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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.

Advent of quantitative polymerase chain reaction and its variants have enabled identification and quantification of seven known Eimeria species of poultry in biological samples. Attempts were made in the present study to identify and quantify three important pathogenic Eimeria species responsible for intestinal coccidiosis in domestic farmed chicken, E. necatrix, E. acervulina and E. maxima in droppings collected from thirty one poultry farms of North Indian states of Haryana, Punjab, Uttar Pradesh and Uttarakhand. The study included broiler, layer and backyard rearing units. Overall occurrence of E. necatrix, E. maxima and E. acervulina was 64.5%. E. necatrix was detected in 55% (11/20) broiler farms, 66.7% (4/6) layer farms and 100% (5/5) backyard rearing units studied. Thus, occurrence of E. necatrix was detected in 64.5% (20/31) farms studied. E. maxima and E. acervulina were detected in droppings of 65% (13/20) broiler farms, 66.7% (4/6) layer farms and 60% (3/5) back yard rearing units. Genome counts of each Eimeria species revealed maximum parasite load of E. necatrix followed by E. acervulina in broiler farms and least in layer farms. The mean parasite load (genome) copies for these parasite species were intermediate for backyard units while E. maxima had the lowest number of genome copies in droppings. Mean E. maxima counts were highest in boiler farms, while it was similar for layer and back yard units. However, statistically no significant differences were observed for parasite load existing either between the broiler, layer or back yard units or between the genome counts of E. necatrix, E. acervulina or E. maxima.

In this report, concurrent infection of Leptospira interrogans serovar icterohaemorrhagiae, Hepatozoon canis and Anaplasma phagocytophilum in a Labrador Retriever dog and its successful therapeutic management have been described. A 3-year-old female dog was presented with a history of anorexia, fever, lethargy, weakness, vomiting, black-coloured faeces and yellowish urine. Clinical and laboratory examination revealed pyrexia, icteric mucous membranes, hepatomegaly, splenomegaly, anaemia, leukocytosis, thrombocytopaenia, hypoalbuminemia and hyperbilirubinemia. Hepatozoon canis and Anaplasma phagocytophilum infections were confirmed by blood smear examination. Leptospira icterohaemorrhagiae was confirmed by microscopic agglutination test (MAT). Animal was stabilised with polyionic fluids, and therapy was started with imidocarb dipropionate (6.6 mg/Kg, SC, single dose), doxycycline (5 mg/kg, PO, bid) for 28 days, N-acetyl cysteine (30 mg/Kg, IV, SID) for 5 days and fipronil (0.25%) spray for topical use. Supportive therapy was done with plasma volume expanders, multivitamins, haematinics and hepatoprotectants. The animal showed complete recovery after 2 months of therapy.

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-β, and IL-4 levels in chickens vaccinated with recombinant protein were significantly increased post-immunization as compared to unimmunized challenged controls (P

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.

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.

Eimeria tenella, the causative agent of caecal coccidiosis, is a pathogenic gut dwelling protozoan which can cause severe morbidity and mortality in farmed chickens. Immune mapped protein-1 (IMP-1) has been identified as an anticoccidial vaccine candidate; in the present study allelic polymorphism was assessed across the IMP-1 coding sequence in E. tenella isolates from four countries and compared with the UK reference Houghton strain. Nucleotide diversity was low, limited to expansion/contraction of a CAG triplet repeat and five substitutions, three of which were non-synonymous. The EtIMP-1 coding sequence from a cloned Indian E. tenella isolate was expressed in E. coli and purified as a His-tagged thioredoxin fusion protein. An in-vivo vaccination and challenge trial was conducted to test the vaccine potential of recombinant EtIMP-1 (rEtIMP-1) and to compare post-vaccination immune responses of chickens to those stimulated by live oocyst infection. Following challenge, parasite replication measured using quantitative PCR was significantly reduced in chickens that had been vaccinated with rEtIMP-1 (rIC group; 67% reduction compared to UC or unimmunised controls; 79% reduction compared to rTC group or recombinant thioredoxin mock-immunised controls, p < 0.05), or the birds vaccinated by infection with oocysts (OC group, 90% compared to unimmunised controls). Chickens vaccinated with oocysts (OC) had significantly higher levels of interferon gamma in their serum post-challenge, compared to rEtIMP-1 vaccinated birds (rIC). Conversely rEtIMP-1 (rIC) vaccinated birds had significantly higher antigen specific serum IgY responses, correlating with higher serum IL-4 (both p < 0.05).

Epidemiology and molecular characterization of Eimeria was carried in goats reared under semi-arid region of west Uttar Pradesh, India. A total of 1285 faecal samples from different goat breeds (Jamunapari, Jakhrana and Barbari) were examined for presence of Eimerian oocysts over a period of eight months along with faecal oocysts count. All raw data of faecal oocyst counts (FOC) were transformed by loge (OPG+ 100) before analysis. All fixed effects like breed, age, months of sample collections along with their interaction were considered in analysis. The overall prevalence of Eimeria infection in goats was 73.85%. Breed wise prevalence in Barbari, Jamunapari and Jakhrana breed was 68.62, 79.70 and 72% respectively. Prevalence observed in 2-6M, 6-12M and >12M was as 70.83, 79.88 and 71.74% respectively. Gender wise prevalence as observed in male and female goats was 71.95 and 74.43% respectively. In oocyst per gram (OPG) data analysis the fixed effects like breed, age, months of sample collection and age versus gender interaction had significant effect on log transformed faecal oocysts counts (LFOC). The overall least square means of OPG was 4.673±0.007 (1403OPG). Of the three goat breeds, Jamunapari had highest OPG (2886OPG) compared to Jakhrana (875OPG) and Barbari (523OPG). Mean OPG in 2-6month age goats was significantly higher than the corresponding values in 6-12 and >12months, significant variation was found among monthly OPG means and wet months showed higher faecal oocysts discharge. Nine Eimeria species were identified infecting goats and E. arloingi and E. ninakohlyakimovae were most frequent and predominant species. Molecular characterization for coccidial infection was conducted using two genes i.e. 18S rDNA and ITS-1 genes which amplified 637bp and 500bp (E. christenseni and E. alijevi) respectively. The ITS1 gene was analysed by sequencing, E. christenseni was found showing nucleotide similarity with E. bovis and E. ellipsoidalis whereas 3' end of the sequence were highly conserved. The ITS1 gene of E. ninakohlyakimovae was found more homologous to E. bovis, E. ellipsoidalis and E. zuernii but for 33rd nucleotide thymidine residue deletion and 5th position G→A mutation. The 18S rDNA sequences of E. ninakohlyakimovae and E. christenseni were studied for evolutionary divergence analysis and maximum divergence was noticed between E. ninakohlyakimovae and E. christenseni (0.0605). The phylogenetic tree showed E. ninakohlyakimovae was placed in same clade with other Eimeria spp. compared, but E. christenseni being placed in a different clade as an out-group.

Highlights • Multivariate analysis revealed comparable poultry clusters in north and south India. • Eimeria species occurrence varied between system clusters. • E. tenella occurrence across systems may underpin region-specific genetic diversity. • E. necatrix was found to be more common in north than south India., Coccidiosis is one of the biggest challenges faced by the global poultry industry. Recent studies have highlighted the ubiquitous distribution of all Eimeria species which can cause this disease in chickens, but intriguingly revealed a regional divide in genetic diversity and population structure for at least one species, Eimeria tenella. The drivers associated with such distinct geographic variation are unclear, but may impact on the occurrence and extent of resistance to anticoccidial drugs and future subunit vaccines. India is one of the largest poultry producers in the world and includes a transition between E. tenella populations defined by high and low genetic diversity. The aim of this study was to identify risk factors associated with the prevalence of Eimeria species defined by high and low pathogenicity in northern and southern states of India, and seek to understand factors which vary between the regions as possible drivers for differential genetic variation. Faecal samples and data relating to farm characteristics and management were collected from 107 farms from northern India and 133 farms from southern India. Faecal samples were analysed using microscopy and PCR to identify Eimeria occurrence. Multiple correspondence analysis was applied to transform correlated putative risk factors into a smaller number of synthetic uncorrelated factors. Hierarchical cluster analysis was used to identify poultry farm typologies, revealing three distinct clusters in the studied regions. The association between clusters and presence of Eimeria species was assessed by logistic regression. The study found that large-scale broiler farms in the north were at greatest risk of harbouring any Eimeria species and a larger proportion of such farms were positive for E. necatrix, the most pathogenic species. Comparison revealed a more even distribution for E. tenella across production systems in south India, but with a lower overall occurrence. Such a polarised region- and system-specific distribution may contribute to the different levels of genetic diversity observed previously in India and may influence parasite population structure across much of Asia and Africa. The findings of the study can be used to prioritise target farms to launch and optimise appropriate anticoccidial strategies for long-term control.

Graphical abstract, Highlights • The seven Eimeria spp. recognised to infect chickens are present globally. • Cryptic Eimeria operational taxonomic units (OTUs) are common in the southern but not northern hemisphere. • Parasite population structure appears to vary between Eimeria spp., The phylum Apicomplexa includes parasites of medical, zoonotic and veterinary significance. Understanding the global distribution and genetic diversity of these protozoa is of fundamental importance for efficient, robust and long-lasting methods of control. Eimeria spp. cause intestinal coccidiosis in all major livestock animals and are the most important parasites of domestic chickens in terms of both economic impact and animal welfare. Despite having significant negative impacts on the efficiency of food production, many fundamental questions relating to the global distribution and genetic variation of Eimeria spp. remain largely unanswered. Here, we provide the broadest map yet of Eimeria occurrence for domestic chickens, confirming that all the known species (Eimeria acervulina, Eimeria brunetti, Eimeria maxima, Eimeria mitis, Eimeria necatrix, Eimeria praecox, Eimeria tenella) are present in all six continents where chickens are found (including 21 countries). Analysis of 248 internal transcribed spacer sequences derived from 17 countries provided evidence of possible allopatric diversity for species such as E. tenella (FST values ⩽0.34) but not E. acervulina and E. mitis, and highlighted a trend towards widespread genetic variance. We found that three genetic variants described previously only in Australia and southern Africa (operational taxonomic units x, y and z) have a wide distribution across the southern, but not the northern hemisphere. While the drivers for such a polarised distribution of these operational taxonomic unit genotypes remains unclear, the occurrence of genetically variant Eimeria may pose a risk to food security and animal welfare in Europe and North America should these parasites spread to the northern hemisphere.

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 (855 bp) 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.

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.

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.

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.

The study was carried out to assess the effect of condensed tannins (CT) supplementation through leaf meal mixture (LMM) on feed intake, humoral [Immunoglobulin G (IgG)], cell mediated immune response (CMI) and faecal egg counts in Haemonchus contortus infected sheep. Eighteen sheep were randomly divided into three groups (negative control-NC, infected control-C and Infected treatment-T) of six animals in each group in a completely randomized block design for a period of 90 days. Twelve H. contortus infected adult sheep were allocated into two equal groups C and T, supplemented with 0 and 1.5 % of CT, respectively. Six non-infected sheep of similar age and body weight of NC group were included in this study to compare their immune response with H. contortus C and CT supplemented T groups. Intake of dry matter and organic matter (g day(-1) and % live weight) was statistically similar (P

Molecular approaches supporting identification of Eimeria parasites infecting chickens have been available for more than 20 years, although they have largely failed to replace traditional measures such as microscopy and pathology. Limitations of microscopy-led diagnostics, including a requirement for specialist parasitological expertise and low sample throughput, are yet to be outweighed by the difficulties associated with accessing genomic DNA from environmental Eimeria samples. A key step towards the use of Eimeria species-specific PCR as a sensitive and reproducible discriminatory tool for use in the field is the production of a standardised protocol that includes sample collection and DNA template preparation, as well as primer selection from the numerous PCR assays now published. Such a protocol will facilitate development of valuable epidemiological datasets which may be easily compared between studies and laboratories. The outcome of an optimisation process undertaken in laboratories in India and the UK is described here, identifying four steps. First, samples were collected into a 2% (w/v) potassium dichromate solution. Second, oocysts were enriched by flotation in saturated saline. Third, genomic DNA was extracted using a QIAamp DNA Stool mini kit protocol including a mechanical homogenisation step. Finally, nested PCR was carried out using previously published primers targeting the internal transcribed spacer region 1 (ITS-1). Alternative methods tested included sample processing in the presence of faecal material, DNA extraction using a traditional phenol/chloroform protocol, the use of SCAR multiplex PCR (one tube and two tube versions) and speciation using the morphometric tool COCCIMORPH for the first time with field samples.

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.

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).

A total of 938 faecal samples (461 cattle calves, 264 buffalo calves, 55 lambs, 116 kids and 42 piglets) from different livestock farms and individual small holdings in six targeted states of India were collected and screened by modified Ziehl-Neelsen staining technique to determine the prevalence of Cryptosporidium spp. and its association with age, sex, season and faecal consistency in domesticated animals. Overall, 16.2 % of the animals were positive for Cryptosporidium infection with prevalence of 16.3, 24.2, 1.8, 3.5 and 19.1 % in cattle calves, buffalo calves, lambs, kids and piglets, respectively. The prevalence of infection was significantly higher (p0.05) in bovines (19.3 % cattle and 33.7 % buffalo) below 1 month of age than in animals between 1 and 3 months of age. But in piglets, it was higher in the age group of 1 to 3 months (22.6 %) than in younger animals (9.1 %). Also, higher prevalence (p0.05) was recorded in females than in males. Seasons had a significant effect (p0.05) on the prevalence of infection in large ruminants, with the highest prevalence in monsoon (cattle 28.8 % and buffalo 36.6 %) followed by pre-monsoon and post-monsoon season. However, in case of sheep and goats, the prevalence was higher (p0.05) in post-monsoon than in monsoon season. A high degree of association was noticed between Cryptosporidium infection and diarrhoea in ruminants screened during the present study. But, in case of pigs, the prevalence was higher in non-diarrhoeic than in diarrhoeic animals. Genotyping of Cryptosporidium spp. based on nested PCR amplification of partial 18S rRNA and its subsequent digestion with SspI, VspI and MboII restriction enzymes revealed prevalence of Cryptosporidium parvum in representative number of positive samples of cattle, buffalo and goats.

The present study was designed to determine the correlations among Anaplasma marginale parasitemia and markers of oxidative stress in crossbred calves. Blood was collected from 11 crossbred calves infected with A. marginale along with 11 healthy crossbred calves as controls for determination of hematology and oxidative stress indicators. Percentage of parasitemia in infected calves varied from 0.8% to 6.0%. The values of hematological indicators and antioxidant enzymes were decreased, whereas erythrocytic lipid peroxidation (LPO) and plasma nitrate (NO) level were significantly (p

The role of Cryptosporidium spp. as a major cause of diarrhoea and gastrointestinal illness of protozoan origin in neonatal calves has been established. Many coprological and serological techniques have been described for detection of the parasites with the limitations of sensitivity and specificity. Polymerase chain reaction (PCR) technique offers a useful alternative to conventional diagnosis of Cryptosporidium spp. in bovines from both clinical and environmental samples. We compared four conventional coprological techniques, viz., direct faecal smear staining (DFSS), normal saline sedimentation staining (NSSS), Sheather's flotation (SF) and Sheather's flotation sedimentation staining (SFSS) with PCR directed against the 18S SSU rRNA gene as standard reference test for the diagnosis of cryptosporidiosis in bovines. Out of 457 faecal samples collected from neonatal bovine calves, specific PCR amplification was achieved in 138 samples, whereas, 65 samples turned positive by DFSS. Normal saline sedimentation staining, SF and SFSS could detect 92, 82 and 109 samples as positive, respectively. Sheather's flotation sedimentation staining was found to be the most sensitive (82.6%) and specific (98.76%) among the coprological techniques. On per sample processing based cost analysis, DFSS was found to be the most economical method (15 cents) followed by NSSS (19.6 cents), SF (23.6 cents) and SFSS (33.9 cents). The time taken for complete processing and diagnosis varied between 70 and 100 min. PCR based diagnosis of a sample took about 7.5–8 h for completion and cost of diagnosis was estimated as approximately 7.604 US$ per sample. Among the conventional coprological methods, SFSS provided the required sensitivity and specificity along with nominal cost for diagnosis on per sample basis, and may be considered as a viable diagnostic alternative when PCR is not an option for a particular laboratory setting, especially in developing countries. This is the first comparative study describing the sensitivity and specificities of four conventional coprological techniques altogether with respect to PCR along with the economic assessment and per sample diagnosis time of all the techniques for the diagnosis of cryptosporidiosis in bovines.

Fasciola giganticafatty acid binding protein (FABP) was evaluated for evoking an effective immune response in mice and rabbits, when delivered as a DNA vaccine in muscle cells. Polyethylenimine (PEI), 25 kDa, branched cationic polymer was used as a delivery vehicle for this DNA in the muscle cells of mice and rabbits. Naked DNA evoked mixed Th1 and Th2 responses in mice. PEI condensed DNA, at amine nitrogen over DNA phosphate (N/P) ratios of 4, 6 and 8 and with various DNA concentrations, failed to evoke a significantly higher antibody response compared to naked DNA in mice. Similarly, the humoral immune response to naked DNA administration in rabbit thigh muscles was poor and no boosting of this antibody response on administration of DNA complexed to PEI was observed. On metacercarial challenge, rabbits failed to show any significant protective immune response in both the naked DNA and PEI–DNA immunized groups. Administration of PEI alone (12.5 μg) in mouse thigh muscles caused significant muscle cytotoxicity but condensation of DNA with PEI had less of a toxic effect on muscle cells, which was inversely related to the N/P ratio. Delivery of plasmid DNA encodingF. giganticaFABP by high molecular weight polyethylenimine (branched, 25 kDa) did not boost the effective immune response in both the animal species, which could either be attributed to cytotoxicity associated with this cationic polymer or muscle cells being unsuitable target cells for PEI condensed DNA delivery.

Epidemiological studies were conducted on Fasciola giagntica infection in different geo-climatic zones (hills, tarai and plains) in six states of north India during January 2001 to December 2004. Prevalence rates of 10.79, 13.90, 2.78 and 2.35 percent were recorded in cattle, buffaloes, sheep and goats, respectively. Animals in the tarai had the highest prevalence of fasciolosis followed by those in the hills and plains, respectively. The prevalence of infection in cattle and buffaloes was highest during the winters (11.84% cattle, 15.57% buffaloes) followed by summers and rains, respectively. However, the seasonal trends in sheep and goats were the reverse, with the peak prevalence during the rains (4.60% sheep, 2.71% goats). Abattoir studies revealed a higher prevalence in buffaloes (31.14%) than in sheep and goats. Screening of Lymnaea auricularia snails revealed that 5.48% of the snails harboured larval stages of F. gigantica. Also, the snails in the tarai had a higher prevalence (7.28%) of infection compared to those in the plains (1.57%). The results of the present study may help to devise an effective control strategy against fasciolosis in north India.

The phylum Apicomplexa includes serious pathogens of humans and animals. Understanding the distribution and population structure of these protozoan parasites is of fundamental importance to explain disease epidemiology and develop sustainable controls. Predicting the likely efficacy and longevity of subunit vaccines in field populations relies on knowledge of relevant preexisting antigenic diversity, population structure, the likelihood of coinfection by genetically distinct strains, and the efficiency of cross-fertilization. All four of these factors have been investigated for Plasmodium species parasites, revealing both clonal and panmictic population structures with exceptional polymorphism associated with immunoprotective antigens such as apical membrane antigen 1 (AMA1). For the coccidian Toxoplasma gondii only genomic diversity and population structure have been defined in depth so far; for the closely related Eimeria species, all four variables are currently unknown. Using Eimeria tenella, a major cause of the enteric disease coccidiosis, which exerts a profound effect on chicken productivity and welfare, we determined population structure, genotype distribution, and likelihood of crossfertilization during coinfection and also investigated the extent of naturally occurring antigenic diversity for the E. tenella AMA1 homolog. Using genome-wide Sequenom SNP-based haplotyping, targeted sequencing, and single-cell genotyping, we show that in this coccidian the functionality of EtAMA1 appears to outweigh immune evasion. This result is in direct contrast to the situation in Plasmodium and most likely is underpinned by the biology of the direct and acute coccidian life cycle in the definitive host.

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 β-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

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 (378 bp) 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.

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.

Beta-tubulin is an important multifunctional protein of eukaryotes abundant in the cytoskeleton and responsible for the formation of tubulin, structures responsible for cell morphology and which aid in motility and intracellular transportation. It has been used as a genotypic marker for studying the evolutionary history and phylogenetic relationships between eukaryotic organisms. Internal transcribed spacers of the ribosomal RNA genes have been widely used for typing inter-species and intra-species variation. An Indian isolate of Eimeria tenella was genotyped following the cloning and sequencing of beta-tubulin and internal transcribed spacer-2 (ITS-2) and compared with other reference isolates of E. tenella. The β-tubulin has 99 % intra-species similarity at the gene level and the functional homology of the protein is very high with more than 95 % amino-acid similarity across the Apicomplexa. The ITS-2 sequence had a similar pattern of nucleotide base arrangement with 99 % homology to Houghton and Nippon strains of E. tenella.

Trypanosomes were observed in the peripheral blood smear of a 37-day-old Indian infant admitted off feeds, with fever and convulsions. Trypanosoma (Herpetosoma) lewisi was identified in the blood. The species identification was confirmed by morphometry, polymerase chain reaction, and sequencing. Human infection with this organism is rare. Only seven cases of this infection have been reported previously in humans. The cases reported are reviewed to develop a composite picture of this disease.

Cryptosporidiosis is an important and established cause of calfhood morbidity in bovines. The present communication reports the prevalence of Cryptosporidium infection among juvenile and adult cattle (6–24 months old) in India based on examination of faecal samples collected from 350 animals across three different agro-climatic regions of the country and further confirmation by a two-step nested PCR assay targeting 18S ssu rRNA gene. A total of 45 samples were positive for Cryptosoridium species by nested PCR assay. The PCR products were subjected to restriction fragment length polymorphism (RFLP) analysis using SspI and VspI restriction enzymes for species differentiation. The results showed that the species involved in all the samples found positive was Cryptosporidium andersoni. The overall prevalence rate was 12.85%, with highest occurrence in the northern states (14.37%) of the country. The animals between age group of 6–12 months were mostly affected (21.67%) and the season wise prevalence of infection was more during the hot and humid monsoon season (20.16%). The results clearly demonstrated that C. andersoni is the major Cryptosporidium species affecting juvenile and adult cattle in three agro-climatically different geographical regions of India. This is the first report on prevalence of C. andersoni in bovines from India the confirmation of which is based on application of nested PCR and PCR-RFLP based molecular tools.

A survey based on PCR assay of 18S SSU rRNA gene revealed a 30.2% infection with Cryptosporidium spp., out of 457 faecal samples collected from neonatal bovine calves across three different regions of India. The PCR-RFLP pattern of the gene in all the positive cases established the species as Cryptosporidium parvum. Highest prevalence was recorded in the monsoon months (37.3%) and in the calves showing acute diarrhoea (32.3%). The calves below 15 days of age were mostly affected (45.1%). The infection was more prevalent in the northern parts (35.4%) of the country than in the eastern or southern parts. Results indicated that C. parvum was the only species of Cryptosporidium prevalent in bovine calves in three different geographical regions of India.

We report the results of investigations that were conducted in a sheep flock in Uttaranchal, India where repeated failure of anthelmintic medication was noted. The study revealed that Haemonchus contortus in sheep had developed resistance to benzimidazoles (fenbendazole, mebendazole and albendazole), imidazothiazole (levamisole) and salicylanide (rafoxanide), while it was fully susceptible to avermectins (ivermectin). Further, the suppression of nematode egg output in faeces of sheep naturally infected with multiple anthelmintic-resistant H. contortus following treatment with ivermectin tablet (0.4 mg/kg body weight (bw), orally), ivermectin injection (1% w/v, 0.2 mg/kg bw, subcutaneously) and ivermectin pour-on (0.5 w/v, 0.5 mg/kg bw) was also studied over a period of 10 weeks post treatment. It was noted that ivermectin tablet after initial clearance of infection (faecal egg count reduction 100%), could not prevent establishment of new patent natural infection for even a single day, while ivermectin pour-on and injection prevented the establishment of new infection for 7 and 14 days post treatment, respectively. Maximum protection period (duration for which mean faecal egg count of sheep reaches 500 eggs per gram of faeces or more) of 68 days was recorded in sheep treated with injectable ivermectin, followed by pour-on (60 days) and oral (53 days) preparations.