Browsing by Author "Rajeev Ranjan Kumar"
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ThesisItem Open Access Epidemiology and anthelmintic resistance of gastrointestinal nematodes in small ruminants with special reference to evaluation and characterization of anthelmintic activity of some herbal plants(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2014-08) Rajeev Ranjan Kumar; Yadav, C.L.The present study was planned to know the distribution of G.I. nematodes, evaluate the efficacy of commonly used anthelmintics against G.I. nematodes of small ruminants and also exploit some herbal plants as an alternative treatment. The epidemiology of G.I. nematodes of small ruminants was studied in district Udham Singh Nagar of Uttarakhand state. Small ruminants were found to be infected with G.I. nematodes throughout the year with slightly higher overall prevalence rate (84.85%) in goats as compared to sheep (81.25%). Higher prevalence of strongyle worms was observed in rainy season (99.81% and 98.60%) and lowest (33.01% and 77.80%) during the month of winter season in both sheep and goat. Higher mean epg of strongyle worms was recorded in goats (605.18) as compared to sheep (579.50) Coproculture studies revealed the presence of H. contortus, O. columbianum, T. colubriformis and S. papillosus throughout the year in both sheep and goats. In the present study, H. contortus was predominat G.I. strongyle worm of small ruminants. The highest (47) pasture larval (L3) burden was observed in the month of August and lowest (7) in February. In sheep and goats, highest (379 and 243) post mortem worm burden was recorded in the month of September. Anthelmintic resistance was detected by FECRT and results showed that sheep of Shamaliti, Kedarkatha and goats of Gwaldum were found resistant to fenbendazole ( FECR%: -170, -10 and 63) and tetramisole (FECR%: 18, 24 and 91) while all sheep and goats were found susceptible to ivermectin with FECR% varied from 97-100%. Post coproculture examination in all animals revealed the presence of 100% H. contortus as resistant nematode. In-vitro trial of herbal plants showed that average highest corrected mortality of 95%, 100%, 100% and 100% of A. indica was observed at 0.25%, 0.5%, 1% and 2%, respectively. However, lowest percent corrected mortality of 0, 0, 20 and 60 of P. persica was recorded at 0.25%, 0.5%, 1% and 2%, respectively. Overall A. indica possed highest anthelmintic activity (corrected mortality-97.0%) followed by T. Copticum (corrected mortality-95%) against H. contortus. Phytochemical analysis revealed that tannins, saponins, alkaloids, flavonoids, reducing sugars, resins, sterols, glycosides and triterpenes were the common constituents found in various extracts of different tested plants. In-vitro trial of various plants indicated that A. indica had highest anthelmintic activity. So that, phytochemicals found in the plant were further fractionated by HPTLC. Phytochemical analysis of A. Indica showed that alkaloids and tannins were found in all extracts (aqueous, diethyl ether and methanol) whereas saponins, triterpenes and reducing sugars were found in diethyl ether and methanol extracts. However, glycosides showed its presence only in methanol extract. Out of all three extracts of A. indica, methanol extract showed maximum in-vitro efficacy against H. contortus followed by aqueous and least by diethyl ether extract. Alkaloids of methanol, aqueous and diethyl ether extracts were fractionated into 7, 3 and 7 peaks, respectively upon HPTLC. The tannins of above extracts were fractionated into 3, 1 and 7 peaks, respectively. On the basis of present study, it is suggests that epidemiological data may be used to formulate effective control strategies against G.I. nematodosis in small ruminants. And avoid the use of resistant drugs for controlling G.I. nematodosis in small ruminants of organized farms in Uttarakhand. Leaves of A. Indica and seeds of T. copticum may be used as an alternative treatment against most pathogenic H. contortus parasite.ThesisItem Open Access Molecular detection of bovine tropical theileriosis in northern India(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2021-09) Harit, Aakanksha; Rajeev Ranjan KumarConsidering 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.ThesisItem Open Access Studies on evaluation of efficacy of herbal plants against gastrointestinal nematodosis in goats(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2016-06) Sastya, Sushmita; Rajeev Ranjan KumarThesisItem Open Access Studies on prevalence of bovine tropical theileriosis and assessment of its economic impact(G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand), 2018-08) Nagar, Ankit; Rajeev Ranjan KumarConsidering the economic importance and increase reports of Bovine Tropical Theileriosis (BTT), the prevalence of the disease in district of Udham Singh Nagar, Uttarakhand was studied host wise, area wise, season wise and month wise from July, 2017 to June, 2018. alongwith evaluation of chemical drugs against Theileria annulata in cattle and assessment of its economic impact. A total of 694 blood samples of bovines (541 from cattle and 153 from buffaloes) were examined and found 338 animals (48.70%) were positive for BTT. Maximum (54.34%) prevalence of BTT was observed in cattle and than in buffaloes (28.75%). Area-wise prevalence of BTT in cattle revealed maximum (100%) in Sitarganj area and the lowest (50%) in Kashipur area. However, in buffaloes, it was recorded maximum (39.39%) in Kichha and the lowest (0%) in Sitarganj areas. Seasonal prevalence of BTT in cattle was recorded maximum (63.73%) in summer followed by rainy (60%) and the lowest (45.62%) in winter season. In buffaloes also it was recorded highest in summer season followed by rainy and then in winter with infection rate of 40.67%, 25.58% and 17.64%, respectively. Monthwise prevalence of BTT in both cattle and buffaloes revealed maximum prevalence in the month of May with infection rate of 70.58% and 47.05%, respectively. However, it was recorded minimum (41.53%) in the month of February in cattle and 0% during January and February in buffaloes. Haematological examination revealed a significant (P<0.05) decrease in haemoglobin concentration (6.28±0.42 g%), packed cell volume (19.14±1.20 %), total erythrocytic count (3.98±0.21 × 106/μl) and increase in TLC (9.31±0.37 × 10³/μl) were recorded in infected animals. However, it was in uninfected group were 10.29±0.38 g%, 31.19±1.09 %, 6.02±0.19 × 106/μl and 8.37±0.24 × 10³/μl, respectively. Significant (P<0.05) lymphopenia (51.90±4.30 %) and neutrophilia (36.00±4.89 %) were observed in infected cattle. However, differential leucocytes count revealed 60.90±1.52 % lymphocytes and 34.80±1.58 % neutrophils in control animals. The increase in monocytes (5.50±1.00 %) and eosinophils (6.60±2.50 %) were observed in infected cattle as compared with uninfected control monocytes (1.80±0.40 %) and eosinophils (2.50±0.57 %). A decrease in MCV (48.04±1.81 fl) and MCH (15.73±0.54 pg) were observed in infected group as compared to control (MCV-51.81±0.77fl and MCH 17.08±0.26 pg) which revealed microcytic RBC’s and low average quantity of haemoglobin present in single erythrocyte, respectively. However, no changes was observed in MCHC (32.78±0.37 g%) in infected group. The anti-theilerial efficacy of various combinations of chemical drugs (buparvaquone +oxytetracycline, chloroquine + oxytetracycline, α,β-Artether + Oxytetracycline and diminazine aceturate +oxytetracycline) was also studied in cattle naturally infected with T. annulata. Buparvaquone showed better efficacy than diaminazine aceturtae, chloroquine and alpha, beta-arteether when used in combination with oxytetracyline at recommended dose. It reduces the intensity of infection from ++++ to + as compared to other drugs but unable to eliminate the parasite from blood of infected animals. The effect of treatment on milk production revealed a total increase of 50.05litre milk yield/animal following treatment with buparvaquone and supportive drugs over a period of 28days. The total economic losses due to BTT were estimated of Rs. 789.85 per animal. On the basis of present study, it can be concluded that prevalence data may be used for formulating an effective control strategies against Bovine Tropical Theileriosis in animals of district Udham Singh Nagar. The findings of evaluation of various chemical drugs suggest that two doses of bupravquone are essentially required for complete elimination of the parasite from the blood of infected animals. The disease is fatal in nature, so that it is suggested that the farmers should go for proper diagnosis of the disease and consult Veterinarians for minimizing the economic losses.