Phylogenetic analysis and haplotype networking of Hepatozoon felis infecting wild animals in Gir National Park, Gujarat, India.

The present study was aimed to determine the phylogenetic relationship, haplotype network, and demographic dynamics of H. felis infecting the endangered Asiatic lions in Gir National Park, Gujarat, India, on the basis of partial 18S rRNA gene. The phylogenetic analysis based on the partial 18S rRNA gene sequences of H. felis exhibited the presence of two distinct genotypes of H. felis (HfG1 and HfG2) infecting the Indian wild felids, viz., the Asiatic lion, Royal Bengal tiger, and Indian leopard. The HfG1 and HfG2 genotypes exhibited 97.6-100% and 99.7-100%, and 96.9-98.7% nucleotide identity within and between themselves, respectively. The HfG1 genotype exhibited a higher genetic diversity as compared to HfG2. A total of 22 molecular signatures were identified in the 18S rRNA gene between these genotypes. Further, analysis of a total 67 sequences of H. felis (13 different host species from 13 countries of Africa, South America, Asia, and Europe) that were downloaded from GenBankTM, generated 30 haplotypes. Among all the haplotypes, Hap_17 (h=12) was the most frequent followed by Hap_12 (h=09) and Hap_4 (h=05). Out of 13 location-wise populations, India (h=12) shared the highest number of haplotypes followed by Japan (h=08), and the least number of haplotypes were found in Hungary (h=02). Population dynamics study involving neutrality tests and mismatch distribution, and genetic differentiation indices, revealed the presence of phylogeographic population structure and a constant population size indicating a uniform gene flow among the populations worldwide. In conclusion, a high genetic diversity along with the presence of two distinct genotypes of H. felis were observed on the basis of 18S rRNA gene sequence analysis.

Genetic and population diversity of Toxocara cati (Schrank, 1788) Brumpt, 1927, on the basis of the internal transcribed spacer (ITS) region.

The present investigation was aimed to study the sequence, phylogenetic and haplotype analyses of Toxocara cati based on the ITS region, along with the genetic diversity, demographic history and population-genetic structure. The maximum likelihood tree based on Kimura 2-parameter model was constructed using the complete ITS region of all the nucleotide sequences (n = 57) of Toxocara spp. and other related ascarid worms available in the GenBank™. It placed all the sequences of T. cati into four major clades designated as T. cati genotypes 1-4 (TcG1-G4). A total of 66 signature nucleotides were identified in the ITS region between genotypes. The median-joining haplotype network displayed a total of 24 haplotypes, with China exhibiting the highest number of haplotypes (h = 20) followed by India (h = 4), and Japan and Russia (h = 1). It indicated a clear distinction between all the four genotypes. The pairwise FST values between all the genotypes indicated huge genetic differentiation (> 0.25) between different T. cati genotypes. Moreover, the gene flow (Nm) between T. cati genotypes was very low. Results of AMOVA revealed higher genetic variation between genotypes (92.82%) as compared to the variation within genotypes (7.18%). The neutrality indices and mismatch distributions for the G1-G4 genotypes, Indian isolates and the overall dataset of T. cati indicated either a constant population size or a slight population increase. The geographical distribution of all the genotypes of T. cati is also reported. This is the first report of genotyping of T. cati on the basis of the ITS region.

Molecular insights into the population structure and haplotype network of Theileria annulata based on the small-subunit ribosomal RNA (18S rRNA) gene.

The present study was conducted to elucidate the population genetic diversity and haplotype network of Theileria annulata based on all available nearly complete 18S rRNA gene sequences in the GenBank™. In total, 52 sequences of the nuclear 18S rRNA gene used to assess the relationship of T. annulata with their country of origin identified 34 haplotypes. Haplotype 4 was widespread, occurring in India, China, Turkey and Iran, while the remaining haplotypes were singleton and unique to one country. Haplotype 4 displayed numerous single haplotypes around it and the stellate shape of the network suggested a rapid population expansion. India exhibited the largest number of haplotypes (h = 25) followed by Turkey (h = 6), China (h = 4), and Iran and Italy (h = 1). No geographical clustering of haplotypes was recorded. Nucleotide diversity was the highest in the Turkish followed by the Indian and Chinese populations. Similarly, haplotype diversity was the highest in China followed by Turkey, and the lowest in India. The overall dataset exhibited a low nucleotide diversity (0.00253 ± 0.00035), but high haplotype diversity (0.917 ± 0.034). It suggested the presence of only minor differences (01-11 nucleotide) between haplotypes which was also evident from the haplotype network. A high level of genetic diversity was documented within the Indian, Chinese and Turkish populations of T. annulata, whereas little genetic differentiation was noticed among these populations with a very high level of gene flow. Analysis of molecular variance (AMOVA) of T. annulata sequences revealed higher genetic variation within countries (83.58%) as compared to the variation among countries (16.42%). Neutrality indices, viz., Tajima's D, Fu and Li's F, Fu's Fs, and R2, along with the unimodal mismatch distributions demonstrated a recent population expansion of T. annulata in India and the overall dataset. However, the non-significant values of Tajima's D, Fu and Li's F, and Fu's Fs for the Chinese population along with a bimodal mismatch distribution signified a constant population size. For the Turkish population, the neutrality and mismatch distribution tests either indicated a constant or a slight increase in population size. The present study provides novel insights into the population genetics and haplotype network of T. annulata based on the 18S rRNA gene for the first time.

Genetic diversity in the Tams1 gene of Theileria annulata (Duschunkowsky and Luhs, 1904) infecting cattle.

The present study describes the genetic diversity in the Tams1 gene (733 bp) of Theileria annulata along with the sequence, phylogenetic and haplotype analyses of the Indian isolates. The phylogenetic analyses displayed distinct clustering of the Indian isolates into three groups suggesting the presence of three genotypes, hitherto designated as T. annulata genotypes 1-3 (G1-G3). Genotype 3 seems to be novel containing only two newly generated sequences. Indian isolates displayed 88.4-100% and 82.2-100% similarity with each other at nucleotide (nt) and amino acid (aa) levels, respectively. However, the newly generated sequences (n = 36) showed 90.5-100% and 84.3-100% identity between them at nt and aa levels, respectively. The most diverse and heterogeneous genotype, G1, exhibited the highest number of polymorphic sites (S = 148), haplotypes (h = 16) and nucleotide differences (k = 43.23) besides haplotype (Hd = 0.903 ± 0.031) and nucleotide (π = 0.059 ± 0.005) diversities. Neutrality indices suggested a respective decrease and increase in population sizes of G1 and G2 genotypes in India. The nucleotide sequence analyses indicated the presence of extensive sequence variations between nucleotide positions 1-124, 194-257 and 396-494. The N-terminus of Tams1 protein displayed a considerable sequence variability with extensive variations in two regions, between amino acid positions 1-39 and 127-172, as compared to the conserved carboxyl terminus.

Sparganosis mimicking a soft-tissue tumor: A diagnostic challenge.

Human sparganosis is a rare but important food borne zoonosis and could be attributed to increased consumption of raw meat of fish, frogs, snakes etc. Sparganosis may involve varied organ systems but subcutaneous sparganosis remains the one of the commonly reported clinical condition. Rarity of this problem reinforces the necessity of sensitising the treating physicians of the differential possibility of this infection in patients with history of practice of consuming raw meat. Expansion of health communication and provision of safe food and water by the civic agencies can be a part of powerful preventive strategies.

Comparative evaluation of Babesia bigemina truncated C-terminal rhoptry associated protein-1 and 200 kDa merozoite protein in indirect enzyme-linked immunosorbent assay.

Babesia bigemina is an intra-erythrocytic apicomplexan protozoon which causes an acute as well as chronic disease in cattle and is transmitted by ixodid ticks throughout the world. Due to low sensitivity of microscopy for detection of the parasite, there is a need for developing effective diagnostic tests that can be used to identify carrier animals in endemic areas. In the present study, C-terminal fragment of rhoptry associated protein-1 (RAP-1/CT) and 200 kDa merozoite protein (P200/CT) of B. bigemina were cloned into pET-32a(+) expression vector and expressed in Escherichia coli as thioredoxin-fusion proteins for use in an indirect ELISA. The rRAP-1/CT and rP200/CT showed no cross reactivity with plasma from cattle infected with other common parasites namely Theileria annulata, Trypanosoma evansi, Cryptosporidium parvum and Anaplasma marginale in the standardized ELISA. Examination of 116 blood samples collected from cattle suspected for haemoprotozoan infections revealed that 17 (14.6%), 46 (39.6%), 52 (44.8%) and 53 (45.7%) were positive for B. bigemina by microscopy, nested PCR, rRAP-1/CT based and rP200/CT based indirect ELISA, respectively. The diagnostic sensitivities of rRAP-1/CT and rP200/CT indirect ELISAs were 97.8% and 91.3%, while the diagnostic specificities were 90% and 84.3%, respectively, when nested PCR was taken as a reference test. An almost perfect agreement (Kappa value -0.859) between rRAP-1/CT ELISA and nested PCR results, and a substantial agreement (Kappa value -0.737) between rP200/CT ELISA and nested PCR were noticed. The findings of the present study suggest that rRAP-1/CT is a better diagnostic candidate antigen than rP200/CT for diagnosis of B. bigemina infection and it may be used in an ELISA for surveillance or diagnosis of B. bigemina infection in bovines.

Genetic characterization and phylogenetic analysis of Sarcocystis suihominis infecting domestic pigs (Sus scrofa) in India.

A total of 57 tissue samples of domestic pigs (Sus scrofa) were collected from the meat outlets of five north Indian states and examined for sarcocystosis by histological and molecular methods. The genomic DNA extracted from five representative positive isolates was subjected to PCR amplification of the partial 18S rRNA gene followed by cloning and sequencing. Sequence analysis of the newly generated Indian isolates recorded 96.9-100.0% identity with published sequences of Sarcocystis suihominis. Two new haplotypes that have not been previously described manifested 99.5-100.0% nucleotide homology within themselves. In the phylogenetic analysis, Indian isolates of S. suihominis grouped together with S. suihominis originating from Italy, and they collectively formed a sister clade with Sarcocystis miescheriana within a clade containing various Sarcocystis spp. of ruminants having felids as final hosts. At the same time, this clade separated from a sister clade containing Sarcocystis spp. of bovid or cervid ruminants using canids as known or surmised definitive host. The current study established the phylogenetic relationship of Indian isolates of S. suihominis with various Sarcocystis spp. as well as with other taxa of Sarcocystidae family based on 18S rRNA gene for the first time.

Genetic characterization of Theileria species infecting bovines in India.

Genetic characterization of Theileria species infecting bovines in India was attempted targeting the 18S ribosomal RNA region of the parasite. Blood samples of bovines (n = 452), suspected for haemoprotozoan infections, from 9 different states of the country were microscopically examined for Theileria species infection. Four Theileria spp. positive blood samples from each state were randomly utilized for PCR amplification of the 18S rRNA gene (approx. 1529 bp) followed by cloning and sequencing. The sequence data analysis of all the 36 isolates revealed that 33 isolates had high sequence similarity with published sequences of T. annulata, whereas 3 isolates (MF287917, MF287924 and MF287928) showed close similarity with published sequences of T. orientalis. Sequence homology within the isolates ranged between 95.8 and 100% and variation in the length of targeted region was also noticed in different isolates (1527-1538 nt). Phylogenetic tree created for T. annulata sequences revealed that a total of 24 Indian isolates formed a major clade and grouped together with isolates originating from countries like China, Spain, Turkey and USA. Remaining 09 isolates clustered in a separate group and were closely related to the TA5 isolate of T. annulata (a new genotype) originating from India and also with the isolates from East Asian countries like Japan and Malaysia. All the three T. orientalis isolates had minimal intraspecific variation (99-100% homology) amongst themselves. Further, in the phylogenetic analysis T. orientalis Indian isolates were found to cluster away from other 14 isolates of T. buffeli/sergenti/orientalis originating from different countries (Australia, China, Indonesia and Spain). However, these 3 isolates clustered together with the T. buffeli Indian isolate (EF126184). Present study confirmed the circulation of different genotypes of T. annulata in India, along with T. orientalis isolates.

Immunoprophylactic evaluation of recombinant gametocyte 22 antigen of Eimeria tenella in broiler chickens.

Gametocyte proteins are being explored as potential vaccine candidates against Eimeria sp. in chicken since they are the components of the resilient oocyst wall. The aim of this study was to investigate the immunoprophylactic efficacy of recombinant Eimeria tenella gametocyte antigen 22 (EtGam22) in chickens against homologous oocyst challenge. Broiler chicks were subcutaneously immunized individually with 100 µg of recombinant EtGam22 adjuvanted with Montanide ISA 71 VG at 7 days of age and boosted 2 weeks later. The immunized chickens were challenged individually with 1 × 104 sporulated oocysts of E. tenella 1 week post-booster immunization. The anti-EtGam22 IgY and serum cytokine response was measured post-immunization. The results showed that the anti-EtGam22 IgY antibody, serum IFN-γ, IL-2, TGF-ß, and IL-4 levels in chickens vaccinated with recombinant protein were significantly increased post-immunization as compared to unimmunized challenged controls (P < 0.05). The peripheral blood lymphocyte proliferation activity was also found significantly higher in EtGam22-immunized group on day 28, i.e., pre-challenge (P < 0.05). Upon homologous oocyst challenge, chickens immunized with rEtGam22 exhibited a significant drop in the total oocyst output per bird (246.78 ± 36.9 × 106, 45.23% reduction) and a significantly higher weight gain (497.7 ± 19.2 g) as compared to unimmunized challenged controls. Taken together, these data indicate that EtGam22 is a potent immunogen for use as a subunit vaccine against cecal coccidiosis in chickens as it induces a diverse and robust immune response involving multiple cytokines and strong antibody titers.

Development of multiplex PCR assay for concurrent detection of tick borne haemoparasitic infections in bovines.

This study describes development and evaluation of a multiplex PCR assay for simultaneous detection of Theileria annulata, Babesia bigemina and Anaplasma marginale infections in bovines. The assay was developed using parasites specific genomic DNA and three sets of PCR primers targeting the Tams1, 18S rRNA and 16S rRNA genes of T. annulata, B. bigemina and A. marginale, respectively. Blood samples collected from a total of 461 bovines, suspected for haemoparasitic infections, were examined microscopically to record the status of infection and simultaneously, genomic DNA extracted from these blood samples were utilized for the optimization and validation of multiplex PCR assay. Microscopic examination of blood samples revealed presence of single and multiple species of haemoparasites in 25.8% and 2.4% samples, respectively. Results of multiplex PCR revealed the presence of single haemoparasitic species infection in 159 cases (34.5%), whereas mixed infection was recorded in 82 (17.8%) samples. Occurrence of individual species infection detected by mPCR in the study was 26.03% (120/461) for T. annulata, 3.25% (15/461) for B. bigemina and 5.20% (24/461) for A. marginale. The detection limit of multiplex PCR assay was at the template dilutions of 10-6, 10-6 and 10-4, which corresponded to 0.1 pg, 0.1 pg and 10.0 pg of DNA for T. annulata, A. marginale, and B. bigemina, respectively. Based on the high diagnostic sensitivity and throughput, multiplex PCR assay developed in the present study could be exploited as a tool to conduct large-scale epidemiological survey for tick-borne haemoparasitic infection of bovines.

Immune response and protective efficacy of Eimeria tenella recombinant refractile body protein, EtSO7, in chickens.

Refractile body protein, SO7, is a highly immunogenic protein which is essentially involved in the early development of Eimeria species infecting the domestic chicken. In the present study, the immune response and protective efficacy of recombinant Eimeria tenella SO7 (rEtSO7) protein was assessed in broiler chickens following homologous oocyst challenge. Broiler chicks were subcutaneously immunized with rEtSO7 antigen adjuvanted with Montanide ISA 71 VG on 7 and 21 days of age and protective efficacy of vaccination was evaluated in terms of body weight gain, lesion score and reduction in oocyst output. The peripheral blood lymphocyte proliferation, serum IgY response, and levels of interferon gamma (IFN-γ), interleukin 2 (IL-2), interleukin 4 (IL-4), tumor growth factor beta (TGF-ß) and nitric oxide (NO) were assessed. The results revealed significant reduction (p < 0.05) in the oocyst output and increased weight gain in immunized birds as compared to unimmunized birds. Significantly increased levels of serum IgY, IFN-γ and proliferation of lymphocytes were evident in rEtSO7 immunized chickens. The results demonstrated that the recombinant protein could effectively elicit the cellular and humoral immune responses in immunized chickens, and provided significant protection against caecal coccidiosis in chickens.

Occurrence and identification of contagious ecthyma in blackbuck.

A carcass of male free ranging adult blackbuck (Antilope cervicapra) was presented for necropsy examination exhibiting thick confluent nodular skin lesions around the mouth and the dry scaly crusts/fissures on the skin of abdomen, thigh and shoulder with subcutaneous haemorrhages. The skin sample around mouth was found positive for orf virus (ORFV) identified by counterimmunoelectrophoresis and PCR. Histopathology of the mouth skin revealed the hyperkeratinization, epidermal sloughing and epithelial hyperplasia showing acanthosis with rete ridges and few eosinophilic intracytoplasmic inclusion bodies in keratinocytes. Further, comparative B2L gene sequence analysis revealed that the virus isolate from blackbuck had shown 97.8-99.6 and 97.6-99.5 % identity at nucleotide and amino acid levels respectively with Indian isolates and maximum identity with ORFV 79/04, an isolate from India. Phylogenetic analysis based on B2L gene also revealed the same evolutionary relationship that it is closely related to Indian isolates. This seems to be the first report of orf in blackbuck from Indian subcontinent.

Benzimidazole resistance in equine cyathostomins in India.

Benzimidazole resistance is a major hindrance to the control of equine cyathostominosis throughout the world. There is a paucity of knowledge on the level of benzimidazole resistance in small strongyles of horses in India. In the present study, allele-specific PCR (AS-PCR) that detects F200Y mutation of the isotype 1 ß-tubulin gene and faecal egg count reduction test (FECRT) were used for detecting benzimidazole resistance in equine cyathostomin populations in different agro-climatic zones of Uttar Pradesh, India. Results of the FECRT revealed prevalence of benzimidazole resistance in cyathostomins in an intensively managed equine farm in the mid-western plain (FECR=27.5%, LCI=0) and in working horses (extensively managed) at three locations in central plains of Uttar Pradesh (FECR=75.7-83.6%, LCI=29-57%). Post-treatment larval cultures revealed the presence of exclusively cyathostomin larvae. Genotyping of cyathostomin larvae by AS-PCR revealed that the frequency of homozygous resistant (rr) individuals and the resistant allele frequency was significantly higher (p<0.001) in the intensively managed farm in the mid-western plain and in working horses at two locations in central plains of the state. The resistant allele (r) frequency in cyathostomins was significantly higher (p<0.05) in Vindhyan and Tarai and Bhabar zones of Uttar Pradesh. The prevalence of benzimidazole resistant allele (r) was significantly higher (p<0.05) in cyathostomins of intensively managed horses (allelic frequency-0.35) as compared to extensively managed horses (allelic frequency-0.22). The widespread prevalence of benzimidazole resistant alleles in equine cyathostomins in Uttar Pradesh, India, necessitates immediate replacement of the drugs of benzimidazole group with other unrelated effective anthelmintics for management and control of equine cyathostomins.

Development of recombinant BgP12 based enzyme linked immunosorbent assays for serodiagnosis of Babesia gibsoni infection in dogs.

Indirect ELISA and dot-ELISA using recombinant BgP12 (rBgP12) were developed for the diagnosis of Babesia gibsoni infected dogs. The complete open reading frame of BgP12 gene (378bp) was cloned in pET-32a(+) expression vector and expressed in Escherichia coli as a soluble thioredoxin (Trx) fusion protein. The purified rBgP12 was used for production of anti-rBgP12 rabbit serum, which recognized a native 12-kDa protein in B. gibsoni infected erythrocyte by Western blot analysis. To evaluate the potential of rBgP12 for the serodiagnosis of B. gibsoni, a panel of serum/plasma samples from dogs infected with B. gibsoni (n=13), uninfected sera (n=13) and sera from dogs infected with other haemoparasites viz., Babesia canis vogeli (n=3), Ehrlichia canis (n=3), Hepatozoon canis (n=1) and Dirofilaria immitis (n=1) were used in ELISA formats. In addition, the performance of rBgP12 based indirect ELISA and dot-ELISA were evaluated using 75 serum/plasma samples collected from suspected dogs, in respect to the nested PCR as reference test. The diagnostic sensitivities of indirect ELISA and dot-ELISA were 94.59% and 89.18%, respectively, while their specificities were 84.21% and 81.57%, respectively. Moreover, both the assays using rBgP12 showed no cross reaction with sera from dogs infected with other common haemoparasites indicating their high specificity. High kappa values of indirect ELISA and dot-ELISA indicated the potentials of these assays with substantial agreement at 95% confidence level. It is concluded that indirect ELISA and dot ELISA using rBgP12 might be used in large scale epidemiological surveys and clinical diagnosis of B. gibsoni infection in dogs.

Genetic diversity within ITS-1 region of Eimeria species infecting chickens of north India.

Coccidiosis, caused by apicomplexan parasites of the genus Eimeria, inflicts severe economic losses to the poultry industry around the globe. In the present study, ITS-1 based species specific nested PCR revealed prevalence of E. acervulina, E. brunetti, E. maxima, E. mitis, E. praecox, E. necatrix and E. tenella in 79.2%, 12.5%, 64.6%, 89.6%, 60.4%, 64.6% and 97.9% poultry farms of north India, respectively. The ITS-1 sequences of different Eimeria spp. from north India were generated and analyzed to establish their phylogenetic relationship. The sequence identity with available sequences ranged from 80 to 100% in E. tenella, 95 to 100% in E. acervulina, 64 to 97% in E. necatrix, 96 to 99% in E. brunetti and 97 to 98% in E. mitis. Only long ITS-1 sequences of E. maxima could be generated in the present study and it had 80-100% identity with published sequences. Two out of the four ITS-1 sequences of E. maxima had mismatches in the published nested primer sequences from Australia, while one sequence of E. necatrix had a mismatch near 3' end of both forward and reverse published nested primer sequences, warranting for the need of designing new set of degenerate primers for these two species of Eimeria. In the phylogenetic tree, all isolates of E. acervulina, E. brunetti, E. mitis, E. tenella and E. necatrix clustered in separate clades with high bootstrap value. E. maxima sequences of north Indian isolates grouped in a long form of E. maxima clade. Complete ITS-1 sequences of E. necatrix and E. mitis are reported for the first time from India. Further studies are required with more number of isolates to verify whether these differences are unique to geographical locations.

Development of loop-mediated isothermal amplification (LAMP) for detection of Babesia gibsoni infection in dogs.

Diagnosis of canine babesiosis, caused by Babesia gibsoni is difficult, especially in chronically infected dogs. A loop mediated isothermal amplification (LAMP) assay was developed and standardized by using four oligonucleotide primers targeting the hypervariable region of 18S rRNA gene (GenBank Acc. no. KC461261). The primers specifically amplified B. gibsoni DNA, while no amplification was detected with DNA from non-infected dogs as well as from dogs infected with Babesia canis vogeli, Hepatozoon canis, Ehrlichia canis and Trypanosoma evansi. The assay could detect 1.35 × 10(-7) parasitaemia and 10(-4) dilution of recombinant plasmid, equivalent to 12 pg of target DNA. All the samples were tested by nested PCR as well as LAMP assay. LAMP was found to be 10 times more sensitive than nested PCR targeting the same gene. Out of 75 suspected field samples, collected from different parts of the country, LAMP could detect B. gibsoni in 43 samples, while nested PCR and microscopy could detect 37 and 23 samples, respectively. High sensitivity, specificity and rapidity of LAMP assay may be exploited for screening large number of samples in a field setting.

Gastrointestinal parasitic infections in chickens of upper gangetic plains of India with special reference to poultry coccidiosis.

Studies on the prevalence of gastrointestinal parasites of chicken reared under backyard and intensive systems were carried out in two north Indian states viz., Uttar Pradesh and Uttarakhand. Out of 58 poultry farms screened for gastrointestinal parasites, 81.03 % were positive for Eimeria spp., 15.52 % for Ascaridia galli, 3.45 % for Hetarakis gallinarum, 1.72 % for Syngamus trachea, 5.17 % for Capillaria spp, 1.72 % for Raillietina spp., 1.72 % for Trichostrongylus tenuis, 1.72 % for Choanotaenia infundibulum and 1.72 % for Strongyloides avium. In broiler farms, the prevalence of Eimeria spp. was higher (88.24 %) as compared to layer farms (71.43 %) and backyard poultry (70 %). Identification of Eimeria spp. using COCCIMORPH software revealed prevalence of E. acervulina, E. tenella, E. necatrix, E. mitis and E. praecox in 94.3, 17.14, 31.44, 85.7 and 2.86 % farms, respectively. However, E. maxima and E. brunetti could not be identified in any of the farms using this software. The prevalence of helminthic infections was higher in poultry farms of Uttarakhand (40.0 %) as compared to Uttar Pradesh (11.62 %) with higher prevalence in backyard poultry (36.4 %), followed by layer farms (28.6 %) and lowest in broiler farms (9.1 %). A. galli was the most common G.I. helminth and it was recorded in free-range (backyard poultry) as well as intensive systems (broiler and layer farms).

Development and evaluation of serodiagnostic assays with recombinant BgSA1 of Babesia gibsoni.

Indirect ELISA, dot-ELISA and double antibody sandwich ELISA (DAS-ELISA) using truncated recombinant BgSA1 (rBgSA1) were developed for detecting Babesia gibsoni infection in naturally infected dogs. Truncated BgSA1 gene of 858 bp, encoding 32 kDa protein was cloned in pET-32a(+) expression vector, expressed in Escherichia coli and the recombinant protein was purified under native conditions. To evaluate the ability of the truncated rBgSA1as serodiagnostic reagent for B. gibsoni infection, a panel of sera/plasma samples from dogs infected with B. gibsoni (n = 13), uninfected sera (n = 13) and sera from dogs infected with other haemoparasites namely, Babesia canis vogeli (n = 3), Ehrlichia canis (n = 3), Hepatozoon canis (n = 1) and Dirofilaria immitis (n = 1) were used. Besides these, 75 samples collected from dogs suspected for babesiosis were used to evaluate the performance of rBgSA1 based serological assays in comparison to nested PCR. Based on the results, the diagnostic sensitivity of indirect ELISA, dot-ELISA and DAS-ELISA were 97.3%, 91.9% and 100%, respectively, when nested PCR was taken as a reference test, while their specificities were 81.6%, 84.2% and 97.4%, respectively. Further, DAS-ELISA had a quantitation limit of 0.03 µg/ml of the rBgSA1. High kappa values of indirect ELISA, dot-ELISA and DAS-ELISA were recorded, indicating that these assays had substantial to almost perfect agreement at 95% confidence level. There was no cross-reactivity with sera from dogs infected with B. canis vogeli, E. canis, H. canis and D. immitis. The results suggest that the indirect ELISA, dot-ELISA and DAS-ELISA with rBgSA1 may be used in large scale epidemiological surveys and clinical diagnosis of B. gibsoni infection in dogs. DAS-ELISA has advantages over indirect or dot-ELISA in the detection of current infection as well as monitoring the parasite burden.

Genetic characterization and phylogenetic relationships based on 18S rRNA and ITS1 region of small form of canine Babesia spp. from India.

Canine babesiosis is a vector borne disease caused by intra-erythrocytic apicomplexan parasites Babesia canis (large form) and Babesia gibsoni (small form), throughout the globe. Apart from few sporadic reports on the occurrence of B. gibsoni infection in dogs, no attempt has been made to characterize Babesia spp. of dogs in India. Fifteen canine blood samples, positive for small form of Babesia, collected from northern to eastern parts of India, were used for amplification of 18S rRNA gene (∼1665bp) of Babesia sp. and partial ITS1 region (∼254bp) of B. gibsoni Asian genotype. Cloning and sequencing of the amplified products of each sample was performed separately. Based on sequences and phylogenetic analysis of 18S rRNA and ITS1 sequences, 13 were considered to be B. gibsoni. These thirteen isolates shared high sequence identity with each other and with B. gibsoni Asian genotype. The other two isolates could not be assigned to any particular species because of the difference(s) in 18S rRNA sequence with B. gibsoni and closer identity with Babesiaoccultans and Babesiaorientalis. In the phylogenetic tree, all the isolates of B. gibsoni Asian genotype formed a separate major clade named as Babesia spp. sensu stricto clade with high bootstrap support. The two unnamed Babesia sp. (Malbazar and Ludhiana isolates) clustered close together with B. orientalis, Babesia sp. (Kashi 1 isolate) and B. occultans of bovines. It can be inferred from this study that 18S rRNA gene and ITS1 region are highly conserved among 13 B. gibsoni isolates from India. It is the maiden attempt of genetic characterization by sequencing of 18S rRNA gene and ITS1 region of B. gibsoni from India and is also the first record on the occurrence of an unknown Babesia sp. of dogs from south and south-east Asia.

Molecular cloning, sequencing, and biological characterization of GRA4 gene of Toxoplasma gondii.

In the present study, GRA4 (dense granule antigen) gene of Toxoplasma gondii was cloned, sequenced, and biologically characterized. The nucleotide sequence data obtained were analyzed and submitted in GenBank database (accession no. EU660037). Analysis of nucleotide sequence of GRA4 gene revealed 99.2 % homology with the published sequence (accession no. M76432). The gene segment (open reading frame) of 1,054 bp was further amplified and re-cloned in expression vector pET-32a. The recombinant protein obtained following the expression in prokaryotic system had a molecular mass of approx. 50 kDa and showed good immunoreactivity with T. gondii sera collected from infected goats. The immunization study of the recombinant protein performed in laboratory mice and live challenge with T. gondii revealed a high level of IgG response against the tachyzoite lysate antigen (TLA) by an indirect ELISA. Protection against T. gondii challenge infection was not evident in immunized mice except for the prolongation of survival period by 2 days. Humoral immune response profile revealed initially a high level of IgG antibody, but at 1 week post-challenge, a sudden drop in the level of the antibody was appreciable. Cytokine profiling by enzyme-linked immunosorbent spot method revealed relatively high level of IFN-γ production by the rodent spleen cells followed by IL-10 and IL-4. Increase in IFN-γ production by spleen cells of immunized mice following TLA stimulation suggested direct correlation to the up-regulated Th1 cells. However, the present immunization trial failed to show any positive relationship with the protection of mice following T. gondii challenge infection.