Selection of a stable reference gene for relative copy number profiling of porcine chromosomal genes using SYBR green qPCR.

Reference gene with stable copy number is essential for normalization in qPCR based copy number assay. Present study aims to identify a suitable reference gene in pigs for qPCR based relative copy number profiling of chromosomal genes. A total of 30 crossbred pigs of both sexes were cyto-screened and gDNA was extracted from the pigs having numerically normal karyotypes. The copy number stability was studied for 7 genes (FSHB, IL4, IGF1R, TCF24, BRMS1L, ARMC1 and SRSF4) selected on the basis of the chromosomal location, reports of single copy and lack of involvement in structural chromosomal abnormalities. The copy number was estimated from Ct values in 3 technical replicates using 6 animals from either sex for each gene. The stability was evaluated from the variations in Ct values using different (Delta Ct, geNorm, BestKeeper and normFinder) algorithms. While the moderate variation was observed among relative copy number stabilities among the genes, comprehensive ranking revealed the most stable gene for normalization (IGF1R > FSHB > TCF24 > IL4 > ARMC1> SRSF4 > BRMS1L) across the samples. The selected reference gene was validated using DNA of cyto-screened pigs to find out ratio of X and Y chromosome fragments using qPCR based copy number analysis.

Stress-inducible expression of AtDREB1A transcription factor greatly improves drought stress tolerance in transgenic indica rice.

The cultivation of rice (Oryza sativa L.), a major food crop, requires ample water (30 % of the fresh water available worldwide), and its productivity is greatly affected by drought, the most significant environmental factor. Much research has focussed on identifying quantitative trait loci, stress-regulated genes and transcription factors that will contribute towards the development of climate-resilient/tolerant crop plants in general and rice in particular. The transcription factor DREB1A, identified from the model plant Arabidopsis thaliana, has been reported to enhance stress tolerance against drought stress. We developed transgenic rice plants with AtDREB1A in the background of indica rice cultivar Samba Mahsuri through Agrobacterium-mediated transformation. The AtDREB1A gene was stably inherited and expressed in T1 and T2 plants and in subsequent generations, as indicated by the results of PCR, Southern blot and RT-PCR analyses. Expression of AtDREB1A was induced by drought stress in transgenic rice lines, which were highly tolerant to severe water deficit stress in both the vegetative and reproductive stages without affecting their morphological or agronomic traits. The physiological studies revealed that the expression of AtDREB1A was associated with an increased accumulation of the osmotic substance proline, maintenance of chlorophyll, increased relative water content and decreased ion leakage under drought stress. Most of the homozygous lines were highly tolerant to drought stress and showed significantly a higher grain yield and spikelet fertility relative to the nontransgenic control plants under both stressed and unstressed conditions. The improvement in drought stress tolerance in combination with agronomic traits is very essential in high premium indica rice cultivars, such as Samba Mahsuri, so that farmers can benefit in times of seasonal droughts and water scarcity.

Evaluation of antioxidants in the kidney of streptozotocin induced diabetic rats.

Diabetes mellitus is one of the most common endocrine metabolic disorders. Dual endocrine deficits of impaired insulin action (insulin resistance) and inadequate insulin secretion create an environment of chronic hyperglycemia and general metabolic disarray. Oxidative stress plays an important role in diabetic pathogenesis. Oxidative stress induced by streptozotocin (STZ) has been shown to damage pancreatic beta cell and produce hyperglycemia in rats. The present study was made to evaluate the antioxidant activity of ethanolic extract of the Evolvulus alsinoides in STZ induced rats. The antioxidant activities were done by using standard protocols. For histopathological analysis, the pancreatic tissues of all experimental groups were fixed with 10 % formalin for 24 h then the samples were stained with hematoxylin-eosin for the microscopic observation. Our results showed the significant decrease in lipid peroxidation and increases in the antioxidant (both enzymatic and nonenzymatic) levels after treatment with standard as well as the E. alsinoides. There is no significant difference between control and plant alone group rats. The histopathology reports also revealed non-toxic effect and protective effect of E. alsinoides in the kidney of STZ induced diabetic rats. Our result indicated that the E. alsinoides extract effectively increased the antioxidant level thereby it prevents oxidative stress during diabetes mellitus and also it showed the protective effect on kidney of STZ induced rats. Hence it can be used to maintain the antioxidant level during diabetes mellitus.

1-[5-(2H-1,3-Benzodioxol-5-yl)-3-(4-methyl-phen-yl)-2-pyrazolin-1-yl]ethanone.

In the title compound, C19H18N2O3, the pyrazoline ring is close to being planar (r.m.s. deviation = 0.035 Å) and subtends dihedral angles of 2.11 (8) and 82.63 (8)° with the p-tolyl and benzene rings, respectively. In the crystal, C-H⋯O and C-H⋯N hydrogen bonds link the mol-ecules, forming a three-dimensional network. A weak C-H⋯π inter-action involving the benzene ring is also observed.

3-(4-Methyl-phen-yl)-4-[(thio-semi-carba-zono)meth-yl]-1,2,3-oxa-diazol-3-ium-5-olate 1,4-dioxane hemisolvate.

The asymmetric unit of the title compound, C11H11N5O2S·0.5C4H8O2, contains one 3-(p-tol-yl)sydnone 4-thio-semi-carba-zone mol-ecule and a half mol-ecule of 1,4-dioxane, which lies abount an inversion centre. The sydnone ring is almost planar, with a maximum deviation of 0.002 (1) Å, and forms a dihedral angle of 46.31 (5)° with the benzene ring. In the crystal, the two components are linked into a tape along [01-1] by N-H⋯O and N-H⋯S hydrogen bonds. The crystal structure is further stabilized by C-H⋯O and C-H⋯π inter-actions, forming a three-dimensional network.

A Protocol for Mitotic Metaphase Chromosome Count Using Shoot Meristematic Tissues of Mulberry Tree Species.

Studies on chromosomal status are a fundamental aspect of plant cytogenetics and breeding because changes in number, size, and shape of chromosomes determine plant physiology/performance. Despite its significance, the classical cytogenetic study is now frequently avoided because of its tedious job. In general, root meristems are used to study the mitotic chromosome number, even though the use of root tips was restricted because of sample availability, processing, and lack of standard protocols. Moreover, to date, a protocol using shoot tips to estimate chromosome number has not yet been achieved for tree species' germplasm with a large number of accessions, like mulberry (Morusspp.). Here, we provide a step-by-step, economically feasible protocol for the pretreatment, fixation, enzymatic treatment, staining, and squashing of meristematic shoot tips. The protocol is validated with worldwide collections of 200 core set accessions with a higher level of ploidy variation, namely diploid (2n = 2x = 28), triploid (2n = 3x = 42), tetraploid (2n = 4x = 56), hexaploid (2n = 6x = 84), and decosaploid (2n = 22x = 308) belonging to nine species of Morus spp. Furthermore, accession from each ploidy group was subjected to flow cytometry (FCM) analysis for confirmation. The present protocol will help to optimize metaphase plate preparation and estimation of chromosome number using meristematic shoot tips of tree species regardless of their sex, location, and/or resources.

Proteomics and in silico approaches to extend understanding of the glutathione transferase superfamily of the tropical liver fluke Fasciola gigantica.

Fasciolosis is an important foodborne, zoonotic disease of livestock and humans, with global annual health and economic losses estimated at several billion US$. Fasciola hepatica is the major species in temperate regions, while F. gigantica dominates in the tropics. In the absence of commercially available vaccines to control fasciolosis, increasing reports of resistance to current chemotherapeutic strategies and the spread of fasciolosis into new areas, new functional genomics approaches are being used to identify potential new drug targets and vaccine candidates. The glutathione transferase (GST) superfamily is both a candidate drug and vaccine target. This study reports the identification of a putatively novel Sigma class GST, present in a water-soluble cytosol extract from the tropical liver fluke F. gigantica. The GST was cloned and expressed as an enzymically active recombinant protein. This GST shares a greater identity with the human schistosomiasis GST vaccine currently at Phase II clinical trials than previously discovered F. gigantica GSTs, stimulating interest in its immuno-protective properties. In addition, in silico analysis of the GST superfamily of both F. gigantica and F. hepatica has revealed an additional Mu class GST, Omega class GSTs, and for the first time, a Zeta class member.

Lateral flow assay for rapid serodiagnosis of bovine leptospirosis.

Background: Leptospirosis is considered to be an economically important disease in bovine. The disease burden is not appropriately monitored due to cumbersome serological tests that could be performed only in established laboratories. This warrants the development of a field level rapid diagnostic test. Aims: The study aimed to develop a lateral flow assay (LFA)-based pen-side diagnostic test to detect antibodies to Leptospira. Methods: LFA strip was prepared with the heat extracted antigen from L. interrogans serovar Pomona. To assess the performance of the developed LFA, a total of 300 bovine serum samples with their clinical histories were used and the initial screening for Leptospira antibodies was performed by the standard microscopic agglutination test (MAT). The sensitivity, specificity, and agreement (kappa value) were calculated between developed LFA and MAT. The stability of LFA was evaluated on days 30, 60, 90, and 120. Results: Out of 300 samples tested, 225 were positive, and 75 were negative on MAT and 208 were positive, and 92 were negative on LFA. The developed LFA had a sensitivity of 90.7% and a specificity of 94.7%. The results of the assay were substantially in agreement with MAT, with a kappa value of 0.79. The LFA strips were stable for 120 days at 4°C. Conclusion: A Lateral flow assay-based rapid pen-side test was developed and its utility to diagnose bovine leptospirosis was evaluated.

Status of Urinary shedders of Leptospires among cattle in Tamilnadu: Implications on zoonosis.

Bovine leptospirosis causes jaundice, mastitis, infertility, abortion, and death of the animal. This research aimed to study the status of urinary shedders of pathogenic Leptospira among the cattle population and identify the infecting serogroup circulating in this region. A total of 305 blood and 305 urine samples were collected from organized farms (n = 44), individually housed animals (n = 81) and animals from the slaughterhouse (n = 180). Microscopic agglutination test was carried out to detect anti­leptospiral antibodies. Dark­field microscopic examination and culture of urine were done to detect and isolate the Leptospira. The isolated Leptospira were identified by cross­agglutination test and gene sequencing. PCR and real­time PCR were carried out to detect leptospiral genomic DNA in urine samples to detect the shedders. The anti­leptospiral antibodies were detected in 6.2% of animals. The Leptospira genomic DNA was detected in 9.2% (28 of 305) of urine samples. Of the 28 Leptospira positive urine samples, 39.2% were from animals with clinical signs suggestive of leptospirosis and 60.8% Leptospira positive samples were from slaughterhouse animals. The Leptospira isolated were identified as Leptospira interrogans serogroup Sejroe and Hebdomadis. The present study demonstrates the need to include leptospirosis in cattle health surveillance programmes to prevent leptospirosis by vaccination, preventing renal carriage.

Influence of multi valent states of vanadium ions in ZnO doped novel calcium fluoro phosphate bio glasses.

ZnO-CaF2-P2O5 glasses doped with different concentrations of V2O5 (ranging from 0 to 1.0 mol %) were prepared. The prepared bio glasses are soaked in SBF for duration of 2, 3, 7 and 10 days in separate plastic containers and then kept in incubator maintained at body temperature 36.5 °C. The influence of valence states of vanadium ions (V4+/V5+) with respect to the structural aspects by means of FTIR and Raman Spectra, elastic properties by means of relevant parameters, the thermal stability by means of DTA studies and other spectroscopic properties by using OA and ESR studies are studied. The raise in wavenumber and comparative areas of the two absorption bands corresponding to electronic transitions 2B2g → E2g, 2B2g → 2B1g respectively in optical absorption spectra of these CZPV glasses clearly indicate that vanadium ions have octahedral co-ordination with tetragonal compression due to modifier action of V2O5in the glass network. The optical absorption and ESR studies have revealed that vanadium ions exist in V4+ states. The characteristic temperatures of these prepared glasses obtained from DTA curves explain modifications taking place in the structure of glass network. The structural changes are explained with the aid of FTIR and Raman studies. The bio active nature of the titled glasses is evident from dissociation and pH studies by SEM &EDS of these glasses before and after immersion into SBF.

Placental expression of miR-21-5p, miR-210-3p and miR-141-3p: relation to human fetoplacental growth.

BACKGROUND/OBJECTIVES: Dysregulation of microRNAs (miRNAs) and their target genes in placental tissue is associated with foetal growth restriction. We aimed to evaluate associations of placental miR-21-5p, miR-141-3p and miR-210-3p expression with maternal, placental and newborn parameters and with placental expression of their potential target genes PTEN, VEGF, FLT and ENG in a set of well-characterized small- (SGA) and appropriate- (AGA) for gestational age full-term singleton pregnancies. SUBJECTS/METHODS: Placental samples (n = 80) from 26 SGA and 54 AGA were collected from full-term singleton pregnancies. Placental transcript abundances of miR-21-5p, miR-141-3p and miR-210-3p were assessed after normalization to a reference miRNA, mir-16-5p by real-time quantitative PCR. Placental transcript abundances of PTEN, VEGF, FLT and ENG were assessed after normalizing to a panel of reference genes. RESULTS: Placental miR-21-5p transcript abundance was negatively associated with placental weight (n = 80, r = -0.222, P = 0.047) and this association was specific to the AGA births (n = 54, r = -0.292, P = 0.032). Placental transcript abundances of miR-210-3p and miR-141-3p were not associated with placental weight or birth weight in all 80 births. However, placental miR-210-3p transcript abundance was positively associated with birth weight specifically in the SGA births (n = 26, r = 0.449, P = 0.021). Placental transcript abundance of miR-21-5p was negatively associated with PTEN transcript abundance (Spearman's ρ = -0.245, P = 0.028) while that of miR-141-3p was positively associated with FLT (Spearman's ρ = 0.261, P = 0.019) and ENG (Spearman's ρ = 0.259, P = 0.020) transcript abundances in all 80 births. CONCLUSION: We conclude that placental miR-21-5p and miR-210-3p may be involved in fetoplacental growth. However, this regulation is unlikely to be mediated through placental expression of PTEN, VEGF, FLT or ENG.

Placental expression of RNU44, RNU48 and miR-16-5p: stability and relations with fetoplacental growth.

BACKGROUND/OBJECTIVES: The current study aimed to identify suitable reference miRNA for placental miRNA expression analysis in a set of well-characterized and fetal-sex balanced small- (SGA) and appropriate- (AGA) for gestational age full-term singleton pregnancies. SUBJECTS/METHODS: In this retrospective study, placental samples (n = 106) from 35 SGA (19 male and 16 female) and 71 AGA (30 male and 41 female) full-term singleton pregnancies were utilized. Placental transcript abundance of three widely used reference miRNAs [miR-16-5p and Small nucleolar RNAs (snoRNAs) RNU44 and RNU48] were assessed by real-time quantitative PCR. Raw cycle threshold (Ct) analysis and RefFinder tool analysis were conducted for evaluating stability of expression of these miRNAs. RESULTS: Raw Ct values of miR-16-5p were similar between SGA and AGA births (P = 0.140) and between male and female births within SGA (P = 0.159) and AGA (P = 0.060) births while that of RNU44 and RNU48 were higher in SGA births (P = 0.008 and 0.006 respectively) and in male births within the SGA group (P = 0.005) for RNU44 and in female births within the AGA group (P = 0.048) for RNU48. Across all 106 samples tested using the RefFinder tool, miR-16-5p and RNU44 were equally stable reference miRNAs. CONCLUSION: We recommend miR-16-5p and RNU44 as suitable reference miRNAs for placental samples from settings similar to our study.

Development of a multiplex polymerase chain reaction for the simultaneous detection of microsporidians, nucleopolyhedrovirus, and densovirus infecting silkworms.

We have developed a novel PCR-based assay for individual and simultaneous detection of three major pathogens (microsporidians, nucleopolyhedrovirus (NPV) and densovirus (DNV)) infecting the silkworm, Bombyx mori. Multiplex PCR, using three primer pairs, two of which were designed from the conserved regions of 16S small subunit ribosomal RNA gene of microsporidians, and polyhedrin gene of NPVs respectively, and a third primer pair designed from the internal sequences of B. mori DNVs (BmDNV), showed discrete and pathogen specific PCR products. The assay showed high specificity and sensitivity for the pathogenic DNA. Under optimized PCR conditions, the assay yielded a 794bp DNA fragment from Nosema bombycis, 471bp fragment from B. mori NPV (BmNPV) and 391bp fragment from BmDNV. Further, this detection method was successfully applied to other silkworm species such as Antheraea mylitta and Samia cynthia ricini, in detecting same or similar pathogens infecting them. This method is a valuable supplement to the conventional microscopic diagnostic methods and can be used for the early detection of pathogens infecting silkworms. Furthermore it can assist research and extension centers for the safe supply of disease-free silkworms to farmers.

Characterization of lipophorin receptor (LpR) mediating the binding of high density lipophorin (HDLp) in the silkworm, Bombyx mori.

In an earlier report, we described the gene encoding a lipophorin receptor (LpR) of the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae), and recombinant expression of the protein. The present study was performed to characterize the corresponding native BmLpR and its binding characteristics. Polyclonal anti-LpR antibody prepared against the cloned receptor fragment from the cytoplasmic domain specifically detected the receptor. Through immunoblotting, ovary and brain membrane protein samples of BmLpR have shown an apparent molecular mass of 105 kDa and 120 kDa under nonreducing and reducing conditions, respectively. Ligand binding of LpR supported the immunoblot results. It bound to high density lipophorin (HDLp) and has shown requirement of Ca(2+) in binding. Further, a dose-dependent inhibition by EDTA was observed in receptor ligand binding. The characteristics of the BmLpR protein confirm the properties of a ligand-receptor interaction similar to that of vertebrate low density lipoprotein receptor (LDLR).

Placental expression of leptin: fetal sex-independent relation with human placental growth.

OBJECTIVES: Leptin (LEP) is a vital placental hormone that is known to affect different aspects of placental function and fetal development. The present study aimed to determine the association of placental LEP transcript abundance with maternal, placental, and newborn parameters. SUBJECTS/METHODS: In this retrospective case-control study, placental samples (n = 105) were collected from small (SGA) and appropriate (AGA) for gestational age full-term singleton pregnancies (n = 44 SGA and n = 61 AGA). Placental transcript abundance of LEP was assessed by real-time quantitative PCR after normalization to a reference gene panel. LEP methylation was measured using a quantitative MethyLight assay in a subset of samples (n = 54). RESULTS: Placental LEP transcript abundance was negatively and significantly associated with placental weight (ß = -3.883, P = 0.015). This association continued to be significant in the SGA group (ß = -10.332, P = 0.001), both in female (ß = -15.423, P = 0.021) and male births (ß = -10.029, P = 0.007). LEP transcript abundance was not associated with LEP methylation levels (Spearman's ρ = 0.148, P = 0.287). CONCLUSION: We conclude that placental upregulation of LEP is an integral and fetal sex-independent component of placental growth restriction, which can be potentially targeted through maternal dietary modifications to improve fetoplacental growth.

Placental expression of ENG, VEGF, and FLT: Gender-specific associations with maternal vitamin B12 status.

OBJECTIVES: Adequate vitamin B12 is a requisite during pregnancy and its deficiency is linked with increased risk for adverse outcomes, likely mediated by impaired placental angiogenesis. Thus, we aimed to test associations of maternal vitamin B12 status with the placental expression of angiogenesis-associated genes ENG, VEGF, and FLT. SUBJECTS/METHODS: In this retrospective case-control study, placental and maternal trimester 1 blood samples (n = 104) were collected from small for gestational age (SGA) and appropriate for gestational age (AGA) full-term singleton pregnancies. Maternal trimester 1 vitamin B12 status was measured. Placentae and neonates were weighed at birth. Realtime quantitative PCR was performed to assess placental transcript abundance of ENG, VEGF, and FLT normalized to a panel of reference genes. Associations of placental transcript abundance of the genes with maternal trimester 1 vitamin B12 status were evaluated. RESULTS: Placental ENG transcript abundance associated negatively with maternal trimester 1 vitamin B12 status (ß = -0.461, P = 0.017, n = 104). This association was specific to the female births (ß = -0.590, P = 0.014, n = 60). Placental VEGF transcript levels were negatively associated with maternal trimester 1 vitamin B12 status only in the female births (ß = -1.995, P = 0.029). Placental FLT transcript levels were not associated with maternal trimester 1 vitamin B12 status. CONCLUSION: Maternal trimester 1 vitamin B12 status was associated negatively with placental ENG and VEGF expression predominantly in the female births. Therefore, we hypothesize that the placenta adapts to low maternal vitamin B12 status by up-regulating angiogenic pathways in a gender-specific manner.

Sequence diversity of major histo-compatibility complex class II DQA1 in Indian Tharparkar cattle: novel alleles and in-silico analysis.

Exon 2 of MHC class II gene codes for the first domain of the molecule that forms the peptide-binding groove and its polymorphism partly explains functional MHC diversity. A 850 bp DQA1 gene fragment spanning from intron I to exon III was typed by sequencing of 40 Tharparkar cattle of various agro-climatic zones of northern India along with 10 Tharparkar crossbreds. On analysis of nucleotide sequences, a total of 30 polymorphic sites (1 insertion and 29 SNPs) were identified in 14 MHC alleles leading to amino acid changes in 5 places in 249 bp (exon 2). Five new BoLa DQA1 alleles were identified and reported. The within group mean distance was highest in Tharparkar herd of Bikaner (0.045) and lowest (0.020) in that of Surathgarh (breeding tract) whereas, between groups mean distance was highest in Bikaner Tharparkar-Suratgarh Tharparkar pair. There was excess of nonsynonymous over synonymous nucleotide substitutions in the present study. The effects of these substitutions were predicted using I-Mutant and Panther online resources. The mean ratio of dN/dS was found to be >1.0 at 12 codons with two mutation hotspots at 13th codon (P = 0.002) and 64th codon (P = 0.01). The phylo-geographic analysis revealed that alleles 5, 7 and 13 formed a different cluster with alleles 7 and 13 grouped by the most frequent allele (BoLa-DQA*1401).

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.

Eccentric placentae have reduced surface area and are associated with lower birth weight in babies small for gestational age.

Placental structure and function determine birth outcomes. Placental mass does not always correlate with fetal birth weight (BW) in uncomplicated pregnancies which raises the possibility of other variables such as placental shape and cord insertion being the determinants of placental efficiency. In total, 160 women with singleton pregnancy, recruited into a pregnancy cohort were studied. Placental weight (PW) was measured and other data were obtained from clinical records. Birth outcomes were classified as small for gestational age (SGA) and appropriate for gestational age (AGA) based on fetal gender, gestational age (GA) and BW. High-resolution images of the chorionic plate were recorded. The shape of the placenta and the insertion of the cord were measured using eccentricity index (EI) and cord centrality index (CCI). Only placentae with eccentrically inserted cords (n=136) were included. The mean BW and PW were 2942 (±435) g and 414 (±82) g with average GA of 38.6 weeks. The mean CCI and EI was 0.483 (±0.17) and 0.482 (±0.16). Neither of these correlated with placental efficiency. However, EI showed negative correlation with placental surface area and breadth. Upon sub-grouping the cohort into SGA (n=32) and AGA (n=104), the SGA babies with the highest EI (third tertile) had significantly lower BW than those with the least eccentric placentae (first tertile). Although eccentric-shaped placentae were present in both SGA and AGA groups, the effect on BW was observed only in the SGA group.

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.