Characterization and distribution of phytoplasma strains associated with temperate stone fruits and their possible natural reservoirs in the north-western Himalayan states of India

European Journal of Plant Pathology - To understand the key soil factors that affect the survival and reproduction of Phytophthora sojae in soil, the number of zoospores and oospores of P. sojae...     

Trends and advances in the diagnosis and control of paratuberculosis in domestic livestock

Paratuberculosis (pTB) is a chronic granulomatous enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP) in a wide variety of domestic and wild animals. Control of pTB is difficult due to the lack of sensitive, efficacious and cost-effective diagnostics and marker vaccines. Microscopy, culture, and PCR have been used for the screening of MAP infection in animals for quite a long time. Besides, giving variable sensitivity and specificity, these tests have not been considered ideal for large-scale screening of domestic livestock. Serological tests like ELISA easily detects anti-MAP antibodies. However, it cannot differentiate between the vaccinated and infected animals. Nanotechnology-based diagnostic tests are underway to improve the sensitivity and specificity. Newer generation diagnostic tests based on recombinant MAP secretory proteins would open new paradigm for the differentiation between infected and vaccinated animals and for early detection of the infection. Due to higher seroreactivity of secretory proteins vis-à-vis cellular proteins, the secretory proteins may be used as marker vaccine, which may aid in the control of pTB infection in animals. Secretory proteins can be potentially used to develop future diagnostics, surveillance and monitoring of the disease progression in animals and the marker vaccine for the control and eradication of pTB.     

A multipronged approach for the detection of leptospirosis in captive sloth bears (Melursus ursinus) in Agra and Bannerghatta sloth bear rescue centers in India

Leptospirosis is an exacerbating factor responsible for the drastic decline of sloth bear population in India. In this study, a multipronged approach based on antigen detection using Polymerase Chain Reaction (PCR) employing G1/G2 and LigBF/LigBR primers, antibody detection using Microscopic Agglutination Test (MAT) and recombinant LigBCon1-5 antigen based Latex Agglutination Test (rLigBCon1-5 LAT), serum biochemistry using hepatic (serum glutamate oxalo acetic transaminase (SGOT) and serum glutamate pyruvic transaminase (SGPT) and renal biomarkers (blood urea nitrogen (BUN) and Creatinine) and gross/histopathological evidence in liver and kidneys were employed to investigate leptospirosis in captive sloth bears. A total of 133 serum samples collected from Agra (n=113) and Bannerghatta (n=20) sloth bear rescue centers were screened using MAT and rLigBCon1-5 LAT. A total of 87 and 78 sera tested positive by MAT and LAT respectively. Pyrogenes was the leading serovar obtained using MAT followed by Icterohaemorrhagiae, Javanica, Grippotyphosa, Canicola and Tarassovi. The relative sensitivity, specificity and accuracy of rLigBCon1-5 LAT in comparison to MAT were 89.66%, 100% and 93.23% respectively. PCR performed on hepatic and renal tissues showed amplicon of 285 and 219 base pairs for G1/G2 and LigBF/LigBR primers respectively. Gross evidence (icteric liver, severely engorged hepatic sinusoids, congested kidneys with necrotic white spots on sub capsular surface), histopathology (severe hepatic degeneration and tubulointerstitial nephritis) and elevated hepatic/renal biomarkers were suggestive of leptospirosis. This study suggests that rLigBCon1-5 LAT can be employed as a pen-side test for detecting leptospirosis in sloth bears.     

Carnein, a serine protease from noxious plant weed Ipomoea carnea (morning glory)

A new serine protease from the latex of Ipomoea carnea spp. fistulosa (Morning glory), belonging to the Convolvulaceae family, was purified to homogeneity by ammonium sulfate fractionation followed by cation exchange chromatography. The enzyme, named carnein, has a molecular mass of 80.24 kDa (matrix-assisted laser desorption/ionization time-of-flight) and an isoelectric point of pH 5.6. The pH and temperature optima for proteolytic activity were 6.5 and 65 degrees C, respectively. The extinction coefficient (epsilon2801%) of the enzyme was estimated as 37.12, and the protein molecule consists of 35 tryptophan, 76 tyrosine, and seven cysteine residues. The effect of several inhibitors such as iodoacetic acid, diisopropylfluorophosphate, phenyl-methanesulfonyl fluoride, chymostatin, soybean trypsin inhibitor, HgCl2, 3S-3-(N-{(S)-1-[N-(4-guanidinobutyl)carbamoyl]3-ethylbutyl}carbamoyl)oxirane-2-carboxylic acid, N-ethyl maleimide, ethylene glycol-bis(alpha-amino ethyl ether)tetraacetic acid, ethylenediamminetetraacetic acid, and o-phenonthroline indicates that carnein belongs to the family of serine proteases. The enzyme is not prone to autolysis even at very low concentrations. The N-terminal sequence of carnein (T-T-H-S-P-E-F-L-G-L-A-E-S-S-G-L-X-P-N-S) exhibited considerable similarity to those of other plant serine proteases; the highest similarity was with alnus AG12, one of the subtilase family endopepetidases.     

Detection of Babesia bigemina DNA in ticks by DNA hybridization using a nonradioactive probe generated by arbitrary PCR

The Dig-labeled probe specific to Babesia bigemina generated from monomorphic RAPD fragment of approximately 873 bp size amplified by a 10 mer CGGTGGCGAA, detected up to 100 ng of template DNA. This nonradioactive probe also detected B. bigemina in preparations of larval tick DNA from two of the five samples on dot-blot hybridization.     

Identification of T-helper and linear B epitope in the hypervariable region of nucleocapsid protein of PPRV and its use in the development of specific antibodies to detect viral antigen

Peste des petits ruminants is a highly contagious viral disease of small ruminants making its diagnosis difficult from the similar symptoms of Rinderpest. Computer based prediction algorithms was applied to identify antigenic determinants on the nucleocapsid (N) protein of PPRV. Specificity and antigenicity of each peptide was evaluated by solid phase ELISA. Six specific peptide sequences were evaluated in multiple antigenic peptide (MAP) form and immune response was evaluated by supplementing universal T-helper epitope human IL-1beta peptide (VQGEESNDK, amino acids 163-171). Out of the six peptides 19mer sequence corresponding to 454-472 region of N protein of PPRV was found to be highly immunogenic and specific to PPRV. Evaluation of overlapping peptides differing in length for this 452-472 region, showed minimum length of 14 amino acid residues were required for the stable affinity binding of antigen-antibody. The results of immunization and indirect ELISA indicated the presence of T-helper epitope at the N-terminal end and linear B epitope at the C-terminal region of 454-472 19mer of nucleocapsid peptide of PPRV-nucleocapsid protein. The antipeptide antibodies developed against this region showed specificity to PPRV antigen differentiating it from RPV when used in indirect ELISA and western blot analysis.     

The plasmid constructs producing shRNA corresponding to the conserved 3D polymerase of Foot and Mouth Disease virus protects guinea pigs against challenge virus

RNA interference (RNAi) has been used as an effective antiviral strategy for its specific silencing of viral gene expression in mammalian cells. In this study, shRNA targeting two regions of Foot and Mouth Disease Virus (FMDV) i.e. 3D and 5'UTR which are very essential in virus replication were evaluated. The constructs were made using h7K RNA polymerase III promoter. We investigated in vivo inhibitory effect of shRNA on FMDV replication in BHK-21 cells and guinea pigs. The results showed that transfection of 3D shRNA could reduce virus growth by three folds when cells were challenged with 10(2) TCID(50) of FMDV. Pretreated guinea pigs with 3DshRNA were protected 80% with 10(3) GPID(50) of FMDV. As a first report in guinea pigs which are recognized animal model for FMD vaccine potency testing, the study suggests that shRNA could be a viable therapeutic approach to control severity of FMD infection and spread.     

Structure-function studies of Bubalus bubalis lingual antimicrobial peptide analogs

Antimicrobial peptides expressed on different epithelial lining are major components of the innate immune system. Based on the deduced amino acid sequence of Bubalus bubalis lingual antimicrobial peptide (LAP) cDNA (Accession No. DQ458768), five overlapping peptides LAP^23–55, LAP^42–64, LAP^21–64, LAP^1–26 and LAP^1–64 were synthesized using solid phase fluorenylmethoxycarbonyl (Fmoc) chemistry. Circular Dichroism spectroscopy of synthesized peptides revealed predominantly β-structure for LAP^23–55, LAP⁴²⁻⁶⁴ and LAP^21–64 with less α-helix in different solutions. Quantitation of secondary structure indicated the highest β-structure for all these three peptides in membrane mimetic SDS solution. The helicogenic solvent TFE could not induce helix in LAP^23–55 however TFE induced helical propensity was observed in LAP^42–64 and LAP^21–64. The quantitation of secondary structure indicated the highest ordered structure for LAP^23–55 followed by LAP^42–64 and LAP^21–64. The antibacterial activity of LAP^23–55 was found to be more potent against Staphylococcus aureus, Listeria monocytogens, Escherichia coli and Salmonella typhimurium followed by LAP^42–64 and LAP^21–64. Minimum inhibitory concentration (MIC) also showed similar trend with lowest value for LAP^23–55 followed by LAP^42–64 and LAP^21–64. Haemolysis and cytotoxicity was observed above 3 fold for LAP^21–64, above six fold for LAP^23–55 and LAP^42–64 of their MIC. The LAP^1–26 and LAP^1–64 could not produce any characteristic CD spectra and did not show any antimicrobial activity, indicating that N- terminal of the peptide negates the antimicrobial activity.     

Characterization of VP2 gene of an Indian Bluetongue virus serotype 2 and its close phylogenetic relationship to the Taiwan isolate

In this study we present the first report on partial amplification, sequencing and phylogenetic relationship of VP2 of the Indian isolate BTV-2. A PCR product of 1135 bp was amplified, cloned and sequenced. About 1063 bp of partial VP2 gene (1792-2854 bp region) of the Indian isolate was subjected to sequence analysis with already published sequences available in the genome database. The percent similarity of 85.2 was observed with Taiwan isolate and 59% with other isolates of BTV-2. However, 46.2% similarity with Australian BTV-1 and no significant similarity were noted with other serotypes. In-silico analysis and restriction enzyme digestion confirmed the presence of conserved SalI site at 2380 bp position in both Indian and Taiwan isolates. Phylogenetic analysis showed that all BTV-2 isolates formed one distinct group in which BTV-2 Indian and Taiwan isolate is more closely related and further demonstrated that BTV’s of the same serotype from different geographical regions were closely related at nucleotide and amino acid level, respectively.     

Identification of host plant resistance to pepper leaf curl virus in chilli (Capsicum species)

Three hundred and seven genotypes belonging to four cultivated and one wild species of Capsicum were screened against pepper leaf curl virus (PepLCV) causing devastating leaf curl disease of chilli (Capsicum annuum). Initial screening was done under field conditions based on coefficient of infection (CI), disease reaction to each genotype was assigned. Subsequently, selfed progenies of eight symptom-less and highly resistant lines were challenged by viruliferous white fly under glasshouse conditions, out of which only three genotypes, viz. GKC-29, BS-35 and EC-497636 showed no symptom. Using scion and root stalk of susceptible genotype (Pusa Jwala), these three putative symptom-less genotypes were further challenged by grafting and alternate grafting. The resistant reactions of GKC-29, BS-35, EC-497636 were confirmed because even after 50 days of successful grafting/alternate grafting, no viral symptom appeared on all the grafted plants of these genotypes. When subjected to PCR amplification with degenerate primers deigned to detect gemnivirus like PepLCV, the three symptom-less genotypes did not show any amplification, suggesting that the resistant reaction in three identified symptom-less resistant sources was because of the absence of viral genome and they are not symptom-less carrier.