Detection of Peste des Petits Ruminants Viral RNA in Fecal Samples of Goats after an Outbreak in Punjab Province of Pakistan: A Longitudinal Study.

Peste des petits ruminants (PPR) is a highly contagious viral disease of domestic and wild small ruminants and thus has serious socioeconomic implications. In Pakistan, during the year 2012-2013, estimated losses due to PPR were worth Rs. 31.51 billions. Close contact between infected and susceptible animals is an important route of transmission of PPR. Therefore, carrier animals play an important role in unnoticed transmission of PPR. The objective of the study was to investigate the detection of PPR virus in goats recovered from PPR. A suspected PPR outbreak was investigated and confirmed as PPR after analysing appropriate samples collected from infected animals using rRT-PCR. A longitudinal study was conducted over the period of 16 weeks to ascertain the detection of PPR virus (PPRV) in faecal samples of recovered goats. Ninety-six (96) faecal samples from each sampling were collected at 4, 8, 12, and 16 weeks after the outbreak. Faecal samples were analysed using rRT-PCR. Of 96 from each sampling a total of 46, 37, 29, and 25 samples were positive for PPR viral genome at 4, 8, 12, and 16 weeks, respectively, after recovery. Attempts were made for the isolation of PPR virus on Vero cells, but results were negative. These results indicated the detection of PPR viral RNA up to 16 weeks after infection. Therefore, these results may help in the future epidemiology of PPR virus shedding and possible role as source of silent infection for healthy animals especially when there is no history of any outbreak in nearby flock or area.

Synthesis of piroxicam loaded novel electrospun biodegradable nanocomposite scaffolds for periodontal regeneration.

Development of biodegradable composites having the ability to suppress or eliminate the pathogenic micro-biota or modulate the inflammatory response has attracted great interest in order to limit/repair periodontal tissue destruction. The present report includes the development of non-steroidal anti-inflammatory drug encapsulated novel biodegradable chitosan (CS)/poly(vinyl alcohol) (PVA)/hydroxyapatite (HA) electro-spun (e-spun) composite nanofibrous mats and films and study of the effect of heat treatment on fibers and films morphology. It also describes comparative in-vitro drug release profiles from heat treated and control (non-heat treated) nanofibrous mats and films containing varying concentrations of piroxicam (PX). Electrospinning was used to obtain drug loaded ultrafine fibrous mats. The physical/chemical interactions were evaluated by Fourier Transform Infrared (FT-IR) spectroscopy. The morphology, structure and pore size of the materials were investigated by scanning electron microscopy (SEM). The thermal behavior of the materials was investigated by thermal gravimetric analysis (TGA) and differential scanning calorimetry (DSC). Control (not heat treated) and heat treated e-spun fibers mats and films were tested for in vitro drug release studies at physiological pH7.4 and initially, as per requirement burst release patterns were observed from both fibers and films and later sustained release profiles were noted. In vitro cytocompatibility was performed using VERO cell line of epithelial cells and all the synthesized materials were found to be non-cytotoxic. The current observations suggested that these materials are potential candidates for periodontal regeneration.

Isolation of buffalo poxvirus from clinical case and variations in the genetics of the B5R gene over fifty passages.

Outbreaks of buffalopox affect udder and teats, which may ultimately lead to mastitis in dairy buffalo and can significantly compromise the production. In this study, we report isolation of buffalo poxvirus and sequence analysis of the B5R gene collected from the buffalo clinically suspected to be poxvirus infected. The virus was isolated on BHK-21 cell line and was passaged for 50 times, B5R gene was amplified and sequenced using gene-specific primers, and analyzed at both nucleotide and amino acid levels. Phylogenetically, the isolate can be classified close to the previously reported Pakistani and Indian isolates with certain level of differential clustering patterns. Three significant putative mutations (I2K, N64D, and K111E) were observed in the B5R protein. The K111E was common with previous human isolate from Karachi, Pakistan in 2005. These mutations differed from poxviruses reported from the neighboring countries. Some deletion mutations were observed which were recovered in upcoming passages. The K111E mutation suggests potential to cause zoonotic infection in human all over the country.

Chitosan-based electrospun nanofibrous mats, hydrogels and cast films: novel anti-bacterial wound dressing matrices.

The development of highly efficient anti-bacterial wound dressings was carried out. For this purpose nanofibrous mats, hydrogels and films were synthesized from chitosan, poly(vinyl alcohol) and hydroxyapatite. The physical/chemical interactions of the synthesized materials were evaluated by FTIR. The morphology, structure; average diameter and pore size of the materials were investigated by scanning electron microscopy. The hydrogels showed a greater degree of swelling as compared to nanofibrous mats and films in phosphate buffer saline solution of pH 7.4. The in vitro drug release studies showed a burst release during the initial period of 4 h and then a sustained release profile was observed in the next upcoming 20 h. The lyophilized hydrogels showed a more slow release as compared to nanofibrous mats and films. Antibacterial potential of drug released solutions collected after 24 h of time interval was determined and all composite matrices showed good to moderate activity against Gram-positive and Gram-negative bacterial strains respectively. To determine the cytotoxicity, cell culture was performed for various cefixime loaded substrates by using neutral red dye uptake assay and all the matrices were found to be non-toxic.

Isolation and characterization of low pathogenic H9N2 avian influenza A viruses from a healthy flock and its comparison to other H9N2 isolates.

Several outbreaks of avian influenza (AI) caused by H9N2 subtype, have been reported in the poultry industry during 1990 around the globe. Currently, H9N2 are endemic in the large area of Middle and Far East, including Pakistan. Since H9N2 AI viruses are sporadically reported from humans, extensive incidence of H9N2 in poultry imposes a great risk for human health. In this context, continuous monitoring of the poultry and determining the genetic nature of these viruses are fundamental to predict any future threat. Thus gene sequences of one isolate of H9N2, isolated from commercial poultry flocks, were analyzed. The results of this investigation, based on hemagglutinin (HA), neuraminidase (NA) and non-structural genes, showed that Pakistani H9N2 isolates are closely related to each other and to other H9N2 isolates from the Middle East. However, several unusual substitutions with unknown functional consequences were observed in HA and NA proteins and thus warrant further investigations for their possible role in viral biology. In conclusion, these findings provide information regarding the genetic nature of H9N2 avian influenza viruses in Pakistani poultry and necessitate the sequencing of more H9N2 viruses from both naturally infected and vaccinated flocks.

Biological characterization and phylogenetic analysis of a novel genetic group of Newcastle disease virus isolated from outbreaks in commercial poultry and from backyard poultry flocks in Pakistan.

Newcastle disease (ND) is a contagious viral disease of many avian species particularly domestic poultry, and is responsible for devastating outbreaks in the poultry industries around the globe. In spite of its importance and endemicity in Southern Asia, data on the genetic nature of the viruses and epizootiological information of the disease is scarce. In this study, six isolates from an emerging wave of ND outbreaks in the north of Pakistan and two isolates from healthy poultry flocks were biologically and genetically characterized. Based on pathogenicity indices such as intracerebral pathogenicity index (ICPI), mean death time (MDT) and cleavage motifs in the fusion protein, all these isolates were classified as virulent. Phylogenetic analysis of the fusion (F), hemagglutinin-neuraminidase (HN) and matrix (M) genes indicated the emergence of a novel genetic group within lineage 5, distinct from isolates previously reported in the region. Several mutations in the neutralizing epitopes and functionally important motifs of the F and HN genes pose a need for re-evaluation of the currently used vaccine and vaccination practices. The characteristics of Newcastle disease virus (NDV) as virulent (F protein cleavage site, ICPI and MDT) in apparently healthy backyard poultry (BYP) explain that BYP can play crucial role in the epizootiology and spread of the disease. The present investigation provides essential information on the genetic nature of NDV circulating in Pakistan and its implication on disease diagnosis and control. Furthermore, these investigations emphasize the importance of continuous surveillance of ND in developing countries.

Detection of paratuberculosis in breeding bulls at pakistani semen production units: a continuous source of threat.

Paratuberculosis is a chronic bowel disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Its secretion through semen highlights the importance of paratuberculosis-free breeding bulls. The breeding and teaser bulls at three semen production units (SPUs) located in Punjab, Pakistan, were screened for the presence of antibodies against MAP. A total of 253 samples were collected from SPUs and a commercially available indirect screen ELISA (Is-ELISA) was applied. Is-ELISA detected antibodies in 20 (24.6%), 16 (22.8%), and 17 (16.6%) samples from SPU-I, SPU-II, and SPU-III, respectively. Collectively, seroprevalence of 20.0% (47/235) in breeding bulls and 33.3% (6/18) in teaser bulls was observed, and thus it poses a potential threat of disease spread to a high number of heifers and cows through artificial insemination. Therefore, this paper highlights the presence of the disease for the first time at SPUs and triggers attempts to ascertain the prevalence of paratuberculosis throughout the country.