Comparative efficacy of live replicating sheeppox vaccine strains in Ovines.

In the present study, two sheeppox vaccines made from strains [sheeppox virus-Srinagar (SPPV-Srin) and Ranipet (SPPV-R)] indigenous to India and adapted to Vero cells were compared in terms of their safety, potency, efficacy and antigenic value with the commercial in-use Roumanian Fanar (SPPV-RF) vaccine, a foreign strain adapted in primary lamb testes cells. The safety test indicated that the SPPV (Sri and RF) vaccines were safe while SPPV-R was not completely attenuated and caused excessive adverse reactions at the passage level tested. The immunized animals showed DTH reaction and resisted virulent SPPV challenge, while control animals developed disease. Specific virus could be detected in the controls and animals immunized with lower dilutions of vaccines after challenge but not in any of the sheep immunized with 1 and 100 doses of each vaccine. All vaccines were found potent and the PD(50) was highest for SPPV (Srin and R) followed by RF. The immunized animals were seroconverted following vaccination with sustained antibody responses after challenge. In conclusion, indigenous SPPV-Srin vaccine was found to be as efficacious as SPPV-R and SPPV-RF vaccines. Thus, there is potential benefit in replacing the currently used commercial vaccine SPPV-RF with indigenous SPPV-Srin vaccine for use in India.

Application of semi-quantitative M gene-based hydrolysis probe (TaqMan) real-time RT-PCR assay for the detection of peste des petits ruminants virus in the clinical samples for investigation into clinical prevalence of disease.

Peste des petits ruminants (PPR) is a contagious, notifiable and economically important transboundary viral disease of small ruminants. In this study, a hydrolysis probe-based real-time reverse transcription-polymerase chain reaction (rt RT-PCR) assay for the detection and semi-quantification of PPR virus (PPRV) nucleic acid was developed using the virus RNA and matrix (M) gene-specific primers with Hex-labelled fluorescent probe and applied for the detection of PPRV in clinical samples to identify outbreaks and to monitor the prevalence of disease. The assay was found specific with a sensitivity detection limit of 0.5 pg of total PPRV RNA. Based on a serial dilution of the live-attenuated PPR vaccine virus, the detection limits were approximately 0.1 and 1 TCID50 for the hydrolysis probe and conventional RT-PCR assays, respectively. The assay was linear within a range of 50 ng to 0.5 pg total virus RNA with an intra-assay coefficient of variation (CV) in the range of 0.91-2.86% and an inter-assay CV ranging between 0.59% and 2.37%. The standardized rt RT-PCR was easily employed for the detection of PPRV nucleic acid directly in the experimental/field clinical samples. This assay detected the PPRV in pre-clinical swab materials as early as the 4th day post-infection (dpi) and up to 17th dpi in nasal, ocular and oral swabs collected from experimentally infected animals. The rt RT-PCR was rapid, specific and 10 times more sensitive than conventional RT-PCR. It is an alternative test to the existing diagnostic assays and could be useful with enhanced applicability in field clinical diagnosis by avoiding the use of expensive commercial real-time PCR reagents. This assay was adopted directly in the detection of PPRV nucleic acid in clinical samples collected from sheep and goats suspected of PPR to monitor outbreak situations and the clinical prevalence of PPR in India.

Sequence and phylogenetic analyses of the structural genes of virulent isolates and vaccine strains of peste des petits ruminants virus from India.

Peste des petits ruminants (PPR) is an acute, highly contagious, notifiable and economically important transboundary viral disease of sheep and goats. In this study, sequence and phylogenetic analyses of structural protein genes, namely the nucleocapsid (N), the matrix (M), the fusion (F) and the haemagglutinin (H) coding sequences of virulent and vaccine strains of PPR virus (PPRV), were undertaken to determine the genetic variations between field isolates and vaccine strains. The open reading frame (ORF) of these genes of the isolates/strains was amplified by RT-PCR, cloned and sequenced. The ORF of N, M, F and H genes was 1578, 1008, 1641 and 1830 nucleotides (nt) in length and encodes polypeptides of 525, 335, 546 and 609 amino acids (aa), respectively, as reported earlier. Comparative sequence analyses of these four genes of isolates/strains were carried out with published sequences. It revealed an identity of 97.7-100% and 97.7-99.8% among the Asian lineage IV and 89.6-98.7% and 89.8-98.9% with other lineages of PPRV at nt and aa levels, respectively. The phylogenetic analyses of these isolates based on the aa sequences showed that all the viruses belonged to lineage IV along with other Asian isolates. This is in agreement with earlier observations that only PPRV lineage IV is in circulation in India since the disease was first reported. Further, sequence analysis of the thermostable/thermo-adapted vaccine strains showed no significant changes in the functional or structural surface protein-coding gene sequences. It is important to monitor the circulation of the PPRV in susceptible animals by H gene-based sequence comparisons in addition to the F gene- and N gene-based approaches to identify the distribution and spread of virus in the regular outbreaks that occur in endemic countries like India.

Expression of P32 protein of goatpox virus in Pichia pastoris and its potential use as a diagnostic antigen in ELISA.

The present study was undertaken to express goatpox virus (GTPV) P32 protein in Pichia pastoris and evaluate its potential use as a diagnostic antigen in ELISA. The amplified P32 gene of GTPV was cloned into pPICZalphaA vector and characterized by PCR, restriction enzyme digestion and sequencing. The characterized linear recombinant plasmids were transformed in Pichia host GSII5 strain by electroporation and the zeocin resistant Pichia transformant containing P32 gene was selected and confirmed by PCR. The expression of P32 protein in Pichia was induced with 0.5% methanol at 30 degrees C. The optimum expression was observed at 72 h post-induction and the yield was 100 mg/L of culture. The expressed protein was precipitated with polyethylene glycol and analyzed by SDS-PAGE and Western blot using GTPV specific serum and GTPV-P32 protein specific monoclonal antibody. Further, the protein precipitated with acetone was evaluated as diagnostic antigen in indirect ELISA in order to replace the whole GTPV. The standardized P32 protein based indirect ELISA had relative specificity and sensitivity of 84.2% and 94.2-100%, respectively when compared with serum neutralization test and whole virus based indirect ELISA. This study showed a potential of the yeast expressed GTPV-P32 protein as safe antigen in ELISA for seroepidemiological study of the capripox infection in sheep and goats, in India as well as capripox enzootic countries.

Expression of Peste des petits ruminants virus nucleocapsid protein in prokaryotic system and its potential use as a diagnostic antigen or immunogen.

In this study, both partial and full-length nucleocapsid (N) gene of Peste des petits ruminants virus (PPRV) were cloned into pET33b vector and expressed in Escherichia coli (BL21) with the objective of replacing live PPRV antigen with recombinant protein in ELISA. The expressed proteins were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot by using a PPRV N protein specific monoclonal antibody. The expressed histidine-tagged fusion proteins were purified using affinity Ni-NTA column and were assessed for their conformation in terms of reactivity by ELISA. The immunogenicity of recombinant proteins was also assessed in rabbits and anti-N antibody response against PPRV was observed in all the immunized rabbits, when tested by competitive and indirect ELISAs. In sandwich ELISA, a mean OD(492 nm) of 1.4 and 0.90 was obtained for crude lysate having expressed the N protein and the PPRV antigen, respectively. Further, the N protein was tested as a coating antigen in competitive ELISA instead of PPRV antigen for serological diagnosis of PPR infection. This indicates the diagnostic potential of the PPRV recombinant N proteins, which are safe and better alternatives to live PPRV antigen in ELISA for clinical or sero-surveillance of PPR in enzootic or non-enzootic countries.

The perceived long-term impact of a psychiatry clerkship on personal growth and clinical skills.

The authors investigated the effects of a psychiatry clerkship that over 14 years has had a constant training philosophy and faculty and has been located in the same acute general hospital setting. In the study, 169 graduates completed a questionnaire on the effects of the clerkship on their knowledge of psychiatry, management of emotional problems in their patients, and personal development. Based on the graduates' responses, the results reveal that the clerkship indeed has had a lasting impact on its former students. Possible implications for recruitment of students into psychiatry are discussed.

Geophagia in a chronic hemodialysis patient.

Geophagia, the deliberate ingestion of earth, is a serious clinical problem, particularly for dialysis patients. This article presents a geophagic patient with end stage renal disease and reviews the etiology, consequences and treatment of this disorder.

Self-mutilation resulting in bacterial meningitis.

Self-mutilation and particularly self-destructive dermatoses are not usually life-threatening. This case involves a man who met the DSM-III R diagnostic criteria for delusional (paranoid) disorder, somatic type. His destructive behavior involving the face and scalp resulted in osteomyelitis and pneumococcal meningitis. He responded to treatment initially, but was later lost to follow-up. No similar case of self-mutilation has been reported.