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Epidemiological Studies on Bovine Tick-Borne Haemoparasitic Diseases in Chennai

Raj kumar Rajupillai Anbu Vijaya Bharathi Mangalanathan Selvaraju Ganapathy Senthil Kumar Alagesan

The present study was aimed to assess the current epidemiological status of bovine tick-borne diseases (TBDs) in and around Chennai by conventional staining method and polymerase chain reaction (PCR). A total of 154 blood smear and whole blood samples were screened by Leishman staining and PCR respectively. PCR assay (50.65%) revealed significantly higher sensitivity in detection of TBDs in clinically suspected cattle than microscopic examination (35.06%). Results showed that Anaplasma spp. (20.78%) was the most prevalent parasite of cattle followed by Theileria spp. (11.69%) and Babesia spp. (2.6%) in Chennai. The infection of Anaplasma spp. (27.77 %), Babesia spp. (5.55 %) and Theileria spp. (11.11 %) were higher in less than 2 year of age group. The prevalence of anaplasmosis was relatively high (21.32%) in cross-bred cattle. However, higher prevalence of babesiosis (5.55%) and theileriosis (22.22%) was noticed in non-descript animals. Statistical analysis using chi-square test indicated a highly significant (P < 0.01) difference between these tests.

Keywords : Epidemiology Tick-borne diseases Cattle Active surveillance PCR Haemoparasites

How to cite: Rajkumar, R., Vijaya Bharathi, M., Selvaraju, G. & Senthil Kumar, A. (2020). Epidemiological Studies on Bovine Tick-Borne Haemoparasitic Diseases in Chennai. International Journal of Livestock Research, 10(2), 36-45. doi: 10.5455/ijlr.20191021101352


Tick-borne pathogens (TBP) are considered to be one of the major hindrances to productivity and health of livestock, globally. The four main pathogens responsible for these losses are the tick-borne protozoa, Babesia and Theileria, and the tick-borne rickettsial disease pathogens, Anaplasma and Ehrlichia. In developing countries like India, TBP spp. can impose considerable economic loss on large and smallholding livestock productivity farming systems: the resulting diseases causing high mortality rates, reduced milk production and loss of body condition (De Castro, 1997). The Theileria species that present in India are T. annulata and T. orientalis and both species are transmitted by Hyalomma anatolicum (Aparna et al., 2011) whereas babesiosis and anaplasmosis are transmitted by Rhipicephalus (Boophilus) microplus. Theileria annulata is widely considered as more pathogenic and associated with greater economic loss (Aparna et al., 2011). In India, bovine babesiosis, is predominantly caused by Babesia bigemina. The incidence of B. bigemina in native, cross-bred cattle and buffaloes has been frequently reported since long (Ghosh and Nagar, 2014). Additionally, B. bovis infection has also been reported in India (Muraleedharan et al., 1984; Add recent reports). Animals that recover from Babesia infection, becomes carriers and parasites go unnoticed on microscopy. Subclinical infections may endure for long periods with infected animals acting as reservoirs (Brown et al., 2006). Cattle that recovered from acute infection of anaplasmosis may develop persistent infection and it is characterized by cyclic low-level rickettsaemia (French et al., 1998). The pathogenic Anaplasma marginale has been reported in multiple states of India, including Odisha, Uttar Pradesh, Punjab, Haryana, Tamil Nadu and Karnataka (Ghosh and Nagar, 2014). Diagnosis of the tick-borne diseases (TBDs) can be challenging due to its different phases and multiple clinical manifestations. Traditional diagnostic techniques (peripheral blood smear examination, cytology and serology) are valuable diagnostic tools for TBDs. However, polymerase chain reaction (PCR) is more sensitive than other conventional methods. Keeping the above facts in view, the present study was undertaken to assess the current epidemiological status of tick-borne haemoparasitic diseases in cattle in Chennai, Tamil Nadu, India by conventional staining methods and PCR.

Materials and Methods

The present active surveillance study was conducted at Madras Veterinary College Teaching Hospital and Blue Cross of India, Chennai, Tamil Nadu, India to assess the current epidemiological status of bovine tick-borne diseases viz. anaplasmosis, babesiosis and theileriosis in and around Chennai. Cattle which were infested with ticks and showing clinical signs of fever (> 40°C), pale or icteric conjunctival and vaginal mucous membrane, panting, dark yellow or bloody urine and enlarged lymph nodes were selected for this active surveillance study. A total of 154 peripheral blood smear and whole blood samples were collected from cross bred (136) and non- descript (18) cattle. Of 154 cattle, 18 samples were received from cattle with less than two years of age, 61 from 1 to 3 calving, 67 from 4 to 7 calving and 8 from more than seven calving. The blood smears were subjected to Leishman staining (Kolawole et al., 2014). Whole blood samples (3 ml) from suspected animals were collected in EDTA vials and stored at – 20ºC until further use. The template DNA was extracted directly from fresh and frozen blood sample by the DNeasy® Blood and Tissue Kit (Qiagen, Germany) and the DNA were stored at -20°C until further use. The Oligo nucleotide primers used in this study are listed in Table 1.

Table 1: List of oligonucleotide primers

Primer sequence (5’ – 3’) Product size Reference
MAR1Bb2 primer set for Anaplasma marginale 265 bp Bilgic et al. (2013)
30-kDa gene for Theileria annulata 721 bp d’Oliveria et al. (1995)
N516 F: 5’ – GTA ACC TTT AAA AAC GT – 3’
N517 R: 5’ – GTT ACG AAC ATG GGT TT – 3’
18S rRNA gene for Babesia spp. 339 bp Birkenheuer et al. (2003)
5-22 F: 5’ – GTT GAT CCT GCC AGT AGT – 3’
1661 R: 5’ – AAC CTT GTT ACG ACT TCT C – 3’

The above-mentioned primers in Table 1 were custom synthesized and obtained from Sigma- Aldrich, Bangalore, India were used in this study. PCR analysis was carried out for the detection of blood parasites viz. A. marginale, Babesia spp., and T. annulata targeting MAR1Bb2, 18S rRNA and 30 kDa genes respectively. The PCR was performed in Biorad thermal cycler. All the reactions were carried out in volume of 25 µl in 0.2 ml PCR tubes. The reaction mixture contains 12.5 µl of PCR master mix-2X (Ampliqon®), 10 pmol of each, template DNA (mention the concentration of DNA instead of volume and 5.5 µl distilled water. Conditions of thermal cycling for different oligonucleotide primers used in this study are listed in Table 2.

Table 2: PCR cyclic conditions for tick-borne pathogens

Cycling conditions
Final Extension Extension Annealing Denaturation Initial denaturation Organism
65C 65°C 56°C 94C 94°C A. marginale
10 min 1 min 30 sec 50 sec 5 min
Repeated for 35 cycles
72°C 72°C 55C 94C 94°C T. annulata
7 min 1 min 1 min 1 min 3 min
Repeated for 30 cycles
72°C 72°C 58C 94C 94C Babesia
5 min 45 sec 45 sec 45 sec 5 min
Repeated for 35 cycles

The amplified PCR products were electrophoresed in an ethidium bromide stained 1.5 per cent agarose gel and visualized in a transilluminator under UV light.

Statistical Methods

The results of the both diagnostic tests were assessed statistically as per the procedure of Snedecor and Cochran (1994). The sensitivity and specificity of diagnostic tests were analyzed as per the methods described by Smith (1994). The concordance and Kappa value were calculated as per the methods delineated by Thrusfield (1995).

Results and Discussion

Peripheral Blood Smear

The overall prevalence of bovine TBDs in Chennai was observed as 35.06 per cent which is in agreement with Soundararajan and Rajavelu, (2006) and Chaudhri et al. (2013) who recorded 32.4 and 27.88 per cent of TBDs in Chennai and eastern Haryana respectively. However, high prevalence of TBDs with 43.1 to 76.85 per cent infection in cattle was also on record (Krishnamurthy et al. 2016 in Shimoga of Karnataka; Ananda et al., 2009 in North Bangalore; and Reetha et al., 2012 in Tamilnadu). In contrary, Bhatnagar et al. (2015) found lower positivity (9%) of tick-borne diseases in Southern Rajasthan. The differences in the overall prevalence rate might be due to variation in the tick infestations and relative potential of tick to transmit parasites, geography and climatic conditions.

In the present study, the prevalence of anaplasmosis was recorded as 20.78 (32/154) per cent which is in accordance with Arunkumar and Nagarajan, (2013) and Reetha et al. (2012) who observed 19.3 and 21.73 per cent respectively, whereas Talukdar and Karim, (2001) have found highest positivity (33%) and Krishnamurthy et al. (2016), Bhatnagar et al. (2015) and Ananda et al. (2014) found lower positivity of 2.79, 3.5 and 12.5 per cent respectively. The higher prevalence of anaplasmosis in suspected cross-bred animals indicates the presence of sub-clinical infection or carrier status of this disease. R. (B.) microplus was reported as the commonest tick species in Tamil Nadu (Koshy et al., 1982). The abundance of biting flies (Tabanus spp. and Stomoxys spp.) due to hot and humid climatic conditions prevailing in the state may augment the mechanical transmission to the naive animals. Babesiosis was observed as 2.6 (4/154) per cent in this study which is in accordance with Bhatnagar et al. (2015) and Nair et al. (2011) who noticed 1.41 and 2.66 per cent respectively, whereas Krishnamurthy et al. (2016) and Vetrivel et al. (2017) found highest positivity of 6.9 and 20.27 per cent respectively. Soundararajan and Rajavelu, (2006) recorded lower prevalence of 0.53 per cent. The lower prevalence of babesiosis might be due to sub-clinical or carrier state of the disease, low number of parasites in circulation and seasonal variations.

In this present study, theileriosis was recorded as 11.69 (18/154) per cent which is in concurrence with Krishnamurthy et al. (2016) and Muraleedharan et al. (1994) who observed 12.5 and 17.7 per cent respectively, whereas Rakha and Sharma, (2003), Anandan, (1991), Nair et al. (2011) and Chaudhri et al. (2013) found higher prevalence of 76, 21.1, 43.33 and 22.88 per cent respectively. Bhatnagar et al. (2015) recorded lower prevalence of 3.8 per cent. The difference might be due to geographical location, climatic conditions, sample size and time period of the study.

Polymerase Chain Reaction

The PCR assay revealed the overall positivity of bovine TBDs as 50.65 per cent in suspected cattle in Chennai.

Agarose gel electrophoresis showing PCR amplification of A. marginale
L1 100 bp DNA ladder L 3,4,5,6, 7,8, 9,10,11,12,14,15 Positive samples
L2 Positive control (265 bp) L13 Negative samples
L16 Negative control