Haryana Vet. (Dec., 2019) 58(2), 190-192 Research Article Table 1 Primers and their sequences used for generic identification MOLECULAR STUDIES ON HEPATOINTESTINAL SCHISTOSOMES IN of blood flukes B.M. MANOHARA, PLACID E. D’SOUZA, G.S. MAMATHA and H. DHANALAKSHMI* Name of Primer sequences (5´-3´) Amplicon Targeted Department of Veterinary Parasitology, Veterinary College, Bengaluru-560024, India primer size gene SrrnS-F TCGAGATTGTCGGGC 328 bp 12S rRNA Received: 26.10.2018; Accepted: 19.08.2019 GATGTAC ABSTRACT SrrnS-R TAGATTCGTCCGGGGG A molecular study was carried out on hepatointestinal schistosomes in cattle for characterization of species. The worms were recovered from AATGTGC the liver and mesentery from the cattle slaughtered at the abattoir from Bengaluru and Hassan district. Polymerase chain reaction was carried out for SCOI-F GGTGGATTTATAGGTCT 1088 bp cytochrome Schistosoma worms by targeting genus specific 12S ribosomal RNA and cytochrome oxidase subunit I and species specific mitochondrial gene TGGGTTAAG oxidase (SPMit) for S. spindale and SI 16S RNA for . All the isolates yielded 328 and 1088 bp DNA fragments specific for Schistosoma subunit I genus and 330 bp specific to S. spindale. No specific amplification was obtained for S. indicum. The sequencing and BLAST analysis of 328 bp amplicon showed homology with S. spindale isolates deposited at GenBank database. Phylogenetic analysis indicated that S. spindale Karnataka SCOI-R TCACAAATGGCCACCACC isolate(Accession no. MG052937) shared 100 per cent homology with isolate of S. spindale from Sri Lanka (EF534282). AAACCAACATTA Fig. 1. PCR analysis of 12S ribosomal RNA (rrnS) S. spindale (Sreenivasa Murthy, 2011) Key words: Abattoir, Cattle, Liver, Mesentery, PCR, S. spindale In this study, the phylogenetic analysis 12S rrnS of S. Schistosomosis in bovines is widely reported as a Schistosoma primers. No template control was maintained with isolate of S. spindale from Sri Lanka (EF534282) and spindale Karnataka were in the same clade as S. spindale disease entity in many parts of Asia and Africa. without a DNA template. DNA extracted from the blood 99 per cent with S. spindale isolate from Bangladesh isolate from Sri Lanka deposited in the GenBank Schistosomosis is one of the most prevalent infectious containing Babesia organisms was used as a negative (EF534281) and Nepal (KR607254). (EF534282) and shared 100 per cent identity and also diseases, endemic in more than 70 countries, mainly control. S. spindale DNA obtained from Dr. Sreenivasa shared 99.99 per cent identity with S. spindale from The present findings were in agreement with the within the developing world (Hovnanian et al., 2010). Murthy, Department of Veterinary Parasitology, Bangladesh (EF534281) and Nepal (KR607254). immunological and molecular studies on cattle and About 530 million of cattle live in areas endemic for Veterinary College, Hyderabad was used as positive Sreenivasa Murthy (2011) from Bengaluru had reported buffaloes in Bengaluru conducted by Sreenivasa Murthy bovine schistosomosis in Africa and Asia while at least 165 control. Primary pairs used and thermal cycles followed that phylogenetic analysis of 12S ribosomal rRNA showed (2011) who reported S. spindale, S. indicum and S. million cattle are infected with schistosomes worldwide are given in Table 1-3. that S. spindale was related to distinct cluster of S. nasale nasalein Bengaluru based on PCR. Sreenivasa Murthy (Bont and Vercruysse, 1997). Schistosomosis is now well from Bangladesh with 660 nucleotide substitutions. Sequence alignment and phylogenetic analysis: The (2011) detected and differentiated S. spindale, S. indicum, recognized as the fifth major helminthosis of domestic PCR products of 12S ribosomal RNA (rrnS) gene of Similarly, Lakshmanan (2014) from Kerala reported that S. nasale by using 12S rrnS and COI genus specific in the Indian sub-continent. Identification of Schistosoma spp. were sequenced at M/s Bioserve biotech, phylogenetic analysis of 28S rRNA of S. spindale from primers which yielded 328 bp and 1088 bp DNA fragments worms based on phenotypic may be difficult due to Pvt Ltd, Hyderabad. The BLAST analysis for the obtained Kerala shared 100 per cent identity with S. spindale from and these products were sequenced and analysed by NCBI overlapping of the characters viz., length of flukes, sequences was performed on GenBank database at Sri Lanka (AY157257). However, further research work is database for the species confirmation and phylogenetic intestinal caeca and number of testes. Molecular National Centre for Biotechnologies Information (NCBI) needed to know the prevalence of Schistosoma spp. in tree was constructed. Similar work was carried out by techniques based on genomes are very useful for the on the website www.ncbi.nlm.gov/ BLAST. The other regions of Karnataka and to implement proper Lakshmanan (2014) from Kerala and Hossain et al. (2015) epidemiological diagnosis as well as for research on sequences were aligned with the published sequences control measures. genetic variation of the parasitic organism. Hence, the deposited in the GenBank database and the phylogenetic from Bangladesh. present study was undertaken for the molecular tree was constructed by maximum likelihood method with Table 2 Primers and their sequences used to identify Schistosoma species characterization of the worms. 1000 bootstrap replicates using MEGA 6.0 software MATERIALS AND METHODS (Tamura et al., 2013). Name of Primer sequences (5´-3´) Amplicon size Targeted gene Species of Reference primer Schistosoma Extraction of genomic DNA: The collected worms from RESULTS AND DISCUSSION SPMit F CTTGGAGTCGGGTTGTTTGAG 330 bp Mitochondrial gene S. spindale Lakshmanan, 2014 liver and mesenteries were subjected to DNA extraction. PCR was carried out by targeting Schistosoma genus SP Mit R CAGACCCTCACACCAACAGTG The DNA was extracted from the worms using QIA amp specific 12S rrnS, COI and species specific SPMit and DNA extraction mini kit (Qiagen, Germany) as per the SI16s RNA genes. All the isolates yielded 328 (Fig. 1) and SI 6sRNAF GAGTTTGTAAATGGAGGCTGAG 606 bp 16sRNA S. indicum protocol described by the manufacturer. 1088 bp (Fig. 2) DNA fragments specific for Schistosoma SI 16sRNAR CCTTATTCAGCCTCTACACCG Attwood et al., 2007 Polymerase chain reaction: A total reaction volume of genus and 330 bp (Fig. 3) specific to S. spindale. No Table 3 25 l pre mixture of PCR consisting of two micro litre of  specific amplification was obtained for S. indicum. Genus PCR cyclical conditions for different genes of Schistosoma spp. forward primer, two micro litre of reverse primer, five specific 328 bp amplicon of both Bengaluru and Hassan Target gene Initial Denat- Annealing Extension No. of Final Reference micro litre of template DNA and 12.5 l of PCR master isolates were selected and were subjected for nucleotide denaturation uration cycles Extension mix (2X) (Genei Laboratories Pvt. Ltd.) and 3.5 l of NFW sequencing. The sequencing and BLAST analysis of 328 Schistosoma genus 94 ºC/5 min 94 ºC/30 sec 50 ºC/1 min 72 ºC/1 min 39 72 ºC/10 min Sreenivasa Murthy, 2011 was constituted. bp amplicon showed homology with S. spindale isolates deposited at GenBank database. Phylogenetic analysis 95 ºC/5 min 95 ºC/1 min 54 ºC/1 min 72 ºC/1 min 35 72 ºC/10 min Hossain et al., 2015 The PCR was carried out in a gradient thermal cycler mitochondrium (SPMit) showed that S. spindale Karnataka isolate (Accession (Eppendorf, Germany) programmed for genus specific Schistosoma indicum 95 ºC/5 min 95 ºC/1 min 52 ºC/1 min 72 ºC/1 min 35 72 ºC/10 min Hossain et al., 2015 number MG052937) (Fig. 4) shared 100 per cent homology 16S rRna (SI 16SrRna) *Corresponding Author: [email protected]

190 191 Haryana Vet. (Dec., 2019) 58(2), 190-192 Research Article Table 1 Primers and their sequences used for generic identification MOLECULAR STUDIES ON HEPATOINTESTINAL SCHISTOSOMES IN CATTLE of Schistosoma blood flukes B.M. MANOHARA, PLACID E. D’SOUZA, G.S. MAMATHA and H. DHANALAKSHMI* Name of Primer sequences (5´-3´) Amplicon Targeted Department of Veterinary Parasitology, Veterinary College, Bengaluru-560024, India primer size gene SrrnS-F TCGAGATTGTCGGGC 328 bp 12S rRNA Received: 26.10.2018; Accepted: 19.08.2019 GATGTAC ABSTRACT SrrnS-R TAGATTCGTCCGGGGG A molecular study was carried out on hepatointestinal schistosomes in cattle for characterization of species. The worms were recovered from AATGTGC the liver and mesentery from the cattle slaughtered at the abattoir from Bengaluru and Hassan district. Polymerase chain reaction was carried out for SCOI-F GGTGGATTTATAGGTCT 1088 bp cytochrome Schistosoma worms by targeting genus specific 12S ribosomal RNA and cytochrome oxidase subunit I and species specific mitochondrial gene TGGGTTAAG oxidase (SPMit) for S. spindale and SI 16S RNA for Schistosoma indicum. All the isolates yielded 328 and 1088 bp DNA fragments specific for Schistosoma subunit I genus and 330 bp specific to S. spindale. No specific amplification was obtained for S. indicum. The sequencing and BLAST analysis of 328 bp amplicon showed homology with S. spindale isolates deposited at GenBank database. Phylogenetic analysis indicated that S. spindale Karnataka SCOI-R TCACAAATGGCCACCACC isolate(Accession no. MG052937) shared 100 per cent homology with isolate of S. spindale from Sri Lanka (EF534282). AAACCAACATTA Fig. 1. PCR analysis of 12S ribosomal RNA (rrnS) S. spindale (Sreenivasa Murthy, 2011) Key words: Abattoir, Cattle, Liver, Mesentery, PCR, S. spindale In this study, the phylogenetic analysis 12S rrnS of S. Schistosomosis in bovines is widely reported as a Schistosoma primers. No template control was maintained with isolate of S. spindale from Sri Lanka (EF534282) and spindale Karnataka were in the same clade as S. spindale disease entity in many parts of Asia and Africa. without a DNA template. DNA extracted from the blood 99 per cent with S. spindale isolate from Bangladesh isolate from Sri Lanka deposited in the GenBank Schistosomosis is one of the most prevalent infectious containing Babesia organisms was used as a negative (EF534281) and Nepal (KR607254). (EF534282) and shared 100 per cent identity and also diseases, endemic in more than 70 countries, mainly control. S. spindale DNA obtained from Dr. Sreenivasa shared 99.99 per cent identity with S. spindale from The present findings were in agreement with the within the developing world (Hovnanian et al., 2010). Murthy, Department of Veterinary Parasitology, Bangladesh (EF534281) and Nepal (KR607254). immunological and molecular studies on cattle and About 530 million of cattle live in areas endemic for Veterinary College, Hyderabad was used as positive Sreenivasa Murthy (2011) from Bengaluru had reported buffaloes in Bengaluru conducted by Sreenivasa Murthy bovine schistosomosis in Africa and Asia while at least 165 control. Primary pairs used and thermal cycles followed that phylogenetic analysis of 12S ribosomal rRNA showed (2011) who reported S. spindale, S. indicum and S. million cattle are infected with schistosomes worldwide are given in Table 1-3. that S. spindale was related to distinct cluster of S. nasale nasalein Bengaluru based on PCR. Sreenivasa Murthy (Bont and Vercruysse, 1997). Schistosomosis is now well from Bangladesh with 660 nucleotide substitutions. Sequence alignment and phylogenetic analysis: The (2011) detected and differentiated S. spindale, S. indicum, recognized as the fifth major helminthosis of domestic PCR products of 12S ribosomal RNA (rrnS) gene of Similarly, Lakshmanan (2014) from Kerala reported that S. nasale by using 12S rrnS and COI genus specific animals in the Indian sub-continent. Identification of Schistosoma spp. were sequenced at M/s Bioserve biotech, phylogenetic analysis of 28S rRNA of S. spindale from primers which yielded 328 bp and 1088 bp DNA fragments worms based on phenotypic may be difficult due to Pvt Ltd, Hyderabad. The BLAST analysis for the obtained Kerala shared 100 per cent identity with S. spindale from and these products were sequenced and analysed by NCBI overlapping of the characters viz., length of flukes, sequences was performed on GenBank database at Sri Lanka (AY157257). However, further research work is database for the species confirmation and phylogenetic intestinal caeca and number of testes. Molecular National Centre for Biotechnologies Information (NCBI) needed to know the prevalence of Schistosoma spp. in tree was constructed. Similar work was carried out by techniques based on genomes are very useful for the on the website www.ncbi.nlm.gov/ BLAST. The other regions of Karnataka and to implement proper Lakshmanan (2014) from Kerala and Hossain et al. (2015) epidemiological diagnosis as well as for research on sequences were aligned with the published sequences control measures. genetic variation of the parasitic organism. Hence, the deposited in the GenBank database and the phylogenetic from Bangladesh. present study was undertaken for the molecular tree was constructed by maximum likelihood method with Table 2 Primers and their sequences used to identify Schistosoma species characterization of the worms. 1000 bootstrap replicates using MEGA 6.0 software MATERIALS AND METHODS (Tamura et al., 2013). Name of Primer sequences (5´-3´) Amplicon size Targeted gene Species of Reference primer Schistosoma Extraction of genomic DNA: The collected worms from RESULTS AND DISCUSSION SPMit F CTTGGAGTCGGGTTGTTTGAG 330 bp Mitochondrial gene S. spindale Lakshmanan, 2014 liver and mesenteries were subjected to DNA extraction. PCR was carried out by targeting Schistosoma genus SP Mit R CAGACCCTCACACCAACAGTG The DNA was extracted from the worms using QIA amp specific 12S rrnS, COI and species specific SPMit and DNA extraction mini kit (Qiagen, Germany) as per the SI16s RNA genes. All the isolates yielded 328 (Fig. 1) and SI 6sRNAF GAGTTTGTAAATGGAGGCTGAG 606 bp 16sRNA S. indicum protocol described by the manufacturer. 1088 bp (Fig. 2) DNA fragments specific for Schistosoma SI 16sRNAR CCTTATTCAGCCTCTACACCG Attwood et al., 2007 Polymerase chain reaction: A total reaction volume of genus and 330 bp (Fig. 3) specific to S. spindale. No Table 3 25 l pre mixture of PCR consisting of two micro litre of  specific amplification was obtained for S. indicum. Genus PCR cyclical conditions for different genes of Schistosoma spp. forward primer, two micro litre of reverse primer, five specific 328 bp amplicon of both Bengaluru and Hassan Target gene Initial Denat- Annealing Extension No. of Final Reference micro litre of template DNA and 12.5 l of PCR master isolates were selected and were subjected for nucleotide denaturation uration cycles Extension mix (2X) (Genei Laboratories Pvt. Ltd.) and 3.5 l of NFW sequencing. The sequencing and BLAST analysis of 328 Schistosoma genus 94 ºC/5 min 94 ºC/30 sec 50 ºC/1 min 72 ºC/1 min 39 72 ºC/10 min Sreenivasa Murthy, 2011 was constituted. bp amplicon showed homology with S. spindale isolates deposited at GenBank database. Phylogenetic analysis Schistosoma spindale 95 ºC/5 min 95 ºC/1 min 54 ºC/1 min 72 ºC/1 min 35 72 ºC/10 min Hossain et al., 2015 The PCR was carried out in a gradient thermal cycler mitochondrium (SPMit) showed that S. spindale Karnataka isolate (Accession (Eppendorf, Germany) programmed for genus specific Schistosoma indicum 95 ºC/5 min 95 ºC/1 min 52 ºC/1 min 72 ºC/1 min 35 72 ºC/10 min Hossain et al., 2015 number MG052937) (Fig. 4) shared 100 per cent homology 16S rRna (SI 16SrRna) *Corresponding Author: [email protected]

190 191 L1: 100 bp ladder; L2: positive control; L3, L4, L5, L6 and L1: 3.5 kb ladder; L2: positive control; L3, L4, L5, L6 and Haryana Vet. (Dec., 2019) 58(2), 193-196 Research Article L7: Samples of S. spindale; L8: negative control. L7: Samples of S. spindale; L8: negative control. PREVALENCE AND RISK ASSESSMENT OF COCCIDIOSIS IN DAIRY ANIMALS OF ARID WESTERN PLAINS OF RAJASTHAN PRAVEEN PANWAR, ABHISHEK GUPTA* and POONAM CHOUDHARY Department of Veterinary Parasitology, College of Veterinary and Science, Bikaner, Rajasthan University of Veterinary and Animal Sciences, Bikaner -334001, India Received: 29.10.2018; Accepted: 21.08.2019 ABSTRACT The present investigation was conducted to determine the prevalence and risk assessment of coccidial infections in dairy animals of Arid Western Plains of Rajasthan. A total of 617 fecal samples (including 235, 188 and 194 samples from native cows, crossbred cows and buffaloes, respectively) were examined for a period of one year and revealed an overall prevalence rate of 25.45% (157/617) for coccidiosis. Risk assessment analysis viz. animal type wise and district wise analysis presented native cattle (30.21%) as the most infected and Jodhpur district (30.29%) as comparatively more conducive, respectively with a highly significant difference (p<0.01). Seasonal dynamics revealed highest prevalence in winter (30.88%) season with a non-significant difference. Quantitative analysis based on the estimation of oocysts per gram (OPG) of faeces revealed mild to moderate severity of Eimeria sp. infection ranging from 400-8300 with an average of 2206.25±518.56. Microscopic examination of sporulated Fig. 3. PCR analysis of mitochondrial gene (SPMit) S. spindale oocyst revealed presence of seven morphotypes of Eimeria viz., E. bovis (35.52%), as the major contributor followed by E. zuernii (18.03%), E. Fig. 2. PCR analysis of COI gene of S. spindale pellita (15.30%), E. auburnensis (12.02%), E. alabamensis (8.20%) and E. subshperica (5.20%) in the decreasing order of prevalence. Keywords: Arid western plains, Assessment, Coccidiosis, Control strategy, Morphotypes L1: 100 bp ladder; L2: positive control; L3, L4, L5, L6 and REFERENCES L7: Samples of S. spindale; L8: negative control. Economically cattle industry is one of the most of Rajasthan (Monika et al., 2017 and Choudhary et al., Attwood, S.W., Fatih, F.A., Fondal, M.M., Alim, M.A., Fadjar, S., Schistosoma spindale EF534282 important units throughout the world. Gastrointestinal 2018). In sequence to it, the present study was undertaken 0.004 Rajapakse, R.P. and Rollinson, D.A. (2007). DNA sequence- 0.008 Schistosoma spindale Karnataka based study of the Schistosoma indicum (: ) illnesses cause main hindrances to production, especially to determine the prevalence, species composition

0.011 Schistosoma spindale EF534281 group: population phylogeny, taxonomy and historical in relation to younger animals, their impact on production (morphotypes) of coccidian species affecting dairy 0.045 Schistosoma spindale KR607254 biogeography. Parasitol.134(14): 2009–2020. usually consists of the consequences of delayed growth animals of Arid western plains of Rajasthan along with the 0.057 Schistosoma cf. indicum KR607240 Bont, J.D.E. and Vercruysse, J. (1997). The epidemiology and control of and even mortality in some cases (Cruvinel et al., 2018). assessment of associated risk factors. cattle schistosomosis. Parasitol. Today. 13: 255-262. 0.078 KR607259 Coccidiosis is a faeco-oral route transmitting intracellular MATERIALS AND METHODS Schistosoma incognitum EF534279 Hossain, M.S., Begum, N., Rima, K.U., Dey, A.R. and Khan, A.H.N.L. 0.076 protozoan disease caused by Eimeria spp. with some FJ897168 (2015).Morphologic and molecular investigation of 0.015 Study area: Present work was performed in Arid western schistosomes from the mesenteric vein of slaughtered cattle. J. recent reports of Isospora spp. (Nalbantoglu et al., 2008) Schistosoma bovis AJ419785 plain zone of Rajasthan, covering Barmer and part of Agri. Vet. Sci. 8(5): 47-53. demanding regular monitoring of coccidial infection. Fasciola sp AB477369 Jodhpur district which includes part of Thar Desert, also Clinical coccidiosis in cattle mainly depends on factors Hovnanian, A., Hoette, S., Fernandes, C.J.C., Jardim, C. and Souza, R. known as the Great Indian Desert. This zone has an 0.30 0.25 0.20 0.15 0.10 0.05 0.00 Relative Times (2010). Schistosomosis associated pulmonary hypertension. like species of Eimeria involved, age of infected animal, average rainfall of 200-370 mm and temperature ranging Intern. J. Clinic. Pract. 165: 25-28. number of oocysts ingested, presence of concurrent Fig. 4. Phylogenetic analysis of 12S ribosomal RNA (rrnS) gene from 8 ºC to 40 ºC (D.O.A., Govt. of Rajasthan, 2016-17). Lakshmanan, B. (2014). Development of improved diagnostics for infections and management practices of the farm. Most nucleotide sequence of Schistosoma spindale The regions are enclosed with desert soils, sand dunes, detection of bovine intestinal schistosomosis. Ph.D. thesis species are considered to have a low pathogenicity; submitted to K.V.A.S.U, Thrissur, Kerala, India. aeolian soil and coarse sand with an erratic and uncertain ACKNOWLEDGEMENTS whereas infection with E. bovis or E. zuernii may cause rainfall witnessing frequent droughts. This paper is a part of the M.V.Sc. thesis submitted to Sreenivasa Murthy, G.S. (2011). Immunological and molecular studies severe disease in calves (Daugschies and Najdrowski, on schistosomes of cattle and buffaloes. Ph.D. thesis submitted Collection of samples: A total of 617 faecal samples KVAFSU, Bidar by the first author. The facilities and to K.V.A.F.S.U., Bidar, Karnataka, India. 2005) while E. alabamensis has been reported to cause (including 235, 188 and 194 samples from native cows, support extended by the ICAR, CAFT in Veterinary Tamura, K., Stecher, G., Peterson, D., Filipski, A. and Kumar, S. (2013). clinical disease in older animals. Animals develop age- Parasitology, Veterinary College, Bengaluru is gratefully MEGA 6: Molecular Evolutionary Genetics Analysis version related species-specific active humoral and cellular crossbred cows and buffaloes, respectively) were acknowledged. 6.0. Molecular boil. Evolution. 30: 2725-2729. immunity rapidly after first antigen contact to the randomly collected season-wise, per rectally or coccidian parasites. It plays a role in the protection of older immediately after defecation from the villages of two cattle that often serve as a source of infection for juvenile districts of Arid western plains of Rajasthan, during winter, animals, which are more susceptible to infection (Farkas et summer and rainy seasons for a period of one year during al., 2007). In spite of these facts there is a paucity in the January 2017 to December 2017. The samples were placed comprehensive data on coccidial infections of dairy in sterile polythene bags, properly labeled with information animals. However, some regional and sporadic reports regarding species, age, sex, deworming history and from different parts of the country have been published location, kept in a cool transport box and brought to the from time to time (Das et al., 2015; Gupta et al., 2016; laboratory for further examination. Murthy and D’Souza et al., 2016 and Nain et al., 2017). In recent past, studies have been conducted to assess the Coprological examination: The faecal samples were first coccidial infection in the dairy animals of different zones subjected to standard qualitative faecal sample *Corresponding author: [email protected] examination by using direct smear method and floatation

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