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20183
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Subject: Veterinary Parasitology × End date: 2018 × Start date: 2018 ×
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    ThesisItemOpen Access
    DEVELOPMENT OF MULTIPLEX POLYMERASE CHAIN REACTION FOR DIAGNOSIS OF AMPHISTOMOSIS AND SCHISTOSOMOSIS IN DAIRY CATTLE
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2018) R. DHIVYA BHARATHI; H. Shameem
    Amphistomosis and schistosomosis are two snail borne trematode infections of dairy cattle in tropical and sub- tropical regions. During the present study three species of amphistomes were identified namely Gastrothylax crumenifer, Fischoederius cobboldi and Paramphistomum cervi and two species of schistosome namely Schistosoma spindale and S. indicum from rumen and mesentery respectively, collected from Municipal Corporation slaughter house, Thrissur. Gradient PCR protocols were standardised with primers targeting cox 2 gene of mitochondrial DNA of amphistomes and schistosomes. An optimum annealing temperature of 61.2°C amplified product size of 618 bp for amphistome and 454 bp for schistosome in multiplex PCR. A minimum concentration of 0.13pg/ µl of amphistome DNA detected by PCR. Cross reaction of amphistome primer was checked by using DNA isolated from common nematodes and schsitosomes. Absence of cross reaction with strongyles and schistosomes confirmed the specificity of mitochondrial primer for amphistomes. Out of 250 faecal samples collected from dairy cattle in and around Thrissur district, 28.4 per cent was found positive for amphistome ova and 4.0 per cent for schistosome ova with an overall parasitic infection of 36.4 per cent by conventional microscopical examination. Ova positive samples were used for standardising multiplex coproPCR. Primer targeting cox 2 gene of mitochondrial DNA of amphistome yielded 618 bp and those targeting mitochondrial DNA of schistosome yielded 454 bp products. Screening of 75 faecal samples from dairy cattle was done to ascertain the sensitivity and specificity of multiplex copro- PCR. Mc Nemars test revealed significant sensitivity of 70 per cent, specificity of 100 per cent, 100 per cent positive predictive value, 62.5 per cent negative predictive value with 80 per cent accuracy. The reliability of test checked by receiver operating characteristics curve indicated good predictability of standardised multiplex copro-PCR. Standardised multiplex copro-PCR could be used for mass screening of dairy cattle and forms a valuable tool in epidemiological studies. Simultaneous detection of the two fluke infection helps in adopting appropriate treatment decisions and control strategies against trematode infections in dairy cattle.
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    ThesisItemOpen Access
    DEVELOPMENT OF COPRO-POLYMERASE CHAIN REACTION FOR DETECTION OF ECONOMICALLY IMPORTANT GASTROINTESTINAL STRONGYLES IN CATTLE
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2018) P. DALBIN BENEDICT; R. Radhika
    The study was conducted for development of copro-PCR for detection of common strongyle species in bovines viz., Haemonchus, Trichostrongylus and Mecistocirrus spp. Three hundred and twenty faecal samples were randomly collected from different areas of Thrissur and the occurrence of strongylosis was studied by different diagnostic methods like floatation, sedimentation and coproculture. Strongyles had an overall occurrence of 36.8 per cent. Concurrent infections with amphistomes (3.4%) and coccidia (1.5%) were also obtained. The strongyle larvae identified on coproculture were Haemonchus spp. (14.68%), Mecistocirrus sp. (8.4%), Trichostrongylus spp. (3.4%), Bunostomum sp. (3.1%) and Cooperia spp. (1.25%). Adult strongyle worms collected from the entrails of cattle from slaughter houses revealed an overall occurrence of 31.88 per cent. The species identified were Haemonchus spp. (8.6%), Trichostrongylus spp. (7.2%), Mecistocirrus sp. (14.4%) and Cooperia spp. (1.4%). Hence the predominant strongyles selected in this study were Haemonchus spp., Mecistocirrus sp. and Trichostrongylus spp. Gradient PCR protocols were standardised with primers targeting the ETS region in H. placei, ITS-2, 28S rRNA partial regions in T. colubriformis and ITS-1, 5.8S, ITS-2 partial regions of rRNA in M. digitatus. The optimum annealing temperature of 60o C was chosen in PCR protocols. Multiplex PCR was standardised for simultaneous detection of the DNA of predominant adult strongyles. Copro-PCR was standardised using copro DNA as template and adult worm DNA as positive control. Multiplex copro-PCR was standardised for simultaneous detection of multiple strongyle infections in the faecal sample of cattle. Ninty five faecal samples were randomly collected from Thrissur and subjected to copro-PCR and multiplex copro-PCR. Detection limits of adult DNA by PCR were determined. A minimum of 0.45 fg/µl of DNA was required for H. placei, 0.34 pg/µl for T. colubriformis and 0.093 ng/µl for M. digitatus. The cross reactivity of primers were checked with DNA isolated from the predominant strongyle species as well as with common ruminant trematodes. No cross reaction were noticed among strongyles and with amphistomes and schistosomes. Sensitivity and specificity of multiplex copro-PCR by Mc Nemar test, using coproculture as the standard were obtained as 66.7 and 98.2 per cent respectively. The diagnosis of strongylosis mainly relies on routine coproscopy and coproculture, which are time consuming and laborious. Development of a rapid molecular diagnostic method for differentiating important species of gastrointestinal strongyles infecting cattle will be a good diagnostic tool and will also help to improve and extend the technology into epidemiological studies, strategic control and prevention of strongylosis. However, multiplex copro-PCR could be employed as a suitable test for simultaneous detection and speciation of strongyle infection in cattle. This will be useful in strategic selective treatment and also signifies the importance of further research on molecular identification of gastrointestinal strongyles.
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    ThesisItemOpen Access
    MOLECULAR CHARACTERISATION OF RHIPICEPHALUS (BOOPHILUS) ANNULATUS AND R. (B.) MICROPLUS USING MITOCHONDRIAL CYTOCHROME C OXIDASE SUBUNIT 1 (COI) GENE AND SECOND INTERNAL TRANSCRIBED SPACER (ITS2)
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD, 2018) AMRUTHA B.M.; AJITH KUMAR K. G.
    The present study was carried out to differentiate closely related Rhipicephalus (Boophilus) species like R. (B.) microplus and R. (B.) annulatus and to characterise these species using molecular tools. Molecular tools like polymerase chain reaction (PCR), sequencing, phylogenetic analysis and divergence analysis were adopted. When a total of 279 cattle were screened for presence of infestation with hard ticks from different geographical locations of Kerala and Karnataka, 213 animals were identified as infested. In Kerala, R. (B.) annulatus was the predominant species (54.26 per cent) followed by H. bispinosa (25.58 per cent), R. (B.) microplus (12.40 per cent) and R. haemaphysaloides (5.42 per cent). R. (B.) microplus was the predominant species (77.10 per cent) in Karnataka followed by H. bispinosa and R. haemaphysaloides with 18.07 per cent and 4.81 per cent prevalence rates respectively. Morphologically identified R. (B.) microplus (13 numbers) and R. (B.) annulatus (14 numbers) were used for the molecular characterization using cytochrome c oxidase subunit 1 (COI) and internal transcribed spacer (ITS2) molecular markers. Some of the R. (B.) microplus male ticks showed presence of a spur in the ventral surface of palpal article I similar to that seen in R. (B.) australis males. Dorsal setae were short and medial alloscutal setae were arranged in 2-3 rows in R. (B.) microplus female ticks. All the PCR products were sequenced and BLAST analysis was performed for confirmation of the species. The phylogenetic analysis of R. (B.) microplus isolates from both Kerala and Karnataka using COI classified them into R. (B.) microplus clade C, comprising R. (B.) microplus isolates from Haryana, India and other neighbour countries like Pakistan, Myanmar and Bangladesh. R. (B.) annulatus isolates from Kerala clustered with other R. (B.) annulatus isolates from Chennai, India, Iraq, Israel and Romania. The phylogenetic tree based on ITS2 failed to distinguish closely related R. (B.) microplus complex as R. (B.) microplus and R. (B.) annulatus isolates were clustered together. Divergence analysis was done further to differentiate R. (B.) microplus and R. (B.) annulatus. The COI gene showed greater value (7.9 per cent) for interspecific divergence compared to ITS2 (3.6 per cent). Thus COI was identified as a better marker in resolving interspecific divergence in comparison to ITS2. The intraspecific divergence for R. (B.) microplus was higher compared to R. (B.) annulatus. When R. (B.) microplus isolates of South India were compared with isolates of R. (B.) microplus clade A, B and C, higher divergence was observed with clade A (11.9 per cent) followed by clade B (7.4 per cent). Least divergence was observed against R. (B.) microplus clade C (2.3 per cent). Thus, the results of the present study revealed that Indian isolates of R. (B.) microplus clade C sensu lato was phylogenetically distant from true R. (B.) microplus clade A sensu stricto.