Molecular prevalence and phylogenetic traits of Babesia bigemina in cattle (Bos taurus) from north Gujarat of India

Introduction

A tick-borne illness that affects both domestic and wild mammals is babesiosis, which is mainly caused by Babesia spp. Cattle tick Rhipicephalus microplus is the main vector of bovine babesiosis, which causes severe economic losses (Suarez and Noh, 2011; Azhar et al., 2023) [5,22] . It causes fever, anaemia, haemoglobinuria, jaundice, loss of appetite, trembling in the muscles, and even death. Cattle in India are frequently infected with Babesia bigemina, which can lead to severe symptoms such as fever, elevated heart and respiration rates, decreased appetite and haemolytic anaemia. In untreated cases, the disease can result up to 30% of deaths (Kumar et al., 2016) [14] . Low parasite counts and the limitations of conventional microscopic techniques make it difficult to detect carrier and subclinical infections (Sharma et al., 2016) [21] . Preventing the risk of spread of disease need early identification and treatment for which the best and most accurate techniques for identifying these infections are molecular techniques, like nucleic acid amplification (Bose et al., 1995) [6] . This study was aimed to investigate the Babesia spp. of cattle in conventional as well as molecular techniques.

Material and Methods

Ethical approval:

Blood collection

Random blood samples were collected from various farms, Gausalas, Panjrapoles, and veterinary clinics of Banaskatha district of North Gujarat. Blood were collected in 5 ml EDTA vacutainer from total 685 cattle of which 298 were Kankrej cattle and 387 were HF crossbred cattle. Before blood collection, cattle were thoroughly examined for tick infestations, disease indicators, and clinical symptoms.

Parasitological method

From the collected blood samples, thin blood smears were made, air dried, fixed in methanol for 5 min and stained with working dilution of 10% Giemsa stain for 30-45 min and washed with tap water, air dried and examined under oil immersion for demonstration of Babesia piroplasms, if any.

DNA Extraction and Sequencing

With a few minor adjustments, genomic DNA was extracted from 200 μl of cow blood using the QIAamp® DNA Blood Mini Kit (Thermo Scientific, Lithuania; Qiagen, Germany) according to the manufacturer's instructions. With a ratio of 1.7 to 2, the optical densities at 260 and 280 nm were compared to determine the concentration and purity of the extracted DNA using a spectrophotometer (Eppendorf, Hamburg, Germany). The extracted genomic DNA was then stored at -20°C for further analysis Genus and species-specific PCR targeting the First denaturation at 94°C for 5 minutes was followed by 35 cycles of denaturation at 94°C for 45 seconds. Annealing was done at 57°C for 45 seconds (for B. bigemina) or 55°C for 30 seconds (for B. bovis) and extension at 72° C for 45 seconds and the final extension was done at 72°C for 4 minutes for 35 cycles. To determine the expected size of amplicons specific to the targeted organism, amplified DNA fragments were subjected to electrophoresis on a 1.2% agarose gel and visualised using a gel documentation system (Life Technologies, USA). To confirm specific amplification, the amplified DNA sequences of Babesia bigemina were sent to Eurofins Genomics India Pvt. Ltd., Bengaluru, for bi-directional Sanger sequencing. The 278 bp Babesia bigemina product's identity was confirmed through the nBLAST tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi). A 100 bp DNA ladder was used to run 2 µl of the PCR result on a 1% agarose gel in order to verify integrity. 10 µl of each primer and 28 µl of the PCR product were sent for sequencing. The sequences were compared to those in GenBank using the BLASTn tool (NCBI, USA), MEGA X, and MegaAlign (DNAstar, USA) for pairwise and phylogenetic distance studies.

Results and Discussion

Overall Prevalence of Babesia spp. Out of 685 total blood samples, 127 (18.54%) showed positive results for Babesia spp. using Giemsa staining, whereas 183 (26.71%) showed positive results using PCR, indicating higher sensitivity of PCR than the gold standard microscopy. Although Kumar et al., 2016 [14] [13,17,18,19,26] . Variations in environmental conditions, animal populations, disease control tactics, sample techniques, and management practices contributed to the increased prevalence in our study. Additionally, the abundance of the vector R. microplus may also be a role. [23] , who found that prevalence rates in cattle were 21.3% by microscopy and 32.4% by PCR. This greater prevalence of babesiosis in crossbred as compared to indigenous cattle was also found by Kaur et al., 2021; Al-Shammari et al., 2024 [3,12] . This discrepancy might be due to genetic differences that lack resistance genes crossbred cattle which increases the susceptibility of tick infestations. These results may be explained by variables including temperature, ecology, tick species, sample gathering techniques, high tick populations, and warm, humid environments that encourage the growth of ticks and piroplasm.

Fig [27] likewise found that older animals had greater prevalence rates, with adults having the highest rates. The prevalence was lowest in the physiologically non-active (not mating, not pregnant, not breastfeeding) group and highest in the immature group, (Ola-Fadunsin et al., 2021) [20] . Babesiosis is more common in older animals; adult lactating cattle are especially susceptible, probably as a result of prolactin hormone and genetic factors (Farooqi et al., 2017) [8] . [11] and a robust innate immunological response (Zintl et al., 2005) [29] . They continue to be vulnerable as their immune systems develop and their passive immunity diminishes (Zia et al., 2019) [28] . Due to immunological memory and higher reactions to re-exposure, older animals that have been exposed to the parasite for a longer period of time have stronger immune defences (Zia et al., 2019) [28] .

Season wise Babesia bigemina infection in cattle

In this study, summer showed the highest prevalence of babesiosis, followed by monsoon and the lowest prevalence was found in the winter in both microscopy and PCR. Microscopic diagnosis revealed the summer prevalence with 66.67% in Kankrej and 70.21% in HF Crossbred, 27.27% and 19.94% were found in monsoon and 6.06% and 10.63% in the winter. PCR technique revealed the higher summer prevalence with 51.72% and 60.8%, 32.76%, 32.76% and 27.2% in the monsoon and 15.51% and 12.00% in the winter. [1,2,7,24,27] where summer showed the highest prevalence ranging from 25% to 50%. This is because of the suitable higher environmental temperature for tick reproduction in summer which increases the incidence. , 2024 [17,18] with a prevalence of 33.62% in Kashmir, and Tariq et al., 2024 [23] with a prevalence of 32.4% in Pakistan. Tick susceptibility may be related to the higher occurrence in cattle due to their skin texture (Jaimes-Dueñez et al., 2018) [10] . According to Maharana et al., 2019 [15] , Gujarat has a 40.97% prevalence, most likely due to the ideal warm and humid climate and high tick density that promotes tick survival and piroplasm development which in turn increases the positive rates of babesiosis. The evolutionary history was inferred using the Neighbor-Joining method. The optimal tree is shown in Fig. 5. Evolutionary analyses were conducted in MEGA11

Acknowledgement

AcknowledgementsThe authors are highly thankful to the Head of Department of Veterinary Parasitology, Principal, College of Veterinary Science and AH, Sardarkrushinagar, and Higher authorities of Kamdhenu University for providing the necessary facilities to the carry out this study.

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