Molecular prevalence of tick-borne haemoprotozoan diseases in cattle of North Eastern state of Tripura, India.

The tick-borne haemoprotozoan diseases are major constraint in the health and production of cattle and causes substantial losses to the livestock industry throughout the world. Tripura is a northeastern state of India characterized by a warm humid tropical climate and high rainfall which is conducive to the multiplication of vector-ticks of haemoprotozoan diseases. A total of 320 tick-infested cattle (22 male and 298 female) of either sex was selected randomly from different district of Tripura for the detection of carrier cattle infected with tick-borne haemoparasites from July, 2017 to June, 2019. Out of 320 tick-infected cattle, 4.69% prevalence of B. bigemina infection in cattle was found while Theleria spp. was not prevalent in the collected blood sample by using polymerase chain reaction. The prevalence of B. bigemina was significantly (< 0.05) higher in cattle more than 3 years of age (6.72%) compared to the 1-3 years age group (3.97%) and < 1 year age groups (1.66%). Sex-wise B. bigemina infection was significantly (< 0.05) seen only in female (5.03%) cattle infested with ticks. The district-wise B. bigemina prevalence (%) in Tripura was as in cattle; Dhalai (0.63), Sipahijala (0.31), Khowai (0.31), Gomati (0.31), South Tripura (0.63), North Tripura (0.31), Unakoti (0.31) and West Tripura (1.87) respectively. The use of PCR assays for the detection of B. bigemina and Theileria spp. in the North Eastern state of Tripura of India showed prevalence of 4.69% and 0%, respectively in tick-infested cattle. This study is the first report about the prevalence of tick-borne haemoprotozoan diseases from cattle of Tripura and suggest that babesiosis is more common disease as compared to theileriosis and is more prevalent in adult female tick-infested cattle in Tripura.

Genetic manipulations in helminth parasites.

Screening of vaccine or drug target in parasitic helminth is hindered by lack of robust tool for functional studies of parasite protein which account for the availability of only a few anti-helminthic vaccines, diagnostic assay and slower pace of development of an anthelmintic drug. With the piling up of parasite transcriptomic and genomic data, in silico screening for possible vaccine/drug target could be validated by functional characterization of proteins by RNA interference or CRISPR/Cas9. These reverse genetic engineering tools have opened up a better avenue and opportunity for screening parasitic proteins in vitro as well as in vivo. RNA interference provides a technique for silencing targeted mRNA transcript for understanding a gene function in helminth as evidence by work in Caenorhabditis elegans. Recent genetic engineering tool, CRISPR/Cas9 allows knock-out/deletion of the desired gene in parasitic helminths and the other provision it provides in terms of gene knock-in/insertion in parasite genome is still to be explored in future. This manuscript discussed the work that has been carried out on RNAi and CRISPR/Cas9 for functional studies of helminth parasitic proteins.

Superoxide dismutase inhibits cytotoxic killing of Fasciola gigantica newly excysted juveniles expressed by sheep invitro.

Fasciola gigantica faces a series of threats from various free radicals produced by the host immune system during its invasion through the abdominal cavity and establishment in the bile duct of ruminants, limiting the fluke viability. The role of the superoxide radical produced by Muzaffarnagari sheep immune effector cells against F. gigantica newly excysted juveniles (NEJs) is highlighted in this study, as is the critical role of superoxide dismutase enzyme (SOD) in dismutation of superoxide radicals derived from host immune effector cells in vitro. Three concentrations of the ovine immune effector cells viz. 2.5, 5, and 10 × 106 cells were tested for their ability to induced cytotoxic killing of the parasite. All the three cell concentrations caused significant (p < 0.01) cytotoxic killing of NEJs in comparison to the control groups. Also, reduction of the immune effector cell concentration directly correlates with the NEJs killing. Attachment of immune effector cells to the parasite tegument in the presence of anti-F. gigantica antibodies was found to be critical in inducing NEJs killing via antibody-dependent cell-mediated cytotoxicity (ADCC). However, the addition of SOD greatly inhibits cytotoxic killing of NEJs, demonstrating the importance of SOD enzyme in fluke survival and parasite evasion of the host immunity. Thus, F. gigantica SOD warrants a promising candidate for immunoprophylactic studies in ruminants against the tropical liver fluke.

Bovine babesiosis in India: Estimation of prevalence by systematic review and meta analysis.

Bovine babesiosis is a serious threat to the livestock sector especially in tropical countries like India. Understanding the epidemiology of the disease in the country is essentially important in strategizing the available methods to effectively control the disease. Keeping this as the background, the present study was undertaken to estimate the pooled prevalence of bovine babesiosis in India. The relevant literature pertaining to bovine babesiosis was identified and a total of 49 studies published between 1983 and 2018 were included in the final systematic review and meta-analysis. Meta-analysis was conducted using meta-package of R software and prevalence estimates were calculated. Bovine babesiosis was reported from 21 states of India with pooled prevalence estimate of 6% (95% CI = 4%-9%) using random effect model. Zone wise analysis revealed highest pooled prevalence in the west zone and north zone (8%) followed by east zone (7%), central zone (6%), south zone (4%) and northeast zone (4%). The results of meta-analysis indicated high variability between studies. In addition, the pooled seroprevalence was high (29%) compared to prevalence of active infection (5%) of bovine babesiosis in India. Further, the pooled prevalence estimate of B. bigemina infection in India was more (7%) compared to B. bovis infection (1%). The estimation of prevalence of active infection and seroprevalence separately will helps to understand the actual disease prevalence in the country. The study indicated the wide prevalence of bovine babesiosis in India which urges for immediate mitigation strategies.

Fasciolosis in India: An overview.

Fasciolosis in ruminants is a relentless constraint in the livestock industry across the world. Immuno-prophylactic vaccines against fasciolosis may not come up in near future, rendering the control of this scourge with chemotherapy and snail population control. With the alarming threats of anti-fasciolid drug resistance reported from certain parts of the world; the control of fasciolosis should be directed towards the development of rapid and reliable diagnostic tools to execute the specific and discrete treatment. Understanding the epidemiology of Fasciola, its genomics and proteomics, host-parasite interplay, and advances in drug design research is vital for improving animal health that would ultimately succour to meet the ever-increasing demand for food. Due to possible differences in immune response depending on the species of the host and parasite, immuno-prophylactic studies in India should aim at achieving protective efficacy in buffalo against F. gigantica as workers from other countries concentrate primarily on vaccination of cattle and sheep against F. hepatica. This manuscript focused on the research that has been carried out in India for understanding the epidemiology, genetic diversity, immuno-diagnosis, and possible control measure in terms of immuno-prophylaxis and drug designing against tropical fasciolosis caused by Fasciola gigantica.

Evaluation of Echinococcus granulosus recombinant EgAgB8/1, EgAgB8/2 and EPC1 antigens in the diagnosis of cystic echinococcosis in buffaloes.

Three recombinant proteins of Echinococcus granulosus including two antigen B sub-units EgAgB8/1 and EgAgB8/2 and Echinococcus protoscolex calcium binding protein 1 (EPC1) were expressed in prokaryotic expression vectors. The diagnostic potential of these three recombinant proteins was evaluated in the detection of cystic echinococcosis in buffaloes in IgG-ELISA. The EgAgB8/1 and EgAgB8/2 recombinant proteins reacted fairly with the hydatid infected buffaloes with sensitivity of 75.0% and 78.6%, respectively and specificity of 75.8% while EPC1 recombinant protein showed higher sensitivity (89.3%) but lower specificity (51.5%). Cross-reactivity of these three antigens was assayed with buffalo sera naturally infected with Explanatum explanatum, Paramphistomum epiclitum, Gastrothylax spp., Fasciola gigantica and Sarcocystis spp. EgAgB8/1 and EPC1 antigens cross-reacted with all these sera while EgAgB8/2 showed no cross-reaction with Sarcocystis spp. and reacted with some of the E. explanatum infected buffalo sera. This study explores the potential of three hydatid antigens viz. EgAgB8/1, EgAgB8/2 and EPC1 for their diagnostic potential in buffaloes positive for cystic echinococcosis.

Molecular characterization of veterinary important trematode and cestode species in the mithun Bos frontalis from north-east India.

Helminth infections in the mithun Bos frontalis, including the liver fluke Fasciola gigantica, hepato-gastric amphistomes Explanatum explanatum, Paramphistomum epiclitum and Calicophoron calicophorum, and the cestodes Echinococcus granulosus and E. ortleppi were studied in north-east India over a 2-year period from 2012 to 2014. Cystic echinococcosis caused by E. granulosus and E. ortleppi was found to be highly prevalent in the mithun, with E. ortleppi being reported for the first time. Molecular markers, including the internal transcribed spacer 2 (ITS-2), 28S rDNA and mitochondrial NADH dehydrogenase sub-unit1 (nad1) were used to confirm the identification of the trematode and cestode species.

Isolation and characterization of Cu/Zn-superoxide dismutase in Fasciola gigantica.

A full-length complementary DNA (cDNA) encoding Cu/Zn-superoxide dismutase was isolated from Fasciola gigantica that on nucleotide sequencing showed a close homology (98.9%) with Cu/Zn-superoxide dismutase (SOD) of the temperate liver fluke, F. hepatica. Expression of the gene was found in all the three developmental stages of the parasite viz. adult, newly excysted juvenile and metacercaria at transcriptional level by reverse transcription-polymerase chain reaction (RT-PCR) and at the protein level by Western blotting. F. gigantica Cu/Zn-SOD cDNA was cloned and expressed in Escherichia coli. Enzyme activity of the recombinant protein was determined by nitroblue tetrazolium (NBT)-polyacrylamide gel electrophoresis (PAGE) and this activity was inactivated by hydrogen peroxide but not by sodium azide, indicating that the recombinant protein is Cu/Zn-SOD. The enzyme activity was relatively stable at a broad pH range of pH 4.0-10.0. Native Cu/Zn-superoxide dismutase protein was detected in the somatic extract and excretory-secretory products of the adult F. gigantica by Western blotting. NBT-PAGE showed a single Cu/Zn-SOD present in the somatic extract while three SODs are released ex vivo by the adult parasite. The recombinant superoxide dismutase did not react with the serum from buffaloes infected with F. gigantica. The role of this enzyme in defense by the parasite against the host reactive oxygen species is discussed.

Genetic characterization of Fasciola gigantica from different geographical regions of India by ribosomal DNA markers.

Ribosomal DNA sequences of the second internal transcribed spacer (ITS-2) and 28S ribosomal DNA (618 bp) of Fasciola gigantica collected from cattle and buffaloes from four different geographical locations of India, were characterized for genotyping. ITS-2 sequence was analyzed in 28 worms that was typical of F. gigantica and differed at six positions, with one of these being a distinguishing deletion (T) at the 327th position in F. gigantica relative to F. hepatica. However, Fasciola specimens also showed intraspecies sequence polymorphism in the ITS-2, with two different ITS-2 sequences existing in the ribosomal DNA (rDNA) array within a single Fasciola worm. One of the sequences was identical to that of F. gigantica and the other showed extensive sequence polymorphism in the ITS-2. Using BspH1-restriction fragment length polymorphism, six variable ITS-2 sequences in F. gigantica were identified within these parasite specimens and were found distributed in these four geographical regions. 28S rDNA sequence of 24 flukes, collected from the above four geographical regions, showed a single nucleotide polymorphism at 284th nucleotide (G/A). Analyzing the sequence data of 28S rDNA of F. gigantica available from some African and Asian countries for this polymorphic 284th nucleotide position, it is proposed that there are two basic lineages of the F. gigantica for 28S rDNA existing in the fluke populations from five African and several Asian countries.