Molecular detection of bovine tropical theileriosis in northern India
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Date
2021-09
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G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand)
Abstract
Considering the economic importance and increasing reports of bovine tropical theileriosis caused by Theileria annulata, from different agro-climatic regions of Northern India, an epidemiological study was studied to determine the state-wise, host-wise, age-wise and season wise prevalence of the disease from July, 2020 to June, 2021 using microscopic thin blood smear examination (TBE) and polymerase chain reaction (PCR) assay. Out of 544 blood samples (383 from cattle and 161 from buffaloes) examined, a total of 158 (29.04%) blood samples were found positive for bovine tropical theileriosis {133 cattle (34.72%) and 25 buffaloes (15.52%)}. State-wise prevalence was found maximum (37.66%) in Haryana (51.06% cattle and 16.66%), followed by 28.63% in Uttarakhand (32.73% cattle and 13.33%), 22.58% in Rajasthan (26.19% cattle and 15% buffaloes) and minimum (21.73%) in Uttar Pradesh (24.05 % in cattle and 16.66% in buffaloes). Overall age-wise prevalence was found maximum (35.22%) in animals of >3 years of age (42.62% in cattle and 18.51% in buffaloes), followed by 27.93% in those between 1-3 years of age (33.33% in cattle and 14% in buffaloes) and minimum (14.85%) in animals <1 year of age (16.90% in cattle and 10% in buffaloes). Seasonal prevalence of bovine tropical theileriosis was found maximum (32.01%) during summer followed by rainy (29.79%) and minimum (19.35%) during winter season. Thin blood smear examination (TBE) for the presence of piroplasm and schizont (KBB) stages, 86 (15.80%) blood samples was positive for theileriosis. KBB was observed in 14 (2.57%), piroplasms in 62 (11.39%) and both KBB and piroplasms in 10 (1.83%). For molecular diagnosis of BTT, allele-specific PCR based on Cytb, β-tubulin and HSP70 gene was conducted. Amplification yielded complete CDS of Cytb, partial HSP70 and β-tubulin gene sequence of length 1092 bp, 275 bp and 450 bp, respectively in all the positive samples. Besides this, nested PCR for T. annulata specific β-tubulin gene was also performed and amplification yielded gene sequence of length 309 bp. The results of the present study showed that PCR assay was 1.83% more sensitive than TBE for large scale epidemiological studies of bovine tropical theileriosis. In order to investigate the genetic variation, Cytb, β-tubulin and HSP70 genes specific PCR products were digested using Alu1, Rsa1 and Taq1 and Alu1 restriction enzymes, respectively. Restriction digestion of Cytb gene specific PCR products generated gene fragments of 365, 225, 207, 105, 98, 82 and 11 bp length. In case of β-tubulin gene, restriction digestion generated four (270bp and 180bp, 286bp and 164bp, 340bp and 110bp, 277bp and 173bp) different patterns. However, in HSP70, Alu1 restriction digestion generated four (143bp, 78bp and 54bp; 107bp, 90bp and 78bp; 182bp, 78bp and 15bp; 125bp, 90bp and 60bp) different patterns and Taq1 restriction digestion generated two (175bp and 110bp, 240bp and 35bp) different patterns. The sequence analysis of all the genes revealed high level of polymorphism when compared with the pre-existing GenBank sequences. All the four isolates of Cytb gene of T. annulata showed four synonymous mutations at codon 78 (Serine), 116 (Threonine), 139 (Leucine), 143 (Phenylalanine) and 290 (Valine) and one non-synonymous mutation at codon 146 (Threonine instead of Alanine). Besides this, Hisar isolate showed a non-synonymous mutation at codon 153 (Alanine instead of Glycine). However, Karnal, Haridwar and Rajasthan isolates showed synonymous mutation at codon 290 (Valine). All the three isolates of β-tubulin gene showed three synonymous mutations at codon 35 (Isoleucine), codon 150 (Tyrosine) and codon 151 (Threonine). Apart from this, Haridwar isolate showed two non-synonymous mutations at codon 37 (Glutamine instead of stop codon) and codon 54 (Phenylalanine instead of Tyrosine). However, Karnal isolate showed a non-synonymous mutation at codon 98 (Leucine instead of Proline) along with three synonymous mutations at codon 35 (Isoleucine), codon 150 (Tyrosine) and codon 151 (Threonine). All the aligned sequences of HSP70 gene showed a non-synonymous mutation at codon 87 (Serine instead of Threonine). However, Hisar, Karnal and UP isolates showed a non-synonymous mutation at codon 51 (Glutamine replaced by stop codon). Phylogenetic analysis of Cytb gene showed two different clades (Haridwar, Karnal and Rajasthan isolates in one clade and Hisar isolate in another clade) under same branch. All the isolates of β-tubulin gene fell into same clade. However, in case of HSP70 gene, Hisar, Karnal and UP isolates shared same clade while Haridwar isolate made a separate node under the same branch. On the basis of present study, it can be concluded that bovines of Northern India are highly susceptible for bovine tropical theileriosis. The findings of various laboratory techniques suggest that AS-PCR may be used to detect latent infection in asymptomatic carriers essentially required for early diagnosis and to save the life of infected animals. The point mutations observed at codon 139, 143 and 146 in Cytb gene of T. annulata may be used to detect buparvaquone treatment failure in animals. As the disease is fatal in nature, so it is suggested that the farmers should consult Veterinarians for proper diagnosis of the disease and minimizing the economic losses.