Cardiovascular outcomes of Nephrotic syndrome in childhood (CVONS) study: a protocol for prospective cohort study.

BACKGROUND: Nephrotic syndrome (NS) is characterized by dyslipidemia which is a well-known risk factor for atherogenesis. Atherosclerosis in childhood is mostly subclinical and endothelial dysfunction is known to precede this. Evidence for screening for endothelial dysfunction and cardiovascular risk factors and early identification of premature onset of atherosclerosis in childhood NS remains tenuous in the absence of well-designed prospective studies addressing cardiovascular comorbidity in NS. The objective of our study is to examine endothelial dysfunction and short-term cardiovascular outcomes in a carefully phenotyped cohort of patients with Nephrotic syndrome as compared to healthy controls. METHODS: In a multi-centric prospective cohort study, 70 Steroid Resistant NS (SRNS), 70 Steroid Sensitive (SSNS) patients along with 70 Healthy Controls are being recruited. After a baseline assessment of functional and structural status of heart (2D Echocardiography), arteries (Carotid Doppler and Intima Media Thickness measurements) and microcirculation [a combination of 2D Echocardiography, Laser Doppler Flowmetry (LDF) and Brachial Artery Flow mediated dilation (FMD) and Nail Fold Capillaroscopy (NFC)], the patients are being investigated for endothelial dysfunction. Venous blood sample (15 ml) is being collected for routine investigations and assay of biochemical endothelial markers through Flow Cytometry. The patients will be followed up at 12 months and 24 months after the recruitment to look for any change from baseline period. DISCUSSION: This study will able to provide a better understanding of the epidemiology of endothelial dysfunction and associated subclinical cardiovascular co-morbidity in childhood NS. Findings on characterization of prevalence of endothelial dysfunction and subclinical markers may be used to design future randomized controlled trials for evaluating the efficacy of preventive and therapeutic interventions in reducing the incidence of cardiovascular disease.

Buffalopox: an emerging and re-emerging zoonosis.

Outbreaks of buffalopox or pox-like infections affecting buffaloes, cows and humans have been recorded in many parts of the world. Since the first outbreak in India, a large number of epidemics have occurred. Unlike in the previous years, generalized forms of the disease are now rare; however, there are severe local forms of the disease affecting the udder and teats, leading to mastitis thereby undermining the productivity of milk animals. The causative agent buffalopox virus (BPXV) is a member of the Orthopoxvirus, and is closely related to Vaccinia virus (VACV), the type-species of the genus. Earlier studies with restriction fragment length polymorphism and recent investigations involving sequencing of the genes that are essential in viral pathogenesis have shown that BPXV is phylogenetically very closely related to VACV and may be considered as a clade of the latter. The review discusses the epidemiology, novel diagnostic methods for the disease, and molecular biology of the virus, and infers genetic relationships of BPXV with other members of the genus.

Upcoming and future strategies of tick control: a review.

Ticks are distributed worldwide and significantly impact human and animal health. Due to severe problems associated with the continuous use of acaricides on animals, integrated tick management is recommended. Increasing public health concern over the tick-borne diseases demands the strategic control of ticks on animals that transmit diseases to human beings. Immunological control of tick vector of Kyasanur Forest Disease (KFD) on cattle and other wild reservoir hosts is one of the possible alternative strategy for reducing the transmission of KFD to man. Chemical-vaccine synergies have been demonstrated and a combination of chemical and vaccine for tick and tick-borne disease control has been identified as a sustainable option. Studies have suggested the possibility of vaccine strategies directed towards both tick control and transmission of pathogens. Besides tick vaccine, use of endosymbionts, which are essential for the survival of arthropod hosts, for the control of tick vectors will be one of the targeted areas of research in near future. India with huge natural resources of herbs and other medicinal plants, the possibilities of developing herbal acaricides is discussed. The future of research directed towards target identification is exciting because of new and emerging technologies for gene discovery and vaccine formulation.

Inhibition of Anatid Herpes Virus-1 replication by small interfering RNAs in cell culture system.

RNA interference (RNAi) mediated by double stranded small interfering RNA (siRNA) is a novel mechanism of post-transcriptional gene silencing. It is projected as a potential tool to inhibit viral replication. In the present paper, we demonstrate the suppression of replication of an avian herpes virus (Anatid Herpes Virus-1, AHV-1) by siRNA mediated gene silencing in avian cells. The UL-6 gene of AHV-1 that codes for a protein involved in viral packaging was targeted. Both cocktail and unique siRNAs were attempted to evaluate the inhibitory potential of AHV-1 replication in duck embryo fibroblast (DEF) cell line. DEF cells were chemically transfected with different siRNAs in separate experiments followed by viral infection. The observed reduction in virus replication was evaluated by cytopathic effect, viral titration and quantitative real time PCR (QRT-PCR). Among the three siRNA targets used the unique siRNA UL-B sequence was found to be more potent in antiviral activity than the cocktail and UL6-A-siRNA sequences.

Three-dimensional structure of mare diferric lactoferrin at 2.6 A resolution.

Lactoferrin is a monomeric glycoprotein with a molecular mass of approximately 80 kDa. The three-dimensional structure of mare diferric lactoferrin (mlf) has been determined at 2.6 A resolution. The protein crystallizes in the space group P 212121with a=85.2 A, b=99.5 A, c=103.1 A with a solvent content of 55 % (v/v). The structure was solved by the molecular replacement method using human diferric lactoferrin as the model. The structure has been refined using XPLOR to a final R -factor of 0.194 for all data in the 15-2.6 A resolution range. The amino acid sequence of mlf was determined using a cDNA method. The final refined model comprises 5281 protein atoms, 2 Fe3+, 2 CO32-and 112 water molecules. The overall folding of mlf is similar to that of other proteins of the transferrin family. The protein folds into two globular lobes, N and C. The lobes are further divided into two domains, N1 and N2, and C1 and C2. The iron-binding cleft is situated between the domains in each lobe. The N lobe appears to be well ordered and is more stable than the C lobe in mlf unlike in other lactoferrins, where the C lobe is the more stable. The opening of the binding cleft in the N lobe of mlf is narrower than those in other proteins of the transferrin family. This is very unusual and is found only in mare lactoferrin. Apart from certain hydrophobic interactions at the mouth of the cleft, one salt-bridge (Lys301 . . . . . . . . Glu216) crosses between the two walls of the cleft. The two lobes are connected covalently by a three-turn alpha-helix involving residues 334-344. The N lobe displays a highly ordered structure with appreciably low temperature factors. The iron coordination is more symmetrical in the N lobe than in the C lobe. There are only 16 intermolecular hydrogen bonds in the structure of mlf.

Calcium ion-induced structural changes in bacteriophage phi X174.

Monoclinic P2(1) crystals of the bacteriophage phi X174 have been incubated with calcium ions (Ca2+) and the induced structural conformational changes studied to 3 A resolution with X-ray crystallographic methods. Three different types of Ca2+ binding sites have been located within the asymmetric unit of the virion. Two sets of sites are associated with the F capsid protein. One set of sites associated with the F protein is in a general position near the icosahedral 3-fold axes of the virus, with the main-chain carbonyl oxygen atoms of residues Gly1321, Asp1421, Met1424 and Ser1426, and the side-chains of Gln1004 and Asp1421 as ligands. The other set of sites associated with the F protein is on the icosahedral 3-fold axes, with the symmetry-related main-chain carbonyl oxygen atoms of Ser1001 and the side-chains of Asn1002 as ligands. The bound Ca2+ induce a conformational change of the amino-terminal residues of the F proteins. A third set of sites, consisting of a pair of Ca2+ on the icosahedral 5-fold axes, are associated with the G spike protein and are concurrently liganded by the symmetry-related carbonyl oxygen side-chains of Asp2117. Concomitant with the binding of Ca2+ to the phage is the rotation of the Asp1209 side-chain of the F protein towards some additional electron density that was not observed in the absence of Ca2+. This density is situated in a shallow depression near the icosahedral 2-fold axes of the virus, and has been tentatively interpreted as a bound glucose molecule that is ordered only in the presence of Ca2+. The putative glucose binding site may be related to the attachment of the virus to cell surface lipopolysaccharides in the initial stages of Escherichia coli infection.

Cloning and sequencing of 28 kDa outer membrane protein gene of Brucella melitensis Rev. 1.

Brucella melitensis is an organism of paramount zoonotic importance. The 28 kDa outer membrane protein (OMP) is one of the immunodominant antigens of B. melitensis. The gene encoding 28 kDa OMP (omp28) has been amplified from B. melitensis Rev. 1 strain. A PCR product of 753 bp, encoding complete omp28 gene of B. melitensis, was obtained. The gene was further cloned and sequenced. The nucleotide sequence of B. melitensis Rev. 1 strain showed substitution of 2 nucleotides from that of 16M strain.

Molecular variability in different Indian isolates of equine herpesvirus-1.

Three abortigenic Indian isolates of equine herpesvirus-1 (EHV-1) (Tohana, Hisar and Bikaner), along with two exotic abortigenic isolates (AB4 and V592) and another EHV-1 isolate (Jind) obtained from a case of perinatal foal mortality, were studied for variability. For this purpose, PCR and restriction endonuclease (RE) digestion techniques were used simultaneously as a DNA fingerprinting system. Nine different regions of EHV-1 virus were amplified by PCR using primer pairs specific for the regions and the products obtained from these regions were subsequently subjected to various restriction endonucleases to further assess the variability in the number of RE sites as well as in their positions. No difference was observed in all the four abortigenic isolates in terms of the size of different PCR products amplified by all the nine primer pairs, except for primer pairs 'E' and 'C'. PCR products obtained with primer pair E revealed that Tohana and Bikaner isolates were most similar while Hisar isolate was like V592 isolate. However, the PCR product obtained from Jind isolate had a size between the PCR products of Hisar and Tohan/Bikaner isolates. The primer pair 'C' used to amplify the region between 1151 to 3679 in 'Gene 1,2,3' clearly differentiated the EHV-1 isolate obtained from a case of perinatal foal mortality from isolates obtained from abortion cases. This primer pair needs to be exploited more extensively for use as a potential marker for differentiating the EHV-1 isolates, mainly the abortion cases from perinatal foal mortality ones. Restriction endonuclease studies done with PCR product of all the isolates with various primer pairs did not reveal any changes in the position or number of RE sites present in the products amplified, indicating no variation in different RE sites within the amplified PCR products. However, this study clarified that all the Indian isolates belonged to the IP group of EHV-1.

Experimental infection of Coxiella burnetii in chicken: clinical symptoms, serologic response, and transmission through egg.

Young laying hens were infected with Coxiella burnetii to study egg transmission, clinical and serologic responses, excretion of the agent in feces, and its persistence in internal organs. No clinical symptoms were noticed. The birds developed good titers in a capillary agglutination test by 13 days postinfection (DPI), which peaked at 30 DPI and therafter declined gradually, becoming negative in some birds around 90 DPI. In vivo and in vitro egg transmission of the agent could not be demonstrated. C. burnetii was recovered at 90 DPI from spleen and liver but not from kidney and ovary.

Sero-epidemiology of Q-fever in poultry.

The sero-epidemiology of Q-fever was studied by capillary agglutination test at 25 poultry farms in the Nainital and Ajmer districts of Uttar Pradesh and Rajasthan. Of 589 birds tested, 78 (13.24%) had Q-fever agglutinins (CAT titers 1:8 to 1:64), involving 16 of the farms. There were more sero-reactors in Ajmer (17.56%) than in Nainital (3.35%). The sero-positive reactors were respectively 19.74% and 5.55% among the age groups above and below 6 months. The breed difference and comparatively high infection rates in poultry attendants of a Q-fever-positive farm are discussed.

An outbreak of fowl cholera due to Pasteurella gallinarum in Uttar Pradesh (India).

An acute outbreak of fowl cholera in one-month-old chickens was investigated. Pasteurella gallinarum was isolated in pure culture from the heart blood of two moribund chicks. One of the isolants on experimental inoculation was found to be nonpathogenic for rabbits, mice, and chickens. It did not provide protection in rabbits against a virulent strain of P. multocida. This seems to be the first record of the isolation of P. gallinarum in India.

Isolation of A/Equi-2 virus during 1987 equine influenza epidemic in India.

Processing of nasal materials from clinical cases during the 1987 influenza epidemic in Northern and Central India resulted in the isolation of two haemagglutinating agents; one each from donkeys and horses at Bhiwani in Haryana State and Ludhiana in Punjab State, respectively. These were typed as Influenza A/Equi-2 viruses by haemagglutination inhibition test. The two isolates were designated as A/Equi-2/Bhiwani/1/87 and A/Equi-2/Ludhiana/1/87. The Bhiwani/87 isolate was confirmed to have H3N8 antigenic structure and was indistinguishable from the Miami/63 strain of A/Equi-2 virus. However, the A/Equi-2 Ludhiana/87 isolate was closely related to the Fontainebleau/79 strain of A/equi-2 virus.

Characterisation of equine influenza isolates from the 1987 epizootic in India by nucleotide sequencing of the HA1 gene.

Two A/Equi-2 (H3N8) isolates were obtained during the 1987 Indian equine influenza epizootic. The sequence of the Ludhiana/87 HA1 gene revealed that this isolate was very similar to recent European and North American isolates of equine influenza. In contrast, the Bhiwani/87 HA1 gene was nearly identical to the Miami/63 prototype H3 sequence. These results support the antigenic analysis previously carried out on these isolates using monoclonal antibodies. However, the finding that Bhiwani/87 is so similar to Miami/63, coupled with the finding that equine H3N8 influenza viruses have previously been shown to evolve along a single lineage, suggests that Miami/63 virus from either a vaccine or laboratory source may be the origin of the Bhiwani/87 isolate.

Poikilotherms as reservoirs of Q-fever (Coxiella burnetii) in Uttar Pradesh.

Water snakes (Natrix natrix), rat snakes (Ptyas korros), cobras (Naja naja), pythons (Python molurus), tortoises (Kachuga sp.), plankton fish (Cirrhina mrigala), frogs (Rana tigrina), toads (Bufo sp.) and monitors (Varanus indicus) were screened for evidence of Q-fever infection by the capillary agglutination test on sera to detect antibodies and/or by attempts to demonstrate Coxiella burnetii in spleen and liver samples. Sero-reactors were observed among water and rat snakes, pythons and tortoises. The organism was isolated from the spleen and liver of the monitor, tortoise and python.

The occurence of coxiellosis among rodents and shrews in the Tarai area of Uttar Praedesh.

Rodents and shrews were screened for serologic evidence of Coxiella burnetii. Attempts were made to isolate the organism from the spleen and liver. Seroreactors: total positive/total tested (% positive), in rats (Rattus rattus, R. norvegicus), ground shrews (Suncus murinus), bandicoots (Bandicota indica, B. bengalensis) and the house mouse (Mus musculus), respectively, were 13/105 (12.38), 6/42 (14.3), 2/15 (13.3) and 1/7 (14.3). Of the eight rickettsial isolants recovered including four from field and household rats, three from ground shrew and one from bandicoots, only three comprising one each from rat, shrew and bandicoot, could be typed as C. burnetii. This appears to be the first record of rodents and an insectivore as reservoirs of C. burnetii in India.

The outbreak of equine influenza (H3N8) in the United Kingdom in 1989: diagnostic use of an antigen capture ELISA.

In July 1989 influenza A/equine-2 (H3N8) was isolated from a nasopharyngeal swab taken from a non-thoroughbred horse exhibiting acute clinical respiratory disease. This was the first isolation of equine influenza virus in the United Kingdom since 1981. Subsequent investigations of acute respiratory disease in horses indicated that the infection was dispersed throughout the UK. However, unlike the previous epidemic of 1979, the first horses from which the virus was isolated had been vaccinated. This outbreak of influenza provided an opportunity to evaluate an antigen capture ELISA, directed against the influenza virus nucleoprotein, as a rapid method for detecting virus in the nasopharyngeal secretions of naturally infected horses.

Application of ELISA in the detection of goat pox antigen and antibodies.

Enzyme linked immunosorbent assay (ELISA) was standardized for detection of goat pox virus (GPV) antibodies and antigen using live and inactivated antigens and hyperimmune serum (HIS), convalescent, post-vaccinal, as also post-challenge sera. The ELISA was most sensitive in detection of antibody when compared with agar gel precipitation (AGP) and counterimmunoelectrophoresis (CIE) tests. There was no complete correlation between the antibody status of vaccinated goats and protective immunity as animals having detectable seroconversion were also solidly immune to virulent challenge. The application of ELISA in pox infections of goats has been discussed.

Single radial immunodiffusion potency test for standardization of indigenous equine influenza vaccine.

Single radial immunodiffusion (SRD) assays were used for measuring the haemagglutinin antigen contents of equine influenza vaccine prepared from an Indian virus isolate. A/Equine-2/Ludhiana/1/87 (H3N8). Five different preparations of the vaccine were standardized by SRD to prepare 913 doses, each containing 20 micrograms HA/ml-1 dose-1. This test also showed influenza virus subtype specificity as no cross reaction was observed between subtype 1 (H7N7) and subtype 2 (H3N8) viruses.

Application of polymerase chain reaction (PCR) for diagnosis of equine herpes virus-1 (EHV-1).

Fifty aborted foetus samples were diagnosed for the presence of equine herpes virus-1 (EHV-1) by polymerase chain reaction (PCR) technique. Specific primer pair for amplification of a particular segment of EHV-1 DNA in gc region having 3 Hae III restriction endonuclease sites was used. A 409 base pair segment obtained as PCR amplification product in 9 samples was digested with Hae III to confirm the presence of EHV-1 as the infectious agent in aborted tissues. It was observed that PCR technique was more sensitive, specific and rapid than the conventional virological diagnostic methods.

Evaluation of guinea pig model for experimental Salmonella serovar Abortusequi infection in reference to infertility.

The present study conclusively revealed the role for Salmonella enterica subspecies enterica serovar Abortusequi in conception failure. None of the 12 guinea pigs conceived when orally exposed to sublethal dose of the pathogen during breeding, while 66.67% of animals in control group were found pregnant during same period of observation under similar conditions. Salmonella carrier animals also had drastic reduction in conception rate (16.67%). During mid pregnancy, S. Abortusequi exposure to guinea pigs through intravaginal, intramuscular and subcutaneous routes induced fetal death followed by resorption. While 2 out of 6 orally inoculated and 3 out of 6 intraperitonially inoculated guinea pigs aborted, in rest of the animals fetal death was followed by meceration and resorption. It was interesting to note that S. Abortusequi could not persist longer than a week in males while in pregnant females it could be detected for >10 weeks after inoculation. In late pregnancy, most of the exposed animals aborted and non aborting animals though had normal parturition, survival rate of their babies was nearly zero in comparison to the control group. The study revealed role for S. Abortusequi in impairing conception, abortion, early fetal deaths, fetal meceration and resorption. Further studies are required to identify factors responsible for increased susceptibility of females particularly during pregnancy.