood glucose and SOD, whereas significantly increased values of total protein, serum creatinine, ALT, AST and MDA as compared to the uninfected dogs. Oxidant-antioxidant imbalance in the infected dogs support the notion that infection is associated with oxidative stress and its course may be linked to the resultant anemia.

The authors are thankful to the Vice-Chancellor, Director of Research and Principal, Veterinary College, NAU, Navsari for providing necessary facilities and fund for the research work.