Advances in diagnostic approaches to Fasciola infection in animals and humans: An overviews.

Fasciolosis, caused by Fasciola hepatica and F. gigantica, is an impediment to the livestock industry's expansion and has a massively negative socio-economic impact due to its widespread prevalence in livestock. It is a waterborne zoonosis affecting human populations in the countries where rural economies are associated with livestock rearing. Conventional diagnosis of Fasciola infection is done by detecting parasite eggs in the faeces of infected animals or by immunological methods. Accurate and quick immunodiagnosis of Fasciola infection in animals and humans is based on the detection of antibodies and specific antigens expressed in the prepatent stage of the parasite. Both molecular and serodiagnostic tests developed thus far have enhanced the reliability of Fasciola diagnosis in both man and animals but are not widely available in resource-poor nations. A pen-side diagnostic test based on a lateral flow assay or a DNA test like loop-mediated isothermal amplification (LAMP) would be simple, fast, and cost-effective, enabling clinicians to treat animals in a targeted manner and avoid the development of drug resistance to the limited flukicides. This review focuses on the recent advances made in the diagnosis of this parasite infection in animals and humans.

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.

Molecular and serological detection of Anaplasma infection in carrier cattle in north India.

Anaplasma marginale infection in cattle (n = 216) in the states of Uttar Pradesh and Uttarakhand, North India was screened by microscopy and nested-polymerase chain reaction (PCR). Two recombinant proteins viz. major surface protein (MSP) 5 and MSP2 of A. marginale were expressed in Escherichia coli and their potential in the detection of antibodies to Anaplasma species in the cattle was evaluated by immunoglobulin G-enzyme linked immunosorbent assay (IgG-ELISA). The MSP5 IgG ELISA results were compared with competitive (c) inhibition ELISA. Microscopy being the least sensitive diagnostic test detected 12.0% of animals positive for A. marginale infection while nested-PCR detected 87.9% of these animals as positive for A. marginale infection. The recombinant MSP5 antigen showed positive reactivity in 170/190 nested-PCR confirmed positive animals (sensitivity 89.5%) with specificity of 77.0%. In comparison, the recombinant MSP2 antigen showed lesser sensitivity and specificity of 79.0% and 69.2%, respectively. The cELISA was more sensitive and specific than IgG-ELISA. However, molecular detection by msp5 nested-PCR was highly sensitive and reliable for detection of carrier cattle for Anaplasma infection. The study indicated that a large cattle population (87.9%) was carrier for A. marginale infection in this region of the country.

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.

Serodiagnosis of Fasciola gigantica Infection in Buffaloes with Native Cathepsin-L Proteases and Recombinant Cathepsin L1-D.

AIM: Serodiagnosis of Fasciola gigantica natural infection in buffaloes with recombinant cathepsin L1-D and native cathepsin-L protease antigens. METHODS: The recombinant cat L1-D antigen was expressed in prokaryotic expression system and native cathepsin-L proteases were purified by alcoholic fractionation from adult F. gigantica flukes. Buffaloes (n  = 325) were screened for anti-Fasciola antibodies with the above antigens in immunoglobulin-G-enzyme linked immunosorbent assay (IgG-ELISA). RESULTS: The recombinant cat L1-D antigen showed positive reactivity with 101/122 necropsy positive animals but 21/122 necropsy confirmed positive animals were negative in this ELISA (sensitivity 82.8%). However, 30/203 (14.8%) necropsy negative animals for Fasciola were seropositive with specificity of 85.2%. With native cat-L protease, 104/122 necropsy confirmed positive animals were ELISA positive but 18/122 necropsy positive animals were seronegative, thereby depicting the sensitivity of 85.2%. But ELISA with this antigen showed 27/203 (13.3%) necropsy negative animals as positive (specificity 86.7%). CONCLUSIONS: Comparative evaluation of both the antigens showed that they are suitable for serodiagnosis of F. gigantica infection in buffalo herds.

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 identification of Mecistocirrus digitatus and Toxocara vitulorum in the mithun (Bos frontalis) from north-east India.

Mecistocirrus digitatus and Toxocara vitulorum are common pathogenic nematode parasites of mithun (Bos frontalis). Species identification by morphological features was confirmed by molecular identification of M. digitatus and T. vitulorum. The internal transcribed spacer-2 (ITS-2) region and beta tubulin gene of M. digitatus were polymerase chain reaction (PCR) amplified and sequenced. ITS-2 sequence analysis showed 100% homology with other isolates of M. digitatus and 83% identity with Haemonchus contortus and H. placei, respectively. Likewise, ITS-1 and ITS-2 sequences of T. vitulorum were PCR amplified and sequenced. Sequence analysis of these internal transcribed spacers from five worms of the parasite from mithun showed no intraspecific variations with T. vitulorum isolates from domestic ruminants.

Report of Amblyomma testudinarium in mithuns (Bos frontalis) from eastern Mizoram (India).

Physical examination of semi-domesticated, free ranging mithuns (Bos frontalis) during an animal health check-up and treatment camp organized at Khuwangleng village in the Champhai district of Mizoram, India and adjacent to Myanmar Border revealed presence of unusually large blood engorged ticks attaching to the dewlap and inner aspects of thighs. On the basis of morphological study, the ticks were found indistinguishable from female Amblyomma testudinarium. Prevalence rate of the tick species in mithuns living in the forests was 9.09 % which was recorded as the highest of all reports made earlier from the North Eastern region of India. Medical and veterinary significance of the tick is discussed in the light of available literature.

Pimply gut condition in mithun (Bos frontalis) calves.

The present observation was recorded at National Research Centre on Mithun, Jharnapani from May 2010 to September 2012. A total of 15 mithun calves, which died in and around Jharnapani, were attended and detailed post-mortem examination was carried out. Out of these, five calves (33.33 %) aging between 1 and 1.5 years exhibiting the condition of chronic wasting and diarrhoea were found positive for pimply gut condition based on gross and microscopic examination. Post-mortem examination revealed extensive nodule formation on the wall of the rectum; however, the entire lumen did not reveal any of adult parasites. In all the cases, there were congestion in the mucous layer and thickening of the intestinal wall. Histopathological examination revealed chronic enteritis with mononuclear cell infiltration comprising mostly of macrophages, lymphocytes and eosinophils. In the muscularis mucosae, encysted larvae were found along with fibrous tissue proliferation. These lesions gave the intestine a nodular appearance as they thickened the wall and projected from the serosal surface. These extensive numbers of nodules in the intestine might have interfered with peristalsis and intestinal absorption which led to chronic wasting and diarrhoea in the calves.

Molecular detection and genetic diversity of Babesia gibsoni in dogs in India.

Babesia gibsoni is a tick borne intraerythrocytic protozoan parasite causing piroplasmosis in dogs and has been predominantly reported in Asian countries, including Japan, Korea, Taiwan, Malaysia, Bangladesh and India. The present communication is the first evidence on the genetic diversity of B. gibsoni of dogs in India. Blood samples were collected from 164 dogs in north and northeast states of India and 13 dogs (7.9%) were found positive for B. gibsoni infection by microscopic examination of blood smears. Molecular confirmation of these microscopic positive cases for B. gibsoni was carried out by 18S rRNA nested-PCR, followed by sequencing. Nested-PCR for the 18S rRNA gene was also carried out on microscopically B. gibsoni negative samples that detected a higher percentage of dogs (28.6%) infected with B. gibsoni. Genetic diversity in B. gibsoni in India was determined by studying B. gibsoni thrombospondin-related adhesive protein (BgTRAP) gene fragments (855bp) in 19 isolates from four north and northeast states of India. Phylogenetic analysis of the BgTRAP gene revealed that B. gibsoni parasite in India and Bangladesh formed a distinct cluster away from other Asian B. gibsoni isolates available from Japan, Taiwan and Korea. In addition, tandem repeat analysis of the BgTRAP gene clearly showed considerable genetic variation among Indian isolates that was shared by B. gibsoni isolates of Bangladesh. These results suggested that B. gibsoni parasites in a different genetic clade are endemic in dogs in India and Bangladesh. Further studies are required for better understanding of the genetic diversity of B. gibsoni prevalent in India and in its neighbouring countries.

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.

Molecular cloning and characterization of arginine kinase gene of Toxocara canis.

Toxocara canis is an important gastrointestinal nematode of dogs and also a causative agent of visceral larva migrans in humans. Arginine kinase (AK) gene is one of the important biomolecule of phosphagen kinase of T. canis which is emerging as an exciting novel diagnostic target in toxocarosis. The present study was carried out to clone and characterize AK gene of T. canis for future utilization as a diagnostic molecule. Total RNA was extracted from intact adult worms and reverse transcription was done with oligo dT primers to obtain complementary DNA (cDNA). Polymerase chain reaction (PCR) was carried out using cDNA as template with specific primers which amplified a product of 1,202 bp. The amplicon was cloned into pDrive cloning vector and clone was confirmed by colony PCR and restriction endonuclease analysis. Sequence analysis of the gene showed 99.8 and 77.9 % homology with the published AK gene of T. canis (EF015466.1) and Ascaris suum respectively. Structural analysis shown that the mature AK protein consist of 400 amino acids with a molecular wt of 45360.73 Da. Further expression studies are required for producing the recombinant protein for its evaluation in the diagnosis of T. canis infection in humans as well as in adult dogs.

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.

Characterization of excretory-secretory antigens of adult Toxocara canis by western blotting.

Toxocara canis is one of the most common helminth worm of dogs which continues to stimulate both public health concern alongside the higher scientific interest. It may cause visceral and ocular damage in humans especially in children. The identification of specific antigens of T. canis is important so as to develop better diagnostic techniques. Excretory-secretory (ES) antigens were prepared by culturing the adult T. canis worms in RPMI 1640 medium without serum supplementation followed by ammonium sulphate precipitation. These antigens were separated using sodium dodecyl sulphate-electrophoresis (SDS-PAGE). Recovered proteins ranged from 30 to 384 kDa. The specific reactivity of the T. canis excretory-secretory (TC-ES) proteins was checked by western blotting. The immuno-reactivity of the naturally infected dog sera with the TC-ES antigens showed five bands at 43, 57,105, 139 and 175 kDa. The immuno-reactivity of the hyper immune serum raised in rabbits against TC-ES antigens was observed with ten polypeptides of 21, 25, 30, 37, 45, 50, 57, 69, 77 and 105 kDa. Common antigens band were observed at 57 and 105 KDa. These antigens merit further evaluation as candidate for use in diagnosis of toxocariasis in humans and adult dogs.

Prevalence of canine toxocariasis in Bareilly, Uttar Pradesh, India.

Toxocara canis is one of the most common parasitic helminth worm of dogs and also a causative agent of zoonotic disease in humans. This pilot study was conducted to determine the presence of T. canis infection in dog population in and around Bareilly, Uttar Pradesh, India. A total of 558 faecal samples both from stray and owned dogs were screened and overall 24.3 % dogs were found positive for T. canis. A comparison between owned and stray dogs suggests that the higher prevalence was observed in the latter group. The age of the dogs had a considerable influence on prevalence, with a much higher proportion of younger dogs being infected. Among the stray dogs, the infection rate is much higher (62.79 %) in pups, as compared to 7.8 % in adult. Similarly, of the owned dogs screened 41.74 % pups were infected while the infection rate in adults was only 3.38 %. The higher rate of prevalence of this parasite in dogs could be the source of soil contamination for transmission of Toxocariasis which is of public health importance in this region.

Evaluation of Gastrothylax crumenifer antigenic preparation in serodiagnosis of paramphistomiasis in sheep.

An evaluation of Gastrothylax crumenifer crude antigen preparation viz., Somatic Antigen (SAg), Excretory Secretory Antigen (ESAg) and Egg Antigen (EAg) in serodiagnosis of disease was undertaken. Test sera samples were obtained from 30 Paramphistomiasis Positive and 30 Gastrothylax free sheep slaughtered at Hazratbal Kashmir. The referral antigenic preparation were evaluated against Paramphistomiasis positive sera, via., control negative sera, using double immunodiffusion test (DID), (IEP) Immunoelectrophoretic assay and ELISA. The performance of referral antigens, as assessed from percent sensitivity and specificity, revealed an increasing trend from DID (Double immunodiffusion-An immunological technique used in the detection, identification and quantification of antibodies and antigens) to IEP (immunoelectrophoresis-A general name for a number of biochemical methods for separation and characterization of proteins based on electrophoresis and reaction with antibodies), followed by ELISA, detecting higher number of sheep positive for paramphistomiasis. In ELISA the ESAg and SAg were evaluated as most reactive antigens with no significant difference and EAg was the least antigenic. In IEP, EAg had the higher sensitivity (60%) and analogous specificity of SAg and ESAg. The formation of the preceptin lines in the proximity to EAg containing wells (cathode end) in IEP was suggestive of higher molecular weight of G. crumenifer specific protein molecules with slower rate of migration. Purification and characterization of G. crumenifer and identification of specific antigenic molecules, particularly in EAg has been suggested for qualitative improvement of diagnostic value of the antigens in the tests used here in.

Th1 and Th2 cytokine gene expression in primary infection and vaccination against Fasciola gigantica in buffaloes by real-time PCR.

Th1 and Th2 cytokine gene expression in buffalo calves during primary infection with Fasciola gigantica as well as in response to immunization with the parasite recombinant fatty acid binding protein (rFABP) and recombinant glutathione S-transferase (rGST) proteins was measured at 14th week of infection by real-time PCR with the double-stranded DNA-binding dye SYBR Green. Experimental animals were randomly distributed into FABP, GST, cocktail, challenge and healthy groups. Animals in groups FABP and GST were immunized with 400 µg rFABP and rGST, respectively, and cocktail group with a mixture of 400 µg each of rFABP and rGST in the neck and thigh muscles. All animals received three immunizations at 3-week interval. Calves were challenged per os with 400 viable metacercariae along with the unimmunized challenge control group 1 month after the last immunization. Expression of various cytokines in response to the immunization and parasite primary infection was measured by real-time PCR. Expression of IL-2 (4.5-fold) and IFN-γ (3.2-fold), followed by IL-6 (1.7-fold) and IL-4 (1.6-fold), with downregulation of TNF-α and IL-10 was observed in response to F. gigantica infection in these animals. However, there was a sharp increase in the expression of the IL-4 (211.93 and 111.81-fold) and IL-6 mRNA (219.22 and 48.29-fold) to GST and FABP immunizations, respectively. A downregulation of the IL-1α, a Th1 cytokine in response to FABP and GST immunization in these calves, was also observed. Overall, a mixed type of Th1 and Th2 cytokine environment was evoked to chronic F. gigantica infection and immunization with the above two recombinant proteins in buffaloes.

Edible vaccines against veterinary parasitic diseases--current status and future prospects.

Protection of domestic animals against parasitic infections remains a major challenge in most of the developing countries, especially in the surge of drug resistant strains. In this circumstance vaccination seems to be the sole practical strategy to combat parasites. Most of the presently available live or killed parasitic vaccines possess many disadvantages. Thus, expression of parasitic antigens has seen a continued interest over the past few decades. However, only a limited success was achieved using bacterial, yeast, insect and mammalian expression systems. This is witnessed by an increasing number of reports on transgenic plant expression of previously reported and new antigens. Oral delivery of plant-made vaccines is particularly attractive due to their exceptional advantages. Moreover, the regulatory burden for veterinary vaccines is less compared to human vaccines. This led to an incredible investment in the field of transgenic plant vaccines for veterinary purpose. Plant based vaccine trials have been conducted to combat various significant parasitic diseases such as fasciolosis, schistosomosis, poultry coccidiosis, porcine cycticercosis and ascariosis. Besides, passive immunization by oral delivery of antibodies expressed in transgenic plants against poultry coccidiosis is an innovative strategy. These trials may pave way to the development of promising edible veterinary vaccines in the near future. As the existing data regarding edible parasitic vaccines are scattered, an attempt has been made to assemble the available literature.

Development of cathepsin-L cysteine proteinase based Dot-enzyme-linked immunosorbent assay for the diagnosis of Fasciola gigantica infection in buffaloes.

Native cathepsin-L cysteine proteinase (28 kDa) was purified from the excretory secretory products of Fasciola gigantica and was used for sero-diagnosis of F. gigantica infection in buffaloes by Dot-enzyme-linked immunosorbent assay (Dot-ELISA). The test detected F. gigantica field infection in these animals with a sensitivity of ∼ 90%. No specific IgG antibody binding was displayed by sera obtained from 76 buffaloes considered to be Fasciola and other parasite-free by microscopic examination of faeces and necropsy examination of liver, rumen and intestine. Additionally, sera from 156 Fasciola-free buffaloes, yet infected with Gigantocotyle explanatum, Paramphistomum epiclitum, Gastrothylax spp., Strongyloides papillosus and hydatid cyst were all negative, indicating that F. gigantica cathepsin-L cysteine proteinase does not cross-react with these helminth parasites in natural infection of the host. The data indicated that cathepsin-L cysteine proteinase based Dot-ELISA reached ∼ 90% sensitivity and 100% specificity with relation to above parasites in the detection of bubaline fasciolosis. The present Dot-ELISA diagnostic assay is relevant to the field diagnosis of F. gigantica infection in buffaloes.