Molecular characterization and serodiagnostic evaluation of the Echinococcus ortleppi recombinant glutaredoxin 1 protein for cystic echinococcosis in buffalo (Bubalus bubalis).

Cystic echinococcosis (CE), caused by the metacestode of Echinococcus granulosus sensu lato (s.l.), adversely affects the physiology of the vital organs in which they grow. Condemnation of meat causes substantial economic loss to the livestock industry. Conventionally the infection is detected by necropsy as serological diagnosis of the infection in livestock is ambiguous. Identification of specific diagnostic antigens would be a substitute for the cyst fluid antigens which lack adequate diagnostic sensitivity and specificity. BLAST analysis supported by the negligible pairwise nucleotide distance of the 389 nt COX1, 489 nt NAD1, and 425 nt ITS1 with the related sequences of E. ortleppi ascertained the association of E. ortleppi with CE in buffaloes. Given the extensive distribution of glutaredoxin 1 in every developmental stage of Echinococcus granulosus s.l that makes it an ideal serodiagnostic antigen for CE, we expressed the 14 kDa E. ortleppi glutaredoxin 1 (rEoGrx1) protein in E. coli BL21 (DE3) and tested a total of 225 sera samples, including 126 sera samples from the necropsy-positive buffalo, by the rEoGrx1 IgG-ELISA. The ELISA could detect a total of 82/126 sera samples as positive. The diagnostic sensitivity and specificity of the rEoGrx1 IgG-ELISA were 65.1 % and 51.5 %, respectively. The protein showed serological cross-reaction against Fasciola gigantica, Toxoplasma gondii, and Sarcocystis sp. The in silico bioinformatics analysis of the E. ortleppi, F. gigantica, and T. gondii glutaredoxin sequences revealed fully conserved amino acids at positions 11 and 21, the substitution of conserved amino acids at positions 14 and 6, and semi-conserved substitutions at positions 3 and 4, respectively. The findings partly explain the molecular basis of the serological cross-reactivity of the protein.

Molecular detection of Coxiella-like endosymbionts in Rhipicephalus microplus from north India.

Apart from the tick-borne pathogens affecting human and animal health, ticks also harbor various non-pathogenic endosymbionts with dynamic ecological interactions. These endosymbionts are unexplored from the Indian ticks; hence this pilot study was conducted. Seventy-nine ticks were collected from Nainital district of Uttarakhand state of north India and were identified as Rhipicephalus microplus morphologically and by molecular analysis. PCR and sequence analysis were carried out to detect the presence of Rickettsia-like, Coxiella-like and Francisella-like endosymbionts in these ticks. Based on the partial 16S rRNA gene sequence, Coxiella-like endosymbiont (CLE) was detected in the adult and other life-cycle stages of ticks with 96.6-97.7% nucleotide sequence identity with the published CLE sequences from GenBank. The phylogenetic analysis revealed that the CLE from R. microplus were clustered with the CLE from other Rhipicephalus species. All these CLE formed distinct clades from the pathogenic Coxiella burnetii. None of the tick samples was found positive for Rickettsia-like and Francisella-like endosymbionts in the present study. We also demonstrated the vertical transmission of CLE from surface sterilized and laboratory reared fully engorged adult females to the eggs and the larvae. However, large scale studies are to be conducted to detect various endosymbionts and endosymbiont-tick associations in the Indian tick species and to explore these associations for tick and tick-borne disease control.

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.

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.