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Sphingomonas sp. Shah is a bacterium that was first isolated from mammalian cell cultures. According to ribotyping data it is very much homologous to the clinically important pathogen Sphingomonas paucimobilis, which has generated pseudo-outbreaks. Using a tissue culture system, Sphingomonas sp. Shah was discovered to induce apoptosis in human lung epithelial carcinoma. Apoptosis of infected cells was determined by numerous criteria including (1) visual alterations in cellular morphology, (2) initiation of nuclear marginalization and chromatin compaction condensation, (3) the attendance of a high percentage of cells with subG1 DNA content, and (4) caspase-3 activation. In the current study we demonstrate the induction of apoptosis in mammalian lung epithelial cells upon infection with Sphingomonas sp. Shah and provide insight into the molecular processes triggering apoptosis.

Cryptococcal infection (CI) is an uncommon fungal disease that poses a particular fatal risk to liver transplant (LT) recipients because of the potential rapid development and dissemination of the disease. Depending on the pathophysiology, CI may manifest with a wide range of clinical presentations that may delay early diagnosis and timely treatment. Additionally, most anticryptococcal therapies may threaten LT recipients owing to the associated hepatotoxicity of these medications. We report a case of a 25-year-old woman who received an LT for cryptogenic cirrhosis and developed rapidly progressive CI with pulmonary, myocardial, and cerebral involvement within a month of transplantation. She presented with severe pulmonary hypertension refractory to medical management and subsequently died despite our efforts. Herein, we review the etiology of cryptococcosis, the natural history of cryptococcal disease, and standard treatments for CI, and we highlight peculiarities of Cryptococcus neoformans infection in solid organ transplant recipients.

Bluetongue (BT), caused by bluetongue virus (BTV), is a vector-borne disease of small ruminants that has the potential to spread across international borders. Despite large populations of susceptible animals and borders with BTV endemic countries, little is known of the disease burden and prevalent serotypes in the province of Balochistan in Pakistan. We conducted a cross-sectional study to determine seroconversion and prevalent serotypes in selected districts of the province using a competitive enzyme-linked immunosorbent assay (cELISA) and real-time polymerase chain reaction (RT-PCR). Sera (n = 876) were collected from clinically healthy sheep and goats originating from the districts of Quetta (n = 300), Mastung (n = 201), Killa Saifullah (n = 75) and Kech (n = 300). None of the study herds (n = 97) were seronegative for BTV, and at the individual level, the overall prevalence of BTV seroconversion was 47.26% (n = 414/876, 95% CI = 43.92%-50.63%). A higher percentage of goats (50.87%, 95% CI = 45.99%-55.73%) were seropositive for anti-VP7 immunoglobulins (IgG) than sheep (44.21%, 95% CI = 39.81%-48.70%). Odds ratios of seroconversion for goats were associated with breed type (χ2 = 16.84, p = .01), parity (χ2 = 23.66, p = .00) and presence of vector (χ2 = 2.63, p = .10), whereas for sheep, it was associated with breed type (χ2 = 13.80, p = .01) and parity (χ2 = 53.40, p = .00). Serotype 8 was the most prevalent (26.82%, 95% CI = 14.75%-43.21%) followed by an equal prevalence of serotypes 2 and 9 (7.31%, 95% CI = 1.91%-21.01%). To the best of our knowledge, this is the first study conducted in Balochistan province and the results indicate that there is a necessity to initiate intervention strategies to control BT disease burden not only in this region of Pakistan but also in adjacent areas of the neighbouring countries, Iran and Afghanistan.

Newcastle disease virus (NDV) live vaccines are supplied in lyophilized form and usually administered through conventional routes (drinking water, spray, or eye drop) following reconstitution in a diluent. Virus inactivation due to physico-chemical properties of the diluent at the time of administration may lead to vaccine failure. The present study aimed to evaluate the survival of NDV live vaccine strain immersed in 5 pH-amended water samples (pH 5.00, pH 6.00, pH 7.00, pH 8.00, and pH 9.00) by sequential determination of virus infectivity on Vero cells for 3 hours. Minimum reduction in virus infectivity was recorded in the water with neutral or slightly alkaline pH, while the virus was relatively less stable at extreme pH conditions. Maximum reduction of infectivity was observed in the water with pH 9.00 in which the virus was completely inactivated within 3 hours. Addition of stabilizers (Cevamune® or skimmed milk) slightly altered the pH and total dissolved solids (TDS) values of the virus-charged water samples. In the stabilizer-added water samples, minimum reduction in infectivity was observed in the water with neutral pH, followed by the ones with a pH of 8.00, 6.00, 5.00, and 9.00. In all types of water samples, T-90 values (time required for 90% reduction in virus infectivity) were highest (485 minutes) at neutral pH (pH 7.00) and lowest (102 to 134 min) at an extreme alkaline condition (pH 9.00). Results of the present study indicate that water with a pH range of 7.00 to 8.00 is suitable for administration of NDV live vaccines. However, the addition of Cevamune® or skimmed milk may have beneficial effects on preserving the infectivity of the virus, even at extreme pH conditions.

Histoplasma capsulatum is typically an indolent disease among immunocompetent patients. However, immunocompromised patients, such as solid organ transplant recipients, are at risk of developing severe histoplasmosis. Yet post-transplant histoplasmosis is a rare pathology, representing less than five percent of invasive fungal infections among transplant recipients. Furthermore, patients tend to present with nonspecific clinical symptoms, complicating timely diagnosis and delaying treatment. Disease features that may be more representative of H. capsulatum infection, such as anemia, leukopenia and pulmonary involvement are often not present until late in the disease course, when the patient is at greater risk of decompensation. Unlike H. capsulatum infections among immunocompetent hosts, extrapulmonary infection among immunocompromised hosts is more the rule than the exception. Treatment with liposomal amphotericin B followed by oral itraconazole is the standard therapy, but special considerations must be made for patients with hepatic and/or renal insufficiency, underlying cardiac abnormalities or malabsorptive pathologies and doses of immunosuppressants will need to be adjusted for drug interactions. Herein we present a case of H. capsulatum infection presenting with generalized lymphadenopathy post-renal transplant. Keywords: Infectious disease, Histoplasma, Transplant, Fungal infection

Japanese encephalitis (JE) is a vaccine-preventable disease caused by the Japanese encephalitis virus (JEV), which is primarily prevalent in Asia. JEV is a Flavivirus, classified into a single serotype with five genetically distinct genotypes (I, II, III, IV, and V). JEV genotype III (GIII) had been the most dominant strain and caused numerous outbreaks in the JEV endemic countries until 1990. However, recent data shows the emergence of JEV genotype I (GI) as a dominant genotype and it is gradually displacing GIII. The exact mechanism of this genotype displacement is still unclear. The virus can replicate in mosquito vectors and vertebrate hosts to maintain its zoonotic life cycle; pigs and aquatic wading birds act as an amplifying/reservoir hosts, and the humans and equines are dead-end hosts. The important role of pigs as an amplifying host for the JEV is well known. However, the influence of other domestic animals, especially birds, that live in high abundance and close proximity to the human is not well studied. Here, we strive to briefly highlight the role of birds in the JEV zoonotic transmission, discovery of birds as a natural reservoirs and amplifying host for JEV, species of birds susceptible to the JEV infection, and the proposed effect of JEV on the poultry industry in the future, a perspective that has been neglected for a long time. We also discuss the recent in vitro and in vivo studies that show that the newly emerged GI viruses replicated more efficiently in bird-derived cells and ducklings/chicks than GIII, and an important role of birds in the JEV genotype shift from GIII to GI.

Silver nanoparticles in the range of 10–40 nm were synthesized chemically and by laser ablation, employed for in vitro antibacterial action against human pathogenic bacterium. Their formation was evidenced by UV-visible spectrophotometer; particle size confirmed by atomic force microscopy, crystal structure determined by X-ray diffraction and chemical composition investigated by Fourier transform infrared spectroscopy. The calculated MIC (minimum inhibitory concentration) of chemically synthesized nanoparticles with 30–40 nm in size are 2.8 µg/mL, 4.37 µg/mL, 13.5 µg/mL and 2.81 µg/mL for E. coli, S. aureus, B. subtillis and Salmonella, respectively. Whereas laser ablated nanoparticles exhibit MIC of 2.10 µg/mL, 2.36 µg/mL and 2.68 µg/mL for E. coli, S. aureus and Salmonella, respectively.

Venomous snakes are important subjects of study in evolution, ecology, and biomedicine. Many venomous snakes have alpha-neurotoxins (&alpha, neurotoxins) in their venom. These toxins bind the alpha-1 nicotinic acetylcholine receptor (nAChR) at the neuromuscular junction, causing paralysis and asphyxia. Several venomous snakes and their predators have evolved resistance to &alpha, neurotoxins. The resistance is conferred by steric hindrance from N-glycosylated asparagines at amino acids 187 or 189, by an arginine at position 187 that has been hypothesized to either electrostatically repulse positively charged neurotoxins or sterically interfere with &alpha, neurotoxin binding, or proline replacements at positions 194 or 197 of the nAChR ligand-binding domain to inhibit &alpha, neurotoxin binding through structural changes in the receptor. Here, we analyzed this domain in 148 vertebrate species, and assessed its amino acid sequences for resistance-associated mutations. Of these sequences, 89 were sequenced de novo. We find widespread convergent evolution of the N-glycosylation form of resistance in several taxa including venomous snakes and their lizard prey, but not in the snake-eating birds studied. We also document new lineages with the arginine form of inhibition. Using an in vivo assay in four species, we provide further evidence that N-glycosylation mutations reduce the toxicity of cobra venom. The nAChR is of crucial importance for normal neuromuscular function and is highly conserved throughout the vertebrates as a result. Our research shows that the evolution of &alpha, neurotoxins in snakes may well have prompted arms races and mutations to this ancient receptor across a wide range of sympatric vertebrates. These findings underscore the inter-connectedness of the biosphere and the ripple effects that one adaption can have across global ecosystems.

Given the global evolutionary dynamics of Newcastle disease viruses (NDVs), it is imperative to continue extensive surveillance, routine monitoring and characterization of isolates originating from natural reservoirs (waterfowls). In this report, we isolated and characterized two virulent NDV strains from clinically healthy mallard (Anas platyrhynchos). Both isolates had a genome of 15,192 nucleotides encoding six genes in an order of 3´-NP-P-M-F-HN-L-5´. The biological characteristics (mean death time: 49.5-50 hr, EID50108.5 ml-1) and presence of a typical cleavage site in the fusion (F) protein (112R-R-Q-K-R↓F117) confirmed the velogenic nature of these isolates. Phylogenetic analysis classified both isolates as members of genotype VII within class-II. Furthermore, based upon the hypervariable region of the F gene (375 nt), isolates showed clustering within sub-genotype VIIi. Similarity index and parallel comparison revealed a higher nucleotide divergence from commonly used vaccine strains; LaSota (21%) and Mukteswar (17%). A comparative residues analysis with representative strains of different genotypes, including vaccine strains, revealed a number of substitutions at important structural and functional domains within the F and hemagglutinin-neuraminidase (HN) proteins. Together, the results highlight consistent evolution among circulating NDVs supporting extensive surveillance of the virus in waterfowl to better elucidate epidemiology, evolutionary relationships and their impacts on commercial and backyard poultry.

Bluetongue (BT) is a vector-borne disease of immense economic importance for small and large ruminants. Despite frequent disease reports from neighboring countries, a little is known about current disease status and prevalent serotypes in Pakistan. We screened a total of 1312 healthy animals for group-specific antibodies and serotype-specific genome for BT virus through competitive ELISA and real-time PCR, respectively. An overall prevalence of group-specific VP7 antibodies [28.81% (n = 378/1312, 95% CI = 26.4–31.4)] was observed. The prevalence was higher in goats [40.75% (n = 194/476, 95% CI = 36.4–45.3)] followed by buffalo [29.34% (n = 81/276, 95% CI = 24.3–34.9)], sheep [18.40% (n = 60/326, 95% CI = 14.5–22.9)] and cattle [17.94% (n = 42/234, 95% CI = 13.56–23.4)]. The odds of seropositivity were more in buffalo of Nili breed (OR = 2.06, 95% CI = 1.19–3.58) as well as those found with a presence of vector (OR = 2.04, 95% CI = 1.16–3.59). Buffalo and cattle with history of abortion [(OR = 3.95, 95% CI = 1.33–11.69) and (OR = 5.89, 95% CI = 1.80–19.27) respectively] were much likely to be infected with the disease. Serotype 8 was detected in all animal species while, serotypes 4 and 6 were detected in sheep, 2, 6 and 11 in goat, and 2 and 16 in buffalo. The study concludes a much frequent exposure of different serotypes of Bluetongue virus (BTV) in small and large ruminants and indicates its expansion to enzootic range worldwide.

A novel contaminant was isolated from Madin Darby Bovine Kidney (MDBK) cells. The organism was unable to grow on standard microbiological media by conventional techniques, but grew well in Dulbecco's Modified Eagle's Medium (DMEM) containing high glucose concentration. The organism formed a white biofilm on the bottom without any signs of turbidity. Upon genome sequence analysis of 16 S rDNA, the contaminant was identified as Sphingomonas sp. Shah, a member of the group α-Proteobacteria. Neutral red dye uptake method confirmed clear cytotoxic potential of the bacterium on A-549 cells. The organism was capable of invading and infecting different mammalian cell lines: MDBK, ZZ-R, 293-T, A549, and HeLa cells. Infected cells showed a variety of cytopathic effects including vacuolation at perinuclear area, cytoplasmic granulation and membrane blebbing. Microscopic analysis of the infected cells revealed the presence of cytoplasmic vacuoles harboring motile organisms. Apparently local serum preparations seem to be the source of this contamination, which is imperceptibly passed on from one culture passage to the other and ultimately leading to serious cytopathic manifestations. J. Cell. Biochem. 116: 934–942, 2015. © 2015 Wiley Periodicals, Inc.

The aim of the present study was to evaluate the anticoccidial efficacy of three different forms of Aloe vera including powder form, aqueous and methanol extracts. Although powdered and aqueous forms of Aloe vera also exhibited anticoccidial activity but its methanol extracts at higher concentration (4g) showed better weight gain and less oocyst per gram count in comparison of Amprolium (anticoccidial drug). We concluded that due to its anticoccidial activity Aloe vera can contribute to minimize the use of growth promoter antibiotics in poultry feed.

Background A widespread epidemic of equine influenza (EI) occurred in nonvaccinated equine population across multiple districts in Khyber Pakhtunkhwa Province of Pakistan during 2015–2016. Objectives and methods An epidemiological surveillance study was conducted from Oct 2015 to April 2016 to investigate the outbreak. EI virus strains were isolated in embryonated eggs from suspected equines swab samples and were subjected to genome sequencing using M13 tagged segment specific primers. Phylogenetic analyses of the nucleotide sequences were concluded using Geneious. Haemagglutinin (HA), Neuraminidase (NA), Matrix (M) and nucleoprotein (NP) genes nucleotide and amino acid sequences of the isolated viruses were aligned with those of OIE recommended, FC-1, FC-2, and contemporary isolates of influenza A viruses from other species. Results HA and NA genes amino acid sequences were very similar to Tennessee/14 and Malaysia/15 of FC-1 and clustered with the contemporary isolates recently reported in the USA. Phylogenetic analysis showed that these viruses were mostly identical (with 99.6% and 97.4% nucleotide homology) to, and were reassortants containing chicken/Pakistan/14 (H7N3) and Canine/Beijing/10 (H3N2) like M and NP genes. Genetic analysis indicated that A/equine/Pakistan/16 viruses were most probably the result of several re-assortments between the co-circulating avian and equine viruses, and were genetically unlike the other equine viruses due to the presence of H7N3 or H3N2 like M and NP genes. Conclusion Epidemiological data analysis indicated the potential chance of mixed, and management such as mixed farming system by keeping equine, canine and backyard poultry together in confined premises as the greater risk factors responsible for the re-assortments. Other factors might have contributed to the spread of the epidemic, including low awareness level, poor control of equine movements, and absence of border control disease strategies.

A total of 300 meat samples comprising mutton, beef, and chicken meat (n = 100) collected from either local butcher shops or large meat outlets situated at various areas of Lahore City located in Punjab province of Pakistan were tested for the isolation of Clostridium perfringens. Prevalence of the organism was highest in the chicken (6 %) followed by mutton (5 %) and beef (1 %). Contamination level was high (10/150) in the samples collected from local butcher shops in comparison to the samples collected from large meat outlets (2/150). All of the raw meat samples were negative for the presence of alpha, beta and epsilon toxins of C. perfringens as detected through ELISA. Out of a total number of 12 isolates only half were capable of producing enterotoxins when cultured in trypticase glucose yeast (TGY) broth. Toxinotyping of the isolates showed that 3 were of type A while one each of the remaining three belonged to type B, C, and D. Antibiotic susceptibility testing of the toxin producing isolates revealed that C. perfringens were susceptible to chloramphenicol, ciprofloxacin, metronidazole, and ceftriaxone. All of the other drugs were relatively less effective with a least activity of amoxicillin against the isolates.

Enteric bacterial human pathogens, i.e., Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Klebsiella pneumoniae, are the major cause of diarrheal infections in children and adults. Their structure badly affects the human immune system. It is important to explore new antibacterial agents instead of antibiotics for treatment. This project is an attempt to explain how gold nanoparticles affect these bacteria. We investigated the important role of the mean particle size, and the inhibition of a bacterium is dose-dependent. Ultra Violet (UV)-visible spectroscopy revealed the size of chemically synthesized gold nanoparticle as 6–40 nm. Atomic force microscopy (AFM) analysis confirmed the size and X-ray diffractometry (XRD) analysis determined the polycrystalline nature of gold nanoparticles. The present findings explained how gold nanoparticles lyse Gram-negative and Gram-positive bacteria.

Ranaviruses infect fish, amphibians, and reptiles. The present study was conducted to compare the persistence of amphibian and reptilian ranaviruses in a pond habitat. The 4 viruses used in this study included 2 amphibian ranaviruses, Frog virus 3 (FV3, the type species of the genus Ranavirus) and an isolate from a frog, and 2 ranaviruses of reptilian origin (from a tortoise and from a gecko). A sandwich germ-carrier technique was used to study the persistence of these viruses in sterile and unsterile pond water (PW) and soil obtained from the bank of a pond. For each virus, virus-loaded carriers were placed in each of the 3 substrates, incubated at 4 and 20°C, and titrated at regular intervals. Serial data were analyzed using a linear regression model to cal- culate T-90 values (time required for 90% reduction in the virus titer). Resistance of the viruses to drying was also studied. All 4 viruses were resistant to drying. At 20°C, T-90 values of the viruses were 22 to 31 d in sterile PW and 22 to 34 d in unsterile PW. Inactivation of all 4 viruses in soil at this temperature appeared to be non-linear. T-90 values at 4°C were 102 to 182 d in sterile PW, 58 to 72 d in unsterile PW, and 30 to 48 d in soil. Viral persistence was highest in the sterile PW, followed by the unsterile PW, and was lowest in soil. There were no significant differences in the survival times between the amphibian and reptilian viruses. The results of the present study sug- gest that ranaviruses can survive for long periods of time in pond habitats at low temperatures.

Objective: To determine the role of water fleas in accumulating avian influenza viruses (AIV) from the surrounding water and to estimate their role as a vector of AIV. Methods: Water fleas were exposed to H4N6 and H5N1 AIV-contaminated water in a closed system. The potential of water fleas to take up and retain the viruses was estimated by quantitative real-time RT-PCR (qRRT-PCR) and titration on cell culture. Results: Contamination trials showed that significantly higher amounts of viral RNA were detectable per gram of water fleas as compared to per milliliter of the surrounding water at 1, 4, and 6 days of incubation. Viral infectivity was only detectable in the water samples collected immediately after mixing the virus in water containing the water fleas, while no virus was detectable in any of the water fleas or water samples collected afterwards. Conclusions: Water fleas are able to accumulate AIV from surrounding water based upon the qRRT-PCR detection of viral RNA. Additional studies are necessary to investigate the inactivation potential of water fleas on viral infectivity.

In order to investigate the potential role of mussels as a vector of influenza A viruses, we exposed zebra mussels (Dreissena polymorpha) to natural lake water containing a low pathogenic H5N1 avian influenza virus. Mussels were kept in water containing virus for 48 hr, then transferred into fresh water for another 14 days. Virus detection in mussels and water samples was performed by quantitative real-time reverse transcriptase-PCR (qRRT-PCR) and egg culture methods. Virus uptake was detected in all of the mussel groups that were exposed to virus. Even after 14 days in fresh water, virus could still be detected in shellfish material by both qRRT-PCR and egg culture methods. The present study demonstrates that zebra mussels are capable of accumulating influenza A viruses from the surrounding water and that these viruses remain in the mussels over an extended period of time.

A germ carrier technique was adapted for the determination of the persistence of influenza viruses in moist environments. The technique was employed with 3 low pathogenic avian influenza viruses (H4N6, H5N1, and H6N8), one human influenza virus (H1N1), and two model viruses (NDV and ECBO) in lake water at five different temperatures (30, 20, 10, 0, and -10°C). Viral quantitation was carried out at regular intervals on cell culture for a maximum duration of 16 weeks. Serial data were analyzed by linear regression model to calculate T-90 values (time required for one log reduction in the virus titer). Persistence of all of the viruses was highest at -10°C followed by 0, 10, 20, and 30°C. At -10°C, the single freeze-thaw cycle resulted in an abrupt decline in the virus titer, followed by long term persistence. Generally, influenza viruses persisted shorter than model viruses while ECBO has the highest survival time in lake water. Individual influenza viruses differed in their persistence at all temperatures. The findings of the present study suggest that AIV can remain infectious in lake water for extended periods of time at low temperatures.

Aim of the present study was to characterise and evaluate probiotic potential of lactobacilli isolated from indigenous poultry. Lactobacilli were isolated from poultry droppings and identified by genus specific polymerase chain reaction and 16S rRNA gene sequencing. Isolates were characterised in vitro by their ability to tolerate low pH and bile salts, phytase activity, antimicrobial activity, antibiotic susceptibility profile, and autoaggregation and coaggregation with poultry gut pathogens. In vivo evaluation of selected isolates was done by their effect on the body weight gain and immune response of broiler chicks. Total of 90, one-day old chicks, were randomly divided in 9 groups and given selected lactobacilli alone and in combinations (108 cfu/bird, daily) from day 7 to day 35. Body weight gain and humoral immune response to New Castle Disease Virus (NDV) vaccine were determined weekly. Three lactobacilli isolates (SMP52, SMP64 and SMP70) were selected as potentially probiotic bacteria on the basis of in vitro characterisation and identified as Lactobacillus crispatus, Lactobacillus casei and L. crispatus, respectively. Chicks supplemented with ‘SMP52’, ‘SMP64’, ‘SMP70’ and ‘SMP64+SMP70’ and a commercial probiotic product (Protexin) showed significantly higher mean weight gain per bird (1,584±35.2, 1,629±30.6, 1,668±34.7, 1,619±29.5 and 1,576±31.7 g/bird, respectively) as compared to negative control group (1,394±26.7 g/bird), on day 35. SMP 70 also showed significantly higher geometric mean titre against NDV vaccine at day 21 as compared to negative control. It is concluded that L. crispatus SMP52, L. casei SMP64 and L. crispatus SMP70 are potential probiotic candidates which alone or in different combinations may increase body weight of broilers.

Background : Rapid industrialization is discharging toxic heavy metals into the environment, disturbing human health in many ways and causing various neurologic, cardiovascular, and dermatologic abnormalities and certain types of cancer. The presence of arsenic in drinking water from different urban and rural areas of the major cities of Pakistan, for example, Lahore, Faisalabad, and Kasur, was found to be beyond the permissible limit of 10 parts per billion set by the World Health Organization. Therefore the present study was initiated to examine the effects of arsenite (As +3 ) on DNA biosynthesis and cell death. Methods : After performing cytotoxic assays on a human epithelial carcinoma cell line, expression analysis was done by quantitative polymerase chain reaction, western blotting, and flow cytometry. Results : We show that As +3 ions have a dose- and time-dependent cytotoxic effect through the activation of the caspase-dependent apoptotic pathway. In contrast to previous research, the present study was designed to explore the early cytotoxic effects produced in human cells during exposure to heavy dosage of As +3 (7.5 µg/ml). Even treatment for 1 h significantly increased the mRNA levels of p21 and p27 and caspases 3, 7, and 9. It was interesting that there was no change in the expression levels of p53, which plays an important role in G 2 /M phase cell cycle arrest. Conclusion : Our results indicate that sudden exposure of cells to arsenite (As +3 ) resulted in cytotoxicity and mitochondrial-mediated apoptosis resulting from up-regulation of caspases. Keywords: apoptosis; epithelial carcinoma; cytotoxicity; arsenite; caspases; Pakistan Responsible Editor: Amin Bredan, VIB Inflammation Research Center & Ghent University, Belgium (Published: 27 April 2015) Citation: Libyan J Med 2015, 10 : 26875 - http://dx.doi.org/10.3402/ljm.v10.26875

This study was undertaken to evaluate the effects of probiotics (Protexin) on the immune response of broiler chicks. The parameters of investigation were hemagglutination inhibition HI titer of antibodie s against avian influenza virus and post field AIV challenge. The findings were compared with the cyclophosphamide treated AIV-vaccinated; untreated and AIV-vaccinated and unvaccinated and control birds this investigation revealed that protexin treated chicks have higher AIV-HI antibody and no AIV post challenge mortality compared to the cyclophosphamide treated and untreated chicks the overall finding of this study clearly demonstrate that the use of this multistrain probiotics has good effect on immune response of broiler chicks.

Background Peste des petits ruminants (PPR) is an endemic and highly contagious disease in small ruminants of Pakistan. Despite the fact that an effective vaccine is available, outbreaks are regularly occurring in the country. Thus so far, the diagnosis has primarily been made based on clinical outcome or serology. This study was carried out to characterize PPRV from an emerging wave of outbreaks from Punjab, Pakistan. Results A total of 32 blood samples from five different flocks were tested with real-time PCR for the presence of PPRV genome. The samples detected positive in real-time PCR (n = 17) were subjected to conventional PCR for the amplification of the nucleoprotein (N) gene. Phylogenetic analysis of the sequenced N genes (n = 8) indicated the grouping of all the sequences in lineage IV along with PPRV strains from Asian and Middle East. However, interestingly sequences were divided into two groups. One group of viruses (n = 7) clustered with previously characterized Pakistani isolates whereas one strain of PPRV was distinct and clustered with Saudi Arabian and Iranian strains of PPRV. Conclusions Results demonstrated in this study expanded the information on the genetic nature of different PPRV population circulating in small ruminants. Such information is essential to understand genetic nature of PPRV strains throughout the country. Proper understanding of these viruses will help to devise control strategies in PPRV endemic countries such as Pakistan.

The persistence of 3 low-pathogenicity avian influenza viruses (LPAIV) (H4N6, H5N1, and H6N8) and one human influenza virus (H1N1) as well as Newcastle disease virus (NDV) and enteric cytopathogenic bovine orphan (ECBO) virus was investigated in lake sediment, duck feces, and duck meat at 30, 20, 10, and 0°C using a germ carrier technique. Virus-loaded germ carriers were incubated in each substrate, and residual infectivity of the eluted virus was quantified on cell culture after regular intervals for a maximum of 24 weeks. Data were analyzed by a linear regression model to calculate T 90 values (time required for 90% loss of virus infectivity) and estimated persistence of the viruses. In general, the persistence of all of the viruses was highest in lake sediment, followed by feces, and was the lowest in duck meat at all temperatures. For the avian influenza virus subtypes, T 90 values in sediment ranged from 5 to 11, 13 to 18, 43 to 54, and 66 to 394 days at 30, 20, 10, and 0°C, respectively, which were 2 to 5 times higher than the T 90 values of the viruses in the feces and meat. Although the individual viruses vary in tenacity, the survival time of influenza viruses was shorter than that of NDV and ECBO virus in all substrates. The results of this study suggest that lake sediment may act as a long-term source of influenza viruses in the aquatic habitat, while the viruses may remain infectious for extended periods of time in duck feces and meat at low temperatures, allowing persistence of the viruses in the environment over winter.

The tenacity of three low pathogenicity avian influenza viruses (AIV; subtypes H4N6, H5N1, and H6N8) was tested at five different temperatures (−10, 0, 10, 20, and 30 C) in distilled water, normal saline, and surface water obtained from Lake Constance. Infectivity of AIV in the samples was quantified at regular intervals by end point titration on Madin-Darby canine kidney cells for a maximum period of 36 wk, and duplicate samples were tested each time. The results showed that the survival time of AIV in all of the water types was inversely proportional to storage temperature. All three viruses showed varying sensitivity to inactivation under each of the experimental conditions. Persistence of the viruses was the longest in distilled water, second longest in normal saline, and shortest in surface water. The virus-inoculated surface water remained infective for a few days at 30 and 20 C, a few weeks at 10 C, and for months at 0 and −10 C.

Purpose: To study the immunosuppressant and immunopotentiating effects of ketoprofen on antibody producing cells.Methods: Mice were given ketoprofen at doses of 1 mg/kg/day and 5 mg/kg/day for seven days. Similarly polyinosinic–polycytidylic acid (Poly IC) and phosphate buffer saline (PBS) were used as positive and negative control, respectively, for seven days. After seven days, the mice were sacrificed and their spleens removed. Simultaneously, blood was withdrawn from the hearts of the mice and serum was separated from the blood. The spleen cells were analyzed by enzyme-linked immunospot (ELISPOT) while the serum was investigated by enzyme-linked immunosorbant (ELISA) to evaluate the effects of ketoprofen on the ability of individual cell to produce antibodies and antibody- mediated immune responses.Results: Ketoprofen significantly (p < 0.001) reduced the ability of individual cells to produce antibodies. There was a significant difference (p < 0.001) in % of spot forming cells of PBS treated negative control group (0.045 %) as against positive control (0.058 %), 1 mg ketoprofen /kg/day (0.037 %) and 5 mg ketoprofen/kg/day (0.032 %) treated groups. The results of ELISA showed a significant (p < 0.005) difference in the absorbance values between negative control, positive control, ELISA control and ketoprofen treated groups. Absorbance was significantly (p < 0.005) reduced in ketoprofen-treated groups.Conclusion: The ability of an individual cell to produce antibodies and antibody-mediated immune responses is suppressed by ketoprofen, suggesting that it is immunosuppressive, and thus indicating its potential application in patients with auto-immune disorders.Keywords: Ketoprofen, Immunomodulatory, Immunosupressive, Antibody, Spot-forming cells, Polyinosinic–polycytidylic acid

The interaction of nickel nanoparticles with Escherichia coli (E. coli) bacteria has been studied. The nickel nanoparticles have been fabricated by continuous wave laser ablation of nickel target and their properties are studied using different characterization techniques. The antibacterial activity of nickel nanoparticles was checked against E. coli bacteria. Escherichia coli were cultured in nutrients broth and different concentrations of nickel nanoparticles were added to bacterial culture solution to investigate the interaction of nickel nanoparticles with bacteria and to check toxicity of the nickel nanoparticles against E. coli. The fabricated Ni nanoparticles have exhibited considerable antimicrobial activity against E. coli.

Background: Newcastle disease (ND) is one of the most deadly diseases of poultry around the globe. The disease is endemic in Pakistan and recurrent outbreaks are being reported regularly in wild captive, rural and commercial poultry flocks. Though, efforts have been made to characterize the causative agent in some of parts of the country, the genetic nature of strains circulating throughout Pakistan is currently lacking. Material and methods: To ascertain the genetics of NDV, 452 blood samples were collected from 113 flocks, originating from all the provinces of Pakistan, showing high mortality (30 – 80%). The samples represented domesticated poultry (broiler, layer and rural) as well as wild captive birds (pigeons, turkeys, pheasants and peacock). Samples were screened with real-time PCR for both matrix and fusion genes (1792 bp), positive samples were subjected to amplification of full fusion gene and subsequent sequencing and phylogenetic analysis. Results: The deduced amino acid sequence of the fusion protein cleavage site indicated the presence of motif (112 RK/RQRR↓F117) typical for velogenic strains of NDV. Phylogenetic analysis of hypervariable region of the fusion gene indicated that all the isolates belong to lineage 5 of NDV except isolates collected from Khyber Pakhtunkhwa (KPK) province. A higher resolution of the phylogenetic analysis of lineage 5 showed the distribution of Pakistani NDV strains to 5b. However, the isolates from KPK belonged to lineage 4c; the first report of such lineage from this province. Conclusions: Taken together, data indicated the prevalence of multiple lineages of NDV in different poultry population including wild captive birds. Such understanding is crucial to underpin the nature of circulating strains of NDV, their potential for interspecies transmission and disease diagnosis and control strategies. Peer Reviewed publishedVersion