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.

Quantification of Eimeria necatrix, E. acervulina and E. maxima genomes in commercial chicken farms by quantitative real time PCR.

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.

Humoral and cytokine response elicited during immunisation with recombinant Immune Mapped protein-1 (EtIMP-1) and oocysts of Eimeria tenella.

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).

Optimization of spinal implant screw for lower vertebra through finite element studies.

The increasing older population is suffering from an increase in age-related spinal degeneration that causes tremendous pain. Spine injury is mostly indicated at the lumbar spine (L3-L5) and corresponding intervertebral disks. Finite element analysis (FEA) is now one of the most efficient and accepted tools used to simulate these pathological conditions in computer-assisted design (CAD) models. In this study, L3-L5 spines were modeled, and FEA was performed to formulate optimal remedial measures. Three different loads (420, 490.5, and 588.6 N) based on three body weights (70, 90, and 120 kg) were applied at the top surface of the L3 vertebra, while the lower surface of the L5 vertebra remained fixed. Models of implants using stainless steel and titanium alloy (Ti6Al4V) pedicle screws and rods with three different diameters (4, 5, and 6 mm) were inserted into the spine models. The relative strengths of bone (very weak, weak, standard, strong, and very strong) were considered to determine the patient-specific effect. A total of 90 models were simulated, and von Mises stress and strain, shear stress, and strain intensity contour at the bone-implant interface were analyzed. Results of these analyses indicate that the 6-mm pedicle screw diameter is optimal for most cases. Experimental and clinical validation are needed to confirm these theoretical results.

Prevalence and risk factors associated with Cryptosporidium spp. infection in young domestic livestock in India.

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 (p<0.05) 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 (p>0.05) was recorded in females than in males. Seasons had a significant effect (p<0.05) 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 (p>0.05) 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.

Correlations among Anaplasma marginale parasitemia and markers of oxidative stress in crossbred calves.

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 < 0.05) augmented in A. marginale-infected animals over healthy group. Parasitemia was positively correlated (p < 0.01) with erythrocytic LPO and plasma NO and negatively correlated (p < 0.01) with hematological indicators and antioxidant enzymes. In addition, erythrocytic LPO was negatively correlated (p < 0.01) with the hemoglobin, erythrocyte count, and packed cell volume. From the present study, it can be concluded that anaplasmosis in crossbred calves is associated with a parasitic load-dependent oxidative damage as indicated by poor antioxidant status and enhanced oxidative stress, which are contributed to severe anemia.

Trypanosoma lewisi or T. lewisi-like infection in a 37-day-old Indian infant.

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.

Unnatural amino acid incorporation onto adenoviral (Ad) coat proteins facilitates chemoselective modification and retargeting of Ad type 5 vectors.

Surface modification of adenovirus vectors can improve tissue-selective targeting, attenuate immunogenicity, and enable imaging of particle biodistribution, thus significantly improving therapeutic potential. Currently, surface engineering is constrained by a combination of factors, including impact on viral fitness, limited access to functionality, or incomplete control over the site of modification. Here, we report a two-step labeling process involving an initial metabolic placement of a uniquely reactive unnatural amino acid, azidohomoalanine (Aha), followed by highly specific chemical modification. As genetic modification of adenovirus is unnecessary, vector production is exceedingly straightforward. Aha incorporation demonstrated no discernible impact on either virus production or infectivity of the resultant particles. "Click" chemical modification of surface-exposed azides was highly selective, allowing for the attachment of a wide range of functionality. Decoration of human adenovirus type 5 (hAd5) with folate, a known cancer-targeting moiety, provided an ∼20-fold increase in infection of murine breast cancer cells (4T1) in a folate receptor-dependent manner. This study demonstrates that incorporation of unnatural amino acids can provide a flexible, straightforward route for the selective chemical modification of adenoviral vectors.

Chemoselective attachment of small molecule effector functionality to human adenoviruses facilitates gene delivery to cancer cells.

We demonstrate here a novel two-step "click" labeling process in which adenoviral particles are first metabolically labeled during production with unnatural azido sugars. Subsequent chemoselective modification allows access to viruses decorated with a broad array of effector functionality. Adenoviruses modified with folate, a known cancer-targeting motif, demonstrated a marked increase in gene delivery to a murine cancer cell line.

Designing suitable sizes of hip implants for Indian patients.

A large number of orthopaedically handicapped patients in India require treatment with suitable low-cost implants of varied sizes and shapes, which has become a crucial problem today. Out of these patients, the case of degenerative hip joints is around one-half million every year, the majority of which are total hip replacements. For most of these cases, the surgery is done with imported implants, which may not be in keeping with the osteological dimensions of Indian patients. To explore this area, a study was done at CMERI on computed tomographic (CT) scan data of the proximal region of the femur bone of patients from different regions of India. Since the problem is prominent with the elderly populations who are above 50 years of age, the CT scan data was collected for patients with age of 50-78 years. In this study, 23 selected osteological dimensions of each femur were measured in a CAD environment from CT scan data of around 100 elderly Indian patients. The measured parameters were statistically analyzed to find out the variations and stepping of dimensions. Depending on these data, the stem of hip implant was designed with different sizes, suitable for Indian patients. The devices were then analyzed in finite element analysis packages individually, as well as by putting the device inside the femur bone, as an actual case. After this analysis, some modifications were required and it was found that after implantation the stress induced in the implant and bone would be well within permissible limits.