Passive immunization against highly pathogenic Avian Influenza Virus (AIV) strain H7N3 with antiserum generated from viral polypeptides protect poultry birds from lethal viral infection

Our studies were aimed at developing a vaccination strategy that could provide protection against highly pathogenic avian influenza virus (AIV), H7N3 or its variants outbreaks. A purified viral stock of highly pathogenic H7N3 isolate was lysed to isolate viral proteins by electrophresing on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by their elution from gel through trituration in phosphate buffered saline (PBS). Overall, five isolated viral polypeptides/proteins upon characterization were used to prepare hyperimmune monovalent serum against respective polypeptides independently and a mixture of all five in poultry birds, and specificity confirmation of each antiserum through dot blot and Western blotting. Antiserum generated from various group birds was pooled and evaluated in 2-week old broiler chicken, for its protection against viral challenge. To evaluate in-vivo protection of each antiserum against viral challenges, six groups of 2-week old broiler chicken were injected with antiserum and a seventh control group received normal saline. Each group was exposed to purified highly pathogenic AIV H7N3 strain at a dose 105 embryo lethal dose (ELD50). We observed that nucleoprotein (NP) antiserum significantly protected birds from viral infection induced morbidity, mortality and lowered viral shedding compared with antiserum from individual viral proteins or mixed polypeptides/proteins inclusive of NP component. The capability of individual viral polypeptide specific antisera to protect against viral challenges in decreasing order was nucleoprotein (NP) > hemagglutinin (HA) > neuraminidase (NA) > viral proteins mix > viral polymerase (PM) > non-structural proteins (NS). Our data provide proof of concept for potential utilization of passive immunization in protecting poultry industry during infection outbreaks. Furthermore conserved nature of avian NP makes it an ideal candidate to produce antiserum protective against viral infection

Delay in diagnosis of tuberculosis in Rawalpindi, Pakistan

<p>Abstract</p> <p>Background</p> <p>Delay in diagnosis and treatment of tuberculosis (TB) may enhance the chances of morbidity and mortality and play a key role in continuous transmission of the bacilli. The objective of this study was to describe health care seeking behavior of suspected TB patients and initial diagnostic work up prior to consultation and diagnosis at National TB Center (NTC).</p> <p>Findings</p> <p>Interviews of 252 sputum smear positive patients were taken from NTC, Rawalpindi. The duration between on-set of symptoms and start of treatment was considered as the total delay and correlated with general characteristics of TB patients. The proportion of males and females were 49.6% and 50.4% with median age of 25 and 24 years respectively. A median delay of 56 days (8 weeks) was observed which was significantly associated with age, cough and fever. More than 50% of the current patients had a history of contact with previously diagnosed TB patients. The majority of patients (63%) visited health care providers within three weeks of appearance of symptoms but only thirty five percent were investigated for TB diagnosis.</p> <p>Conclusion</p> <p>Cough and fever are being ignored as likely symptoms of TB by patients as well as health care providers resulting in delay. Engaging private practitioners through public private mix (PPM) approach for expansion of TB diagnosis and increasing public awareness could be more beneficial to reduce delay.</p

A Study of DNA Protective Ability of Peels of Different Citrus Species

The aim of the present study was to assess the DNA protection ability, free radicals scavenging activity, and phytochemical constituents of peel extracts of various species of citrus. Results showed the presence of carbohydrates, amino acids, glycosides, tannins, steroids and alkaloids. DNA protection assay showed maximum protection with methanolic extracts of peels of C. limon, C. reticulata, C. aurantium and C. sinensis as compared to C. limetta which showed less protection. DPPH assay used to assess the antioxidants showed the highest activity in the methanolic extract of peels of C. reticulata and C. aurantium with 75% and 86% respectively at 10 mg/ml. It is therefore concluded that C. limon, C. reticulata, C. aurantium and C. sinensis in terms of DNA protection and C. reticulata and C. aurantium in terms of free radicals scavenging activity are the best ones and these are the potential candidates to be used for further studies

Cloning, Expression and Genetic Immunization Studies of Mycobacterium tuberculosis Gene esat6

Abstract.-Early secreted antigenic target protein 6 (esat6) is one of the genes present on region of difference 1 (RD1) of Mycobacterium tuberculosis (M. tb) genome. This RD1 is a characteristic of virulent strains of M. tb and Mycobacterium bovis and this is one of the major differences between the disease causing strains and Bacillus-Calmat Guerin (BCG) vaccine strains. Studies have proved the presence of large number of memory T cells in M. tb infected individuals and these memory cells are reactive towards Esat6 antigen, which highlighted the importance of this gene especially in early infections. In this study, numbers of esat6 gene constructs were made in order to get a suitable construct to be used as good DNA vaccine. First esat6 gene construct was made in pND vector without Kozak sequence upstream the gene, second construct was made in pcDNA3.1 vector with Kozak sequence upstream the gene, third construct was made again in pND vector with Kozak sequence, fourth construct was made with Kozak sequence upstream and GGG downstream the ATG as a second codon of gene first in pcDNA3.1 and later in pND vectors respectively which was designated as construct five. Sixth construct was a fusion and in pcDNA3.1 vector with Kozak upstream the gene and epitope V and poly histidine tail sequence provided by vector down stream the gene through inframe cloning of esat6 gene with sequences provided by vector by removing stop codon through PCR based primers. Seventh and final construct was prepared in pND14 vector also as a fusion construct and gene was cloned under tissue plasminogen activator sequence in an in-frame through PCR based primers. All these constructs were subjected to 293T human embryonic kidney cell lines to evaluate their level of expression. Although none of the constructs gave detectable level of expression in cultured cells when tested through Western blots (WB) but tpa-esat6-pND14 construct was selected as potential DNA vaccine candidate to inject intramuscularly and interadermally to balb/c mice along with controls to obtain detectable response in vivo. Animals were tested nine weeks post vaccination and found positive against tpa-esat6-pND14 vaccine through WB and multiplex micro bead immunoassay (MMIA)

Peri-parturient rise in faecal nematode egg counts with reference to Haemonchus contortus in Bulkhi ewes in Northern Punjab, Pakistan

The occurrence of the peri-parturient rise (PPR) in fecal egg count (FEC) phenomena in Bulkhi ewes and its subsequent impact on naive lambs reared in a traditional semi-intensive husbandry system were monitored at the Small Ruminants Research Station, National Agricultural Research Centre, Islamabad, Pakistan. In this study, two ewe groups, pregnant/lactating (n=37) and open/non-prcgnant (n=37), were observed for the PPR phenomena in the sub-tropical area of Pakistan. A significant difference (P\u3c 0.01) was noted for FEC, individual larval culture, packed cell volume (PCV) and haemoglobin (Hb) level in the pregnant/lactating ewes as compared to the open ewes throughout the study. Faecal examination showed consistently higher, predominantly Haemonchus contortus, FEC with lower PCV and Hb level in the pregnant/lactating ewes. The sharp increase in FEC occurred two weeks before lambing and persisted for 12 weeks after lambing. The results showed that the PPR in FEC was associated with both gestation and lactation which provided a large number of third-stage infective larvae (L3) on pasture. These larvae were considered the primary source of infection for the lambs. Thus, infection in lambs showed an initial rise in FEC after four weeks when highly susceptible lambs were allowed to graze along with their dams on the same naturally contaminated pastures. A control measure to consider would be to deworm ewes before lambing or in early lactation to reduce pasture contamination and infection of lambs. Copyright 2009 Zoological Society of Pakistan