Collection of cysts

Larval stages of the parasite were collected from buffalo from a slaughterhouse under the jurisdiction of the Kolkata Municipality Corporation, West Bengal, India. The cysts were grouped into six different morphotypes as follows. Cysts with a white germinal layer with protoscoleces and clear cystic fluid were designated as S1. Cysts with transparent germinal layer without protoscoleces and clear cystic fluid were termed S2. A designation of S3 was given to those cysts that contained protoscoleces, germinal layer and turbid cystic fluid. Cysts with a white germinal layer and clear cystic fluid without protoscoleces were categorized as S4. Those with slightly yellowish and thickened germinal layer, clear fluid without protoscoleces were termed S5. And finally, cysts with no protoscoleces and calcified and turbid cystic content were designated as S6. Ten samples of S1 cysts were collected while eight cysts each were collected from the remaining S2, S3, S4, S5 and S6 categories.

Isolation of protoscoleces and the germinal layer

Protoscoleces and the germinal layer from both the fertile and the sterile cysts were isolated, rinsed in sterilized phosphate-buffered saline (PBS) (pH 7.2) three times and stored until further use (Eckert et al., Reference Eckert, Gemmell, Meslin and Pawlowski2001).

DNA extraction

Genomic DNA was isolated from protoscoleces and the germinal layer using the B10 101^® system GNOME^® (Qbiogene/MP Biomedicals, Irvine, California, USA) according to the protocol provided within the kit. The same kit and methodology were used for both sterile and fertile cysts. The genomic DNA was dissolved in 25 μl Tris–EDTA (TE) buffer (pH 8.0).

RNA extraction and cDNA preparation

Total RNA was isolated from fresh protoscoleces and the germinal layers using TRI Reagent^® (Sigma, St Louis, Missouri, USA). Each sample was incubated for 45 min at 37°C with RQ1 RNase-free DNase (Promega, Madison, Wisconsin, USA) and heat inactivated at 65°C for 10 min using DNase-stop buffer (Promega). Total RNA was quantified after determination of optical density at 260 nm. A total of 3 μg of RNA was used for cDNA preparation for each isolate. The cDNA was synthesized from each RNA sample using high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, California, USA). cDNA samples were kept at − 70°C until further use.

Primer design and semi-quantitative RT-PCR analysis

Primers were used to amplify actin II (housekeeping gene) and 14-3-3 protein using Primer Premier Version 5 (PREMIER Biosoft International, Palo Alto, California, USA). All primers were designed from available actin II sequence information (accession number L07774) and 14-3-3 protein (accession number AY 265907). The designs of primers were:

• Actin II forward: 5′-CGAGCAGGAAATGATAACG-3′

• Actin II reverse: 5′-GAA CAG GGC TTC AGG ACA-3′

• 14-3-3 gene forward: 5′-ATG TCT TCT CTC AGT AAG CGC G- 3′

• 14-3-3 gene reverse: 5′-CTT TTC AAG TTG CGC TCA GTT C-3′.

Polymerase chain reaction (PCR) was performed using GeneAmp^® PCR System 9700 (Applied Biosystems) in a final 25 μl volume containing 200 μm of each deoxynucleoside triphosphate (dNTP) (Genei, India), 10 × PCR buffer, 2.5 mm MgCl₂, 10 pmol of each gene-specific primer and 1 unit of Taq DNA polymerase (Fermentas, Glen Burnie, Maryland, USA) and an equal volume of cDNA was used as template for each isolate in reaction buffer. Briefly, the PCR process comprised an initial denaturing step (95°C for 5 min.); followed by 35 cycles of 95°C for 45 s (denaturation), annealing at 56°C (Eg14-3-3) or 61°C (actin II) for 45 s and elongation at 72°C for 90 s; and a final extension step (72°C for 10 min). The specificity and size of the amplified product was assessed by electrophoresis in 2% (w/v) Tris acetate–EDTA agarose gel stained with ethidium bromide. The amplified product was quantified directly using GelQuant software version 2.7 (DNR Bio-Imaging Systems Ltd., Israel). Relative quantification of expression of Eg14-3-3 was normalized against actin II (housekeeping gene or internal control) in each isolate. The transcription levels of the selected 14-3-3 gene were compared with the housekeeping gene and relative quantification was determined as a ratio.

Statistical analysis

Data were expressed as mean ± standard error (SE) and evaluated for their statistical significance of difference with analysis of variance (ANOVA). P values of less than 0.05 were considered to be statistically significant.

Analysis of E. granulosus genotype

Genotyping of all isolates was accomplished by mitochondrial adenosine triphosphatase (ATPase) subunit 6 (ATP6) sequence, as previously described (Gudewar et al., Reference Gudewar, Pan, Bera, Das, Konar, Rao, Tiwari and Bhattacharya2009).

In silico analysis

DNA sequences were aligned with other reported 14-3-3 isoforms of E. granulosus available in a public database using CLUSTAL W analysis by MEGA 4 (Tamura et al., Reference Tamura, Dudley, Nei and Kumar2007). The predicted amino acid sequence of 14-3-3 was deduced on the basis of genetic codes available at the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov). Clustering of the 14-3-3 isoforms was done by the unweighted pair group method with arithmetic mean (UPGMA) using the above-mentioned programme (Tamura et al., Reference Tamura, Dudley, Nei and Kumar2007).