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ThesisItem Open Access Molecular studies on the prevalence of Tick borne Haemoprotozoan and Rickettsial infections in Canine and vector ticks(Sher-e-Kashmir University of Agricultural Sciences & Technology, Jammu (J&K), 2023-03-31) Sharma, Sabhyata; Yadav, AnishThe present study was conducted from August, 2020 to October, 2022 to determine the prevalence of haemoprotozoan and rickettsial infections in clinically suspected canines and vector ticks of Jammu region.The overall prevalence of tick borne haemoprotozoan and rickettsial infections in canine population of Jammu region was recorded as 24.3% (n= 92/378). The overall prevalence of Babesia canis vogeli was 10.58% (40/378) followed by Ehrlichia canis (7.67%, 29/378), mixed infections (3.70%, 14/378), Babesia gibsoni (2.11%, 8/378) and Hepatozoon canis (0.26%, 1/378). Out of total prevalence, Babesia canis vogeli was recorded with higher prevalence in dogs followed by Ehrlichia canis, Babesia gibsoni and Hepatozoon canis. Categorically, among the age groups of dogs, the prevalence of haemoprotozoan and rickettsial infection was significantly (p˂0.05) high in 6-12 months (52.77%) of age group as compared to 1year and above (14.22%) age group of canine. Monsoon recorded significantly (p˂0.05) highest infection rate (27.40%) as compared to winter (6.90%). As per breed, German shepherd were shown non-significantly (p>0.05) higher prevalence of 39.7%. Males showed non-significantly higher infection (25.5%) as compared to females (20%). Haematological alterations in infected group of canines revealed significant drop in mean values of haemoglobin count, platelet count, packed cell volume count and Total erythrocyte count as compared to healthy control group of canines.A blood sample was considered as positivefor Ehrlichia canis when amplified with the size of 380bp product. Similarly, product size of 602bp, 671bp and 666 bp were yielded by Babesia canis vogeli, Babesia gibsoni and Hepatozoon canis, when amplified by targeting 18S rRNA genefor all three of the parasites. A total of four PCR products comprising Babesia canis vogeli and Ehrlichia canis, representing different isolates of Jammu region were sent for sequencing. The sequence information received was analysed using Basiclocal alignment search tool (BLAST, NCBI) and DNA Star, Laser gene software. The submitted sequences of Babesia canis vogeli and Ehrlichia canis belonging to Jammu region are under process with NCBI. Identification using standard keys and tick mounting procedures revealed that all the ticks collected from 257 tick infested dogs were identified as Rhipicephalus sanguineus. The prevalence of tick infestation was recorded significantly (p<0.05) higher among 6-12 months (82.29%) of age group in monsoon season (86.98%). German shepherd breeds (86.79%) were at higher risk of gaining infection while males (77.34%) were recorded with significantly higher tick infestation as compared to females (48.36%). Standardisation and optimization of PCR on ticks revealed the presence of DNA of Babesia canis vogeliand Ehrlichia canis, in vector ticks, collected from suspected canines of Jammu region.At the last phase of the study period, duplex PCR was also standardized for simultaneous detection of Babesia canis vogeli and Ehrlichia canis was performed using the similar primers which we used in molecular studies for blood and vector tick samples, respectively at the annealing temperature of 52ºC.ThesisItem Open Access Epidemiological studies and molecular characterization of gastrointestinal helminthosis in migratory sheep and goats of Jammu region(Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu(UT of J&K), 2022-08-22) Sofi, Omer Mohi U Din; Godara, RajeshThe prevalence of gastrointestinal (GI) helminths in migratory sheep and goats of Jammu region was found as 40.13 and 41.1% based on faecal (n=2350) and abattoir (n=1000) examinations, respectively.The study revealed1 trematode (amphistome), 1 cestode (Moniezia) and 3 nematodes (strongyle, Strongyloides and Trichuris). Strongyleshad the highest prevalence (15.19%) followed by amphistomes (7.49%), Strongyloides (4.81%), Trichuris (3.87%) and Moniezia (3.15%). Mixed infections were observed in 5.74% of examined animals. Seasonally, the highest prevalence was found in monsoon (55.93%) followed by post monsoon (47.21%), summer (38.9%) and the least in winter (19.63%).The study revealed 5strongyle species based on morphology and measurements of different characteristics of L3 strongyle larvae i.e. Trichostrongylusaxei, Haemonchuscontortus, Ostertagiaostertagi, NematodirusbattusandCooperiaoncophora.Prevalence of Haemonchuscontortus (44.3%) was significantly (p<0.05) higher followed by Trichostrongylusaxei(27.4%), Nematodirusbattus (16.4%), Ostertagiaostertagi(7.6%) and the least for Cooperiaoncophora(4.3%).Five genera of strongyles were identified and clear amplification was noticed in Haemonchuscontortus(318bp), Ostertagiaostertagi(285bp), Trichostrongylusaxei (269bp), Cooperiaoncophora (420bp) and Nematodirusbattus (387bp) based on ITS-2 sequence with flanking partial 18S rDNA and 5.8S rDNA region using PCR.Total annual economic loss due to amphistomosis in sheep for the study flock was estimated as Rs. 462384.0 (US $6188.22) and may be concluded as loss of Rs. 1235 per animal per annum. Evaluation of the total annual economic recovery by sustainable control of amphistomosis in sheep was estimated as Rs. 107745.0 (US $1441.99) and may be concluded as recovery of Rs. 981 per animal per annumThesisItem Open Access Detection of ivermectin resistance and its mitigation in Rhipicephalus (Boophilus) microplus of Jammu region(Sher-e-Kashmir University of Agricultural Sciences & Technology of Jammu (J&K), 2022-01-25) Nazim, Kaifa; Katoch, Dr. RajeshStatus of ivermectin resistance was studied in 16 isolates of Rhipicephalus (Boophilus) microplus collected from 7 districts of Jammu region using Larval Immersion Test (LIT). The regression graphs of probit mortality of tick larvae plotted against log values of concentrations of ivermectin were utilized for the determination of slope mortality, lethal concentrations (LC50 and LC95) and resistance factor (RF). Based on the RF values obtained, resistance level was categorized as I, II, III and IV. Out of these 16 isolates examined, 15 were found resistant against ivermectin. Level II resistance was detected in eleven isolates, whereas four isolates (Doda, Sumbli, Kishtwar and Bhaderwah) exhibited level I resistance. Only one isolate (Padder) was found susceptible. Effect of two (MK-571 and Cyclosporin-A) ATP-binding cassette (ABC) transport inhibitors on the efficacy of ivermectin against R. (B.) microplus was studied. Pre-exposure to a single pre-determined sub-lethal concentration of these two synergists has led to reduction in LC50 values of ivermectin in different field isolates of R. (B.) microplus. MK-571 showed the highest increase in toxicity against ivermectin in the field isolates of R. (B.) microplus. The highest synergistic factors for MK-571 and Cyclosporin-A, were recorded in RS Pura isolate (2.51 and 1.78, respectively), whereas the lowest value was found for Kishtwar isolate (1.7 and 1.23, respectively). Efficacy of extracts (acetone, chloroform and ethanol) of hull of Juglans regia and rhizomes of Curcuma longa were tested against unfed larvae of R. (B.) microplus using LIT. For J. regia, based on LC50 values, the highest acaricidal activity was observed in ethanolic extract (LC50 of 1.43%) as compared to chloroform (1.52%) and acetone (1.22%). Among the three extracts of C. longa used, the highest acaricidal activity was observed in ethanolic extract with LC50, LC90, LC95 and LC99 values calculated as 2.43, 9.12, 13.26 and 26.77%, respectively.ThesisItem Open Access Molecular characterization of Eimeria spp. infecting poultry and evaluation of ionophore resistance against caecal coccidiosis in Jammu region(Sher-e-Kashmir University of Agricultural Sciences & Technology of Jammu (J&K), 2022-05-02) Khursheed, Aiman; Yadav, Dr. AnishFaecal examination of 192 poultry farms, from three agroclimatic zones of UT of Jammu and Kashmir, by simple salt flotation revealed 115 farms positive for different Eimeria species with an overall prevalence of 59.89%. Categorically, among the farm management, the prevalence of coccidiosis was significantly (p<0.05) high in unorganized farms (67.50%) as compared to organized farms (47.22%). Monsoon recorded significantly (p<0.05) highest infection rate (85.41%) followed by post monsoon (70.83%), winter (45.83%) and summer (37.50%). As per agroclimatic conditions, subtropical zone (78.12%) and intermediate zone (57.81%) showed significantly (p<0.05) high prevalence as compared to temperate zone (43.75%). The mean oocyst output was significantly (p<0.05) high during monsoon irrespective of agroclimatic zone and farm management. Morphometric identification using COCCIMORPH software revealed seven Eimeria species i.e. E. tenella, E. necatrix, E. mitis, E. acervulina, E. maxima, E. praecox and E. brunetti from Jammu region. However, E. tenella was the most predominant species observed in all the three zones of Jammu region. Molecular characterization targeting ITS-1 gene revealed six species with band size of 278bp in E. tenella, 321bp in E. acervulina, 383bp in E. necatrix, 145bp in E. maxima, 193bp in E. mitis and 116 bp in E. praecox. Eighteen ITS-1 sequences of different Eimeria spp. with accession numbers MZ983636.1, OL989151.1, OL505606.1, OL505603.1, MZ983633.1, OK019726.1, OL957473.1, OL505604.1, OL505607.1, OL695839.1, OK011553.1, OL957472.1, OL695840.1, OL669324.1, OL602187.1, OL669328.1, OK030703.1 and OM501135.1 were submitted to NCBI. The ITS-1 sequences of different Eimeria spp. generated were analyzed to establish their phylogenetic relationship. The sequence homology with available sequences in GenBank revealed 98.16%-100% identity for E. tenella, 94.97%-100 % for E. mitis, 98.75%-100% for E. acervulina, 96%-100% for E. maxima, 98.20%-100 % for E. praecox and 91.24%-100 % for E. necatrix. In the phylogenetic tree, all the ITS-1 sequences of E. acervulina, E. mitis, E. tenella, E. maxima, E. praecox and E. necatrix clustered in separate clades, irrespective of their geographical location. The sequences of E. tenella of Srinagar isolate formed a separate clade whereas other four E. tenella sequences of Jammu, Kathua, Udhampur and Poonch isolates were placed in the clade formed by the ITS-1 sequences of E. tenella. Drug resistance of four field isolates (FI) of Eimeria tenella namely FI-I, FI-II, FI-III and FI-IV collected from Jammu, Poonch, Udhampur and Srinagar, respectively of UT of Jammu and Kashmir were assessed against commonly used ionophores i.e. salinomycin @ 60 ppm and maduramicin @5 ppm by calculating the global index (GI) based on five parameters viz., weight gain, feed conversion ratio, oocyst index, lesion score and mortality for individual ionophore compound. Results of present investigation revealed that resistance was found in FI-I against salinomycin and maduramicin (GI: 29.52% and 48.64%, respectively). The FI-II exhibited limited efficacy against both salinomycin and maduramicin (GI: 70.59% and 76.25%, respectively). In case of FI-III, salinomycin exhibited partial resistance (GI: 56.62%) whereas maduramicin showed limited efficacy (GI: 73.32%). The result regarding FI-IV showed resistance against salinomycin (GI: 42.97%) and partial resistance against maduramicin (GI: 59.26%).ThesisItem Open Access Gastrointestinal parasitism in buffaloes of Jammu region(Sher-e-Kashmir University of Agricultural Sciences & Technology, Jammu (J&K), 2021-11-26) Bhargava, Yash; Katoch, RajeshThe present study was undertaken to determine the gastrointestinal parasitism in buffaloes of Jammu region based on fecal examination (n=1680). Most prevalent parasite recorded was amphistome (7.9%) followed by Buxtonella spp. (7.5%), coccidia (5.8%), strongyles (5.6%), ascarids (0.3%) and Moniezia spp. (0.06%). The mixed infections were found in 12.3% of samples examined. Monsoon season showed significantly higher (p<0.01) prevalence (54.3%) in comparison to summer (50.2%), post-monsoon (33.6%) and winter (20.2%) seasons. In males, the infection rate was higher (44.3%) than females (39.2%). The animals aged up to 6 months showed significantly higher (p<0.01) prevalence (54.3%) in comparison to animals above 6 months to 1 year (40.3%) and above 1 year (38.3%) age groups. The irrigated area showed significantly higher (p<0.01) prevalence rate (49.9%) in comparison to rain-fed area (29.3%). Based on the morphological characteristics of sporulated oocysts, nine species of Eimeria were identified as E. bareillyi (28.6%), E. zurenii (18.0%), E. bovis (14.0%), E. subspherica (12.8%), E. canadensis (9.0%), E. alabamensis (6.2%), E. ellipsoidalis (5.4%), E. auburnensis (3.3%) and E. cylindrical (2.7%). Molecular characterization of strongyle larvae revealed Ostertagia and Trichostrongylus targeting ITS-2 gene fragments at 293 bp and 237 bp, respectively.ThesisItem Open Access Molecular Studies on the Prevalence of Tick borne Haemoprotozoan and Rickettsial infections in Bovines and Vector Ticks(Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu (J&K), 2020-02-25) Kour, Rabjot; Yadav, Dr. AnishOverall prevalence of tick borne haemoprotozoan and rickettsial infection was studied in 278 clinically suspected bovines of Jammu region using conventional microscopy and molecular techniques (PCR). The animals found to be positive for Anaplasma spp., Babesia spp.and Theileria spp. were 54.76%, 32.14% and 2.38%, respectively. The overall prevalence was recorded to be 30.21% (n=84). Categorically, the prevalence was highest in unorganised farms (36.87%) as compared to organised farms (21.18%). Agewise, animals > 3 years of age revealed significantly (p<0.05) higher prevalence as compared to < 1 years of age. The prevalence of the infection was highest in monsoon season (46.57%) followed by post monsoon (40.00%), summer (23.07%) and winter (5.00%) and the difference was significant (p<0.05). Cross bred animals exhibited significantly (p<0.05) higher prevalence (33.62%) with respect to indigenous breeds (15.38%). Female cattle (30.82%) showed higher prevalence than male cattle (16.66%) which was non significant (P> 0.05). Molecular characterization revealed presence of Babesia bigemina, Anaplasma marginale and Theileria annulata in blood samples of bovine in Jammu region. Five sequences with accession numbers MN566925.1, MN567603, MN566924.1, MH733242.1 and MN567602.1 have been submitted to NCBI, gene bank and one sequence of Theileria annulata is under process. There was a significant decrease in Hb, PCV and RBC count due to severe anaemia in animals. The only tick spp. found in the present study was Rhiphicephalus microplus. The overall prevalence of ticks was recorded to be 61.87%. PCR analysis in ticks revealed 26.66% positivity for tick borne haemoprotozoan and rickettsial infection. Results also revealed 10 tick samples positive for Anaplasma marginale whereas Babesia bigemina was detected in 6 samples of vector ticks.ThesisItem Open Access Antimicrobial properties of Chrysomyia megacephala larvae in chronic wound healing(Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu (J&K), 2019-11-26) Tariq, Mufeeda; Borkataki, SankuThe present study was conducted to elucidate the effectiveness of antimicrobial properties of Chrysomyia megacephala larvae in healing of contaminated excisional wound in Wistar rats. A total of 36 male Wistar rats were taken for the study. Wounds were created on all and induced with pure colonies of bacteria Staphylococcus aureus. The animals were then divided into three groups according to the treatment given i.e. Control (C), Antibiotic (A) and Maggot (M) treated groups with 12 animals each. Colonization of larvae to be used for medicinal purpose was done on three different diets i.e. sheep liver, fish meat and liver extract. The variable wound kinetic parameters revealed significant reduction in wound area with maximum contraction (90%), early elimination of bacterial bioburden (3.33±0.02) and significant reduction in wound pH (6.14±0.09) in two weeks in maggot treated group when compared to control and antibiotic treated groups. Colonization of larvae of Chrysomyia megacephala in three different diets showed total duration of life cycle on fish meat was shorter (240.88±28.09h) as compared to sheep liver(295.63±13.82h) and liver extract (557.38±11.7h) at the temperature of 27±2o C and 70% RH. Biochemical analysis of excretions and secretions (ES) of L1 larvae were found to be alkaline with the mean pH values of 8.83±0.17 and total protein was found to be 1.54±0.16mg/ml. Protein profiling of the same was done by SDS-PAGE which revealed several polypeptides of different molecular weights,among which polypeptides of 25 and 35 kDa were more prominent. In vitro antimicrobial activity of ES was tested using optical density method and showed its antimicrobial activity against S. aureus.ThesisItem Open Access Molecular studies on Cryptosporidium and Giardia species in cattle calves and therapeutic evaluation of Curcuma longa against experimental cryptosporidiosis in mice(Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu (J&K), 2019-10-15) Ganai, Alveena; Yadav, Dr. AnishThe prevalence studies were based on identification of Cryptosporidium oocyst and Giardia cyst in cattle calves of Jammu region by examining 614 faecal samples subjected to Diethyl ether sedimentation technique (DEES) followed by modified Ziehl-Neelsen staining technique and lugols iodine wet mount technique for identification of Cryptosporidiumspp.andGiardia spp. infection. The study revealed an overall positivity of 144 animals (23.45%) for cryptosporidiosis and of 43 animals (7.00%) for Giardia infection. Highest prevalence of Cryptosporidium spp. was recorded in Samba (28.57%) district, whereas highest prevalence of giardiosis was recorded in Kathua (7.78%) district of Jammu region. The highest prevalence of Cryptosporidium infection was recorded in < 1 month of age animals (43.24%) than other age group whereas highest prevalence of Giardia infection (11.2%) was recorded in 1-6 month age group. Diarrhoeic animals revealed significantly higher prevalence (29.51%) of Cryptosporidium infection and (9.02%) Giardia infection as compared to non diarrhoeic animals. Highest prevalence of Cryptosporidium infection was recorded (34.65%) in winter months from December to February but in case of Giardia infection it was recorded highest (10.34%) in monsoon months from July to September. Cattle calves having mucus in the faeces showed significantly higher prevalence (37.33%) of cryptosporidiosis whereas in case of giardiosis faecal samples with no mucus/blood showed significantly higher prevalence (7.62%). Male animals showed non significantly higher incidence of Cryptosporidium infection (37.77%) and Giardia infection (12.77%) as compared to females. Genetic characterisation of 120 samples (40 samples per age group) found positive by mZN was carried using molecular techniques. Nested PCR of 18S small subunit (SSU) rRNA gene of Cryptosporidium spp. amplified a product of 830 bp band size whereas β-giardin gene of Giardia spp. amplified a product of 511 bp band size.RFLP analysis of nested PCR product of Cryptosporidium spp. by three restriction enzymes namely SspI, VspI and MboIIwas carried which resulted in higher prevalence of C. parvum(74.07%) in young animalswhereas, 16.66% were positive for C. andersoniand 9.25% were found to be positive for C. bovis.For subtyping of C. parvum,DNA of 17 positive samples GP60 gene was amplified and sequenced andyielded clear band at 850 bp. Molecular phylogenetic analysis of these sequences showed that these C. parvum isolates belonged to one subtype family, i.e., IIa. Fifteen of the C. parvum specimens belonged to the subtype IIaA15G2R1, whereas two specimens belonged to subtype IIaA14G2R1. The 511 bp nested product of Giardia spp. when digested by Hae III restriction enzymes, zoonotic Giardia intestinalis Assemblage B with 1.6% prevalence and Assemblage E with 14.16% prevalence in young cattle calves of Jammu region was recorded.Administration of curcumin, obtained from ethanolic extract of Curcuma longa as therapeutic agent at dose rate of 8 mg/ kg body weight for five days resulted in decreased oocyst production in experimentally infected mice. Although higher body weight gain was recorded in treated groups, but it was not significantly affected. The findings of the present study clearly suggest that molecular characterization and subtypes/assemblages of Cryptosporidium spp. and Giardia spp. established zoonotic potential of C. parvum and G. intestinalis infecting cattle of Jammu region. Curcuma longa showed promising anticryptosporidial effects in reduction of oocyst count in experimentally infected mice and highlights the significance of further trial in other susceptible host like cattle.ThesisItem Open Access ANTHELMINTIC RESISTANCE IN HAEMONCHUS CONTORTUS OF SMALL RUMINANTS IN JAMMU(Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu, 2018) Saleem, Taniya; Katoch, RajeshThe emergence of anthelmintic resistance on government and private farms (2 each) in Jammu, India was investigated using Faecal egg count reduction test (FECRT) for fenbendazole (FBZ) and levamisole (LEV), Egg hatch assay (EHA) for benzimidazole (BZ) and Egg hatch paralysis assay (EHPA) for LEV. For FECRT, a total of 240 naturally infected animals (Sheep and Goats) of 3-6 months of age were divided randomly in 3 groups of 20 animals each. At each farm, Group 1 and Group 2 animals were given FBZ (@ 5.0 mg/kg body weight) and LEV (@ 7.5mg/kg body weight) orally as drench, respectively and Group 3 animals were kept untreated. Faecal samples were collected per rectum 14 days post-treatment. FECR after treatment with FBZ and LEV were found to be 58 and 44 percent (farm 1), 61 and 49 percent (farm 2), 51 and 60 percent (farm 3), 68 and 86 percent (farm 4) respectively. On EHA, the ED50 values for the isolates of Haemonchus contortus were 0.171, 0.163, 0.178 and 0.156 µg of TBZ/ml in farm 1, 2, 3 and 4 respectively. The ED50 values on EHPA for the isolates of H. contortus were 4.445, 1.050, 0.698 and 0.282μg/mL of LEV in farm 1, 2, 3 and 4, respectively. The H. contortus larvae isolated from animals were genotyped for detection of mutation in the β-tubulin isotype 1 gene using AS-PCR. A total of 144 larvae, 36 from each farm were used in the study. In farm 1, the prevalence of different genotypes varied significantly (p<0.001) with 80 percent ‘rr’ type, 11 percent ‘rS’ and 8 percent ‘SS’ types. In farm 2, the types of genotypes varied significantly (p<0.001) with 72 percent ‘rr’, 17 percent ‘rS’ and 11 percent ‘SS’. In farm 3, 89 percent were found to be of ‘rr’ genotype, 8 percent of ‘rS’ genotype and 3 percent of ‘SS’ genotype. In farm 4, the prevalence was predominated by ‘rS’ genotype (50 percent) followed by ‘rr’ genotype (36 percent) and ‘SS’ genotype (14 percent). The results of AS-PCR from the pasture larvae showed the overall predominance of ‘rr’ genotype (60 percent) followed by ‘rS’ (33 percent) genotype and ‘SS’ (7 percent) genotype. Thus prevalence of BZ resistant (r) allele was significantly (p<0.001) higher (76.5 percent) as compared to the 23.5 percent prevalence of BZ susceptible (S) allele.
