Evaluation of maize varieties via multivariate analysis: Roles of ionome, antioxidants, and autophagy in salt tolerance.

Salt stress presents a major obstacle to maize (Zea mays L.) production globally, impeding its growth and development. In this study, we aimed to identify salt-tolerant maize varieties through evaluation using multivariate analysis and shed light on the role of ionome, antioxidant capacity, and autophagy in salt tolerance. We investigated multiple growth indices, including shoot fresh weight, shoot dry weight, plant height, chlorophyll content, electrolyte leakage, potassium and sodium contents, and potassium-to-sodium ratio in 20 maize varieties at the V3 stage under salt stress (200 mM NaCl). The results showed significant differences in the growth indices, accompanied by a wide range in their coefficient of variation, suggesting their suitability for screening salt tolerance. Based on D values, clustering analysis categorized the 20 varieties into four distinct groups. TG88, KN20, and LR888 (group I) emerged as the most salt-tolerant varieties, while YD9, XD903, and LH151 (group IV) were identified as the most sensitive. TG88 showcased nutrient preservation and redistribution under salt stress, surpassing YD9. It maintained nitrogen and iron levels in roots while YD9 experienced decreases. TG88 redistributed more nitrogen, zinc, and potassium to its leaves, outperforming YD9. TG88 preserved sulfur levels in both roots and leaves, unlike YD9. Additionally, TG88 demonstrated higher enzymatic antioxidant capacity (SOD, POD, APX, and GR) both at the enzyme and gene expression levels, upregulation of autophagy-related (ATG) genes (ZmATG6, ZmATG8a, and ZmATG10), and increased autophagic activity. Overall, this study offers insights into accurate maize varieties evaluation methods and the physiological mechanisms underlying salt tolerance and identifies promising materials for further research.

Simulations and Experimental Analysis of a High Viscosity Inkjet Printing Device Based on Fabry-Pérot Resonator.

The study investigates the effect of changing various input parameters on the pressure responses at acoustic cavities of a droplet-based acoustic printing device consisting of a Fabry-Pérot (FP) resonator and a standing wave-source chamber. The standing wave of the acoustic radiation pressure at the FP resonator is analyzed. The behavior of the standing wave and acoustic radiation force at the FP resonator is presented and compared with the measured results by varying the position of the standing wave-generating plate. The pressure changes inside the standing wave-source chamber are investigated and discussed to determine the reason for the sudden high-pressure drop at the FP resonator. Furthermore, the effects of inserting the nozzle and droplet inside the FP resonator on the standing wave and acoustic radiation force are analyzed. Experimental analysis is performed by collecting acoustic pressure data at the outlet of the FP resonator. The simulated and measured pressure drop behaviors are compared. The presented numerical approach can be used to set optimal design guidelines for obtaining a higher acoustic pressure inside the acoustic cavities of droplet-based acoustic jetting and other acoustofluidic devices.

Rhomboid protein 2 of Eimeria maxima provided partial protection against infection by homologous species.

Rhomboid-like proteases (ROMs) are considered as new candidate antigens for developing new-generation vaccines due to their important role involved in the invasion of apicomplexan protozoa. In prior works, we obtained a ROM2 sequence of Eimeria maxima (EmROM2). This study was conducted to evaluate the immunogenicity and protective efficacy of EmROM2 recombinant protein (rEmROM2) and EmROM2 DNA (pVAX1-EmROM2) against infection by Eimeria maxima (E. maxima). Firstly, Western blot assay was conducted to analyze the immunogenicity of rEmROM2. The result showed that rEmROM2 was recognized by chicken anti-E. maxima serum. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assay revealed apparent transcription and expression of EmROM2 at the injection site. qRT-PCR (quantitative real-time PCR), flow cytometry and indirect ELISA indicated that vaccination with rEmROM2 or EmROM2 DNA significantly upregulated the transcription level of cytokines (IFN-γ, IL-2, IL-4, IL-10, IL-17, TGF-ß and TNF SF15), the proportion of CD8+ and CD4+ T lymphocytes and serum IgG antibody response. Ultimately, a vaccination-challenge trial was performed to evaluate the protective efficacy of rEmROM2 and pVAX1-EmROM2 against E. maxima. The result revealed that vaccination with rEmROM2 or pVAX1-EmROM2 significantly alleviated enteric lesions, weight loss, and reduced oocyst output caused by challenge infection of E. maxima, and provided anticoccidial index (ACI) of more than 160, indicating partial protection against E. maxima. In summary, vaccination with rEmROM2 or pVAX1-EmROM2 activated notable humoral and cell-mediated immunity and provided partial protection against E. maxima. These results demonstrated that EmROM2 protein and DNA are promising vaccine candidates against E. maxima infection.

Antibiotic susceptibility and drug prescription pattern in uropathogenic Escherichia coli in district Muzaffarabad, Azad Jammu and Kashmir, Pakistan.

The present research study was done to determine the correlation between antibiotic susceptibility and drug prescription patterns in empirical treatment of uropathogenic Escherichia coli (E. coli) in two hospitals of district Muzaffarabad, Azad Kashmir, Pakistan. One hundred uropathogenic E. coli clinical isolates were collected from UTI patients who attended the Combined Military Hospital (CMH) and Abbas Institute of Medical Sciences (AIMS), district Muzaffarabad, AJK. All isolates were subjected to antibiotic susceptibility against seven commonly prescribed antibiotics by Kirby-Bauer disk diffusion method. However, all the E. coli isolates were susceptible to Imipenem. Eight percent and 10% of isolates from CMH and AIMS were found to be resistant against Nitrofurantoin drug respectively. Similarly, 94% and 74% isolates from CMH and 60% and 64% isolates from AIMS were found to be resistant against Co-trimoxazole and Coamoxiclave, respectively. Pipemedic acid resistance was also detected in 76% and 60% isolates from AIMS and CMH, respectively. Ciprofloxacin resistance was also observed in 54% and 36% isolates from AIMS and CMH, respectively. The finding of the study revealed that both hospitals have different drug susceptibility pattern against uropathogenic E. coli, which may be associated with patterns of drug prescription in empirical treatment of urinary tract infections. There is a vital need for appropriate development of hospital-specific antibiograms to determine appropriate empiric therapy of urinary tract infections.

Type III secretion system confers enhanced virulence in clinical non-O1/non-O139 Vibrio cholerae.

Vibrio cholerae O1 infections mainly are responsible for significant mortality and morbidity amongst children, however, non-O1/non-O139 V. cholerae have also been reported to cause mild to severe infections because of their virulence potential. The pathogenic mechanisms of non-O1, non-O139 isolates are not as clearly understood as for that of O1 and O139 isolates. Type three secretion system (TTSS) is also considered one of the important virulent factors and during the current study, we investigated the role of TTSS in association with non-O1/non-O139 clinical isolates. We report that the presence of TTSS in non-O1/non-O139 V. cholerae clinical isolate (D13) from a child confers more virulence compared to the one lacking it (D15) in another clinical case during the small cholera epidemic. Moreover, the antibiotic susceptibility profiles of D13 and D15 indicate that they are multiple drug resistance (MDR) isolates. The sequence analysis for TTSS cluster was carried out for D13 and compared with the TTSS positive reference Vibrio parahaemolyticus RIMD2210633 and V. cholerae AM19226 non-O1/non-O139. Furthermore, the pathogenic potential of D13 & D15 was also explored in simple and economical invertebrate host model, Galleria mellonella and the results revealed that TTSS+ve isolate (D13) was more virulent compared to TTSS-ve isolate (D15). We suggest that this distinct genetic difference, seen in natural variants D13 and D15, is also reflected by the clinical picture of the former in contributing towards the severity of disease symptoms and this finding was further validated by assessing virulence potential of both isolates using inexpensive G. mellonella infection model.

Green Synthesis of Silver Nanoparticles Using Jurinea dolomiaea and Biological Activities.

Green syntheses of nanoparticles using plant materials are of tremendous scope. Here we report advantageous green synthesis for silver nanoparticles (AgNPs) using aqueous-root extract of Jurinea dolomiaea and AgNO3. Color change of solution and UV-Vis absorption at 444 nm indicated the formation of AgNPs. XRD confirmed their face centered cubic structure (fcc) with average particle size of 24.58 nm. SEM analysis showed their spherical, cubic and triangular structures. FT-IR indicated the presence of functional groups of reducing and stabilizing phytochemicals. Methanol-root extract of J. dolomiaea revealed high flavonoid (445 mg RE/g) and phenolic contents (92 mg GAE/g). Methanol-extract showed high antioxidant potency (IC50 = 0.494 µg/mL), rationally due to its high phenolic and flavonoid contents. These AgNPs showed the highest and equal antimicrobial activities against Escherichia coli and Pseudomonas aeruginosa (Inhibition zone 11.0 mm) whereas, methanol-roots extract showed equal and intermediate activities (Inhibition zone 8.0 mm) against both pathogens but aqueous extract showed poor activities (Inhibition zone 6.0 mm) against these both pathogens. AgNPs are playing a major role in the field of nanotechnology and nanomedicine due to their antimicrobial and drug delivery efficacy as well as reasonable tolerance in human biology.

Biogenic Synthesis of AgNPs with Saussurea lappa C.B. Clarke and Studies on Their Biochemical Properties.

Nanotechnology has become an irreplaceable need and green synthesis of nanoparticles offers several advantages over physical and chemical methods. Medicinal plants are the main reservoirs of drugs and drug candidates. We report the biogenic synthesis of silver nanoparticles (AgNPs) using aqueous root extract of Saussurea lappa. Verification and characterization of these nanoparticles were done by UV-visible spectroscopy, XRD-analysis and Scanning Electron Microscopy and FT-IR. Extract-loaded-AgNPs showed the highest inhibition zone against Escherichia coli (11.0 mm) and intermediate against Pseudomonas aeruginosa (9.0 mm). The methanolic root extract of S. lappa alone, also moderately inhibited Pseudomonas aeruginosa (9.0 mm) and showed lower activity (6.0 mm) against Escherichia coli. Its aqueous roots extract inhibited (6.0 mm) the growth of tested organisms. Methanolic extract showed antioxidant potency (IC50 = 0.814 µg/mL). Experiments revealed the presence of phenols and flavonoids in the roots of Saussurea lappa. These findings provide promising interest to exploit Saussurea lappa for the biogenic synthesis of AgNPs and their biological applications.

Safety of fenbendazole in common peafowl (Pavo cristatus).

The present study was undertaken to find out the safety levels of fenbendazole in common peafowl. This bird, raised on aviaries and zoos, can be severely parasitized with Ascaridia galli (enteric worms) and Syngamus trachea (gapeworm) along with other parasitic worms. Fenbendazole is a highly effective benzimidazole-class anthelmintic in animals. The objective of this work was to provide target animal safety data in young peafowl and to demonstrate reproductive safety in adult birds. During the experimental study, diets containing fenbendazole at 0, 100, 200 and 300 ppm were fed for 21 days (three times the normal treatment duration). Data for feed consumption, feed conversion rate, and body weights were recorded for each bird in each group. Drug concentrations in different tissues of birds were determined to correlate concentrations with clinical observations, clinical pathology, and histologic findings. There were no morbidities or mortalities after study day 21. Additionally, there were no statistically significant treatment-related differences among above mentioned parameters. Analysis of fenbendazole concentrations in kidney, liver, leg/thigh, and breast muscle and skin with associated fat revealed that, even at the highest dose level used and with no feed withdrawal, fenbendazole concentrations were relatively low in these tissues. These findings indicate that fenbendazole has a relatively wide margin of safety in young peafowl and that the proposed dose of 100 ppm in the feed for 7 consecutive days is well within the margin of safety. In the reproductive safety study, five breeder peafowl farms fed fendbendazole at 100ppm for 7 days and collected data on hatching percentage of peahen eggs before and after treatment. Reproductive performance in peahen was not adversely affected.

Integration of Nucleic Acid Extraction Protocol with Automated Extractor for Multiplex Viral Detection.

The isolation of nucleic acids (NA) is the preliminary step to carry out genetic studies and DNA biosensor development. The presence of inhibitors in the purified NA interferes with the downstream application. These salts and other organic contaminations particularly challenge the analytical sensitivity of DNA biosensors. The detailed study was carried out to optimize the factors which might affect viral nucleic acid purification. The results suggested that 6 M guanidinium hydrochloride salt concentration was critical for NA isolation. The inverse relation has been found in the pH of the lysis buffer and quality and quantity of NA. The NA yield was relatively stable at pH 4­5. It has been observed that the use of carrier RNA was indispensable for viral genome isolation. The addition of ethanol to lysate in 1:1 ratio greatly improved NA recovery. The elution efficiency of DNase and RNase free water, 1× TE buffer and 1× PCR buffer was compared. The carrier RNA was best eluted in DNase and RNase free water and 1× TE buffer. It was further demonstrated that this method can be automatized for high throughput detection. A simple experiment was conducted to optimize the different parameters of an automated NA extractor to simultaneously extract HBV DNA and HCV RNA. The purified NA was successfully amplified in PCR and RT-PCR to verify the reliability of the established protocol. Thus a semi-automated system for the simultaneous detection of multiple viruses has been demonstrated.

Nanomaterial Induced Immune Responses and Cytotoxicity.

Nanomaterials are utilized in a wide array of end user products such as pharmaceuticals, electronics, clothes and cosmetic products. Due to its size (< 100 nm), nanoparticles have the propensity to enter through the airway and skin, making its path perilous with the potential to cause damages of varying severity. Once within the body, these particles have unconstrained access to different tissues and organs including the brain, liver, and kidney. As a result, nanomaterials may cause the perturbation of the immune system eliciting an inflammatory response and cytotoxicity. This potential role is dependent on many factors such as the characteristics of the nanomaterials, presence or absence of diseases, and genetic predisposition. Cobalt and nickel nanoparticles, for example, were shown to have inflammogenic properties, while silver nanoparticles were shown to reduce allergic inflammation. Just as asbestos fibers, carbon nanotubes were shown to cause lungs damage. Some nanomaterials were shown, based on animal studies, to result in cell damage, leading to the formation of pre-cancerous lesions. This review highlights the impact of nanomaterials on immune system and its effect on human health with toxicity consideration. It recommends the development of suitable animal models to study the toxicity and bio-clearance of nanomaterials and propose safety guidelines.

CLINICAL FEATURES AND OUTCOME ANALYSIS OF INTRACRANIAL HYDATID CYSTS.

BACKGROUND: Hydatid cyst of the brain is serious zoonotic parasitic infections which have profound health consequences if left untreated. The surgical excisions of the cysts are rewarding for both the patient the neurosurgeon. METHODS: The study was conducted prospectively at Department of Neurosurgery Hayatabad Medical Complex Peshawar from January 2013 to December 2014. Patients with a diagnosis of intracranial hydatid cysts were included, clinical and radiological features recorded, intervention and postoperative outcome were analysed. RESULTS: Eleven patients with a male to female ratio of 1.7:1. Mean age was 12.4 (SD ± 6.5) years with median GCS on arrival of 10 (SD ± 2.5). Clinical features were headache (81.8%), vomiting (90.9%), seizures (36.4%), focal deficits (54.5%) and papilloedema (72.7%). The median GCS on discharge was 13 (SD ± 1.1) while GOS at 1 month follow up was 4 (SD ± 0.7). The bivariate analysis showed inverse correlation (R² = -0.68; p = 0.02) between duration of symptoms and outcome while GCS on admission was positively correlated (r(s) = 0.75; p = 0.007) with the outcome. There was no mortality. CONCLUSION: Despite its rarity the clinical features are non-specific while radiological features help in establishing diagnosis. Earlier diagnosis and prompt intervention is the key to favourable outcome.

Role of aflatoxin toxicity on transmissibility and pathogenicity of H9N2 avian influenza virus in turkeys.

The study was conducted to investigate the role of aflatoxin on the infectivity and transmissibility of H9N2 AI virus. The experiment was performed on 80 non-vaccinated turkeys, divided into 4 groups of 20 birds each. Group A was kept as non-infected and a non-treated negative control; Group B was inoculated intratracheally with H9N2 AI virus (1 × 10(7) EID50) at 4 weeks of age; Group C was fed on a diet containing 0.5 ppm aflatoxin from Day 1 through the entire experiment period and Group D was fed on diet containing 0.5 ppm aflatoxin as for Group C but inoculated intratracheally with H9N2 AI virus (1 × 10(7) EID50) at the fourth week of age and then mixed with naïve birds. Infected and contact birds showed clinical signs of different severity, showing the most prominent disease signs in birds of the aflatoxin + H9N2 group. All infected birds showed virus shedding, however, the pattern of virus shedding was different for birds of the aflatoxin + H9N2 group showing pronounced virus secretion. Similarly, efficient transmission of virus was observed between infected and contact birds, but more prominent virus transmission was seen in those birds inoculated and fed aflatoxin-treated diet. Moreover, significantly lower antibody titres against H9N2 AIV were observed in birds fed aflatoxin-treated diet, indicating an immunotoxic nature of aflatoxin as the reason for poor seroconversion. Similarly, decreased IFNγ mRNA expression and higher mortality (35%) suggest an immunotoxic and immunosuppressive effect of aflatoxin leading to enhanced pathogenesis of H9N2 viruses in aflatoxin-fed birds. The immunosuppressive nature of aflatoxin might delay influenza virus clearance and this may be one of the reasons for increased pathogenicity of H9N2 LPAI viruses in turkeys under field conditions.

DNA Mediated Vaccines Delivery Through Nanoparticles.

Vaccination has led to the eradication of those diseases which had once claimed millions of lives worldwide; however, it is accompanied with a number of dis-advantages especially safety issues until the entry of DNA vaccines. The DNA vaccines have been emerged as the best remedy for problematic diseases being capable of producing humoral and cellular immune responses as well as the safest vaccines so far. However, the magnitude of immune responses produced in primates is lower than that in experimental animals. There are several reasons described theoretically for this limited efficacy and a number of novel approaches have been applied to boost their immune responses, e.g., use of more efficient promoters and coding optimization, addition of traditional or genetic adjuvants, electroporation, intradermal delivery and various prime-boost strategies. One of these strategies is controlled antigen administration of plasmid DNA through microspheres and nanoparticles. This approach is accompanied with a number of advantages to overcome the limitations of traditional delivery systems in terms of stability, solubility and pharmacology. Furthermore, the surface structure of a virus highly resembles with a nanoparticle because of their geometrical regularities and nanoscale dimensions; therefore, the engineering of nanoparticles is based upon principles of natural virus attack which will be the best tool for vaccination. There is evidence that these immune responses can be augmented by properly structured nanosized particles (nanoparticles) that may avoid DNA degradation and facilitate targeted delivery to antigen presenting cells. Adsorption, formulation or encapsulation with particles has been found to stabilize DNA formulations. The use of nanoparticles for DNA vaccine delivery is a platform technology and has been applied for delivery of a variety of existing and potential vaccines successfully.

Genomic epidemiology of Vibrio cholerae O1 associated with floods, Pakistan, 2010.

In August 2010, Pakistan experienced major floods and a subsequent cholera epidemic. To clarify the population dynamics and transmission of Vibrio cholerae in Pakistan, we sequenced the genomes of all V. cholerae O1 El Tor isolates and compared the sequences to a global collection of 146 V. cholerae strains. Within the global phylogeny, all isolates from Pakistan formed 2 new subclades (PSC-1 and PSC-2), lying in the third transmission wave of the seventh-pandemic lineage that could be distinguished by signature deletions and their antimicrobial susceptibilities. Geographically, PSC-1 isolates originated from the coast, whereas PSC-2 isolates originated from inland areas flooded by the Indus River. Single-nucleotide polymorphism accumulation analysis correlated river flow direction with the spread of PSC-2. We found at least 2 sources of cholera in Pakistan during the 2010 epidemic and illustrate the value of a global genomic data bank in contextualizing cholera outbreaks.

Applications of nanoparticles for DNA based rabies vaccine.

Rabies is a fatal encephalomyelitis. Most cases occur in developing countries and are transmitted by dogs. The cell culture vaccines as associated with high cost; therefore, have not replaced the unsafe brain-derived vaccines. In the developing countries these brain-derived rabies vaccines still can be seen in action. Moreover, there will be a need for vaccines against rabies-related viruses against which classical vaccines are not always effective. The worldwide incidence of rabies and the inability of currently used vaccination strategies to provide highly potent and cost-effective therapy indicate the need for alternate control strategies. DNA vaccines have emerged as the safest vaccines and best remedy for complicated diseases like hepatitis, HIV, and rabies. A number of recombinant DNA vaccines are now being developed against several diseases such as AIDS and malaria. Therefore, it can be a valuable alternative for the production of cheaper rabies vaccines against its larger spectrum of viruses. In this review we report published data on DNA-based immunization with sequences encoding rabies with special reference to nanotechnology.

Detection of Pseudomonas aeruginosa based on magnetic enrichment and nested PCR.

Pseudomonas aeruginosa is a common opportunistic pathogen in clinics. The species-specific ecfX gene of Pseudomonas aeruginosa has high specificity. In this experiment, we are intended to develop a new method for the detection of Pseudomonas aeruginosa based on magnetic enrichment and nested PCR, and the specific ecfX gene of Pseudomonas aeruginosa was used as the detection object. The genomic DNA of Pseudomonas aeruginosa was extracted using amino-modified magnetic nanoparticles (MNPs). The ecfX gene was amplified by nested PCR and the product of PCR was detected by agarose gel electrophoresis. The results showed that the optimal annealing temperature was 64 degrees C and 62 degrees C respectively in the first and the second rounds of PCR. The lowest concentration of Pseudomonas aeruginosa that could be detected was 10 cfu/mL. The method provides a reliable, timely and accurate technology for early detection of Pseudomonas aeruginosa. Furthermore, the method can shorten the procedure and time from DNA extraction to detection, which made automation more convenient.

Magnetic beads-based chemiluminescent assay for ultrasensitive detection of pseudorabies virus.

A rapid, ultrasensitive and economical Pseudorabies virus (PRV) detection system based on magnetic beads (MBs) and chemiluminescence was developed in this paper. The carboxyl functionalized MBs (MBs-COOH) were covalently coupled with aminated DNA probes for capturing PRV biotinylated amplicon, the product of polymerase chain reaction (PCR). Agarose gel electrophoresis analysis approved the reliability of biotinylated amplicon. The MBs composites were incubated with alkaline phosphatase labeled streptavidin (ALP-SA) and chemiluminescene was determined by subsequently adding 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD). The optimal conditions of the PRV detection method were 10 microM for probe concentration, 50 degrees C for hybridization temperature and 30 min for hybridization time. The limit of detection (LOD) was as low as 100 amol/5 pM of amplicon which proved that this approach for PRV detection was ultrasensitive.

TRAIL mediated apoptosis ruling and anticancer trigger by fine-tuned nano spheres of Fagonia cretica methanolic extracts as novel cancer regime.

Fagonia cretica L. is a tropical plant of family Zygophyllaceae with wide range of medicinally important secondary metabolites. The low cellular uptake of the polar compounds in the extract of the plant limits its biological application. In present study efficacy of F. cretica modified bioactive nano-formulations for in vitro modulation of TRAIL mediated extrinsic apoptotic pathway as cancer therapy was investigated. F. cretica methanolic extracts were formulated at nano-scale for green synthesis of silver nanoparticles, albumin conjugation and liposomes encapsulation to enhance targeted bioactivity against cancer. Physical characterization of the synthesized nanoparticles was done by SEM, EDX and Zeta potential analyzer. In vitro cell viability assay MTT was done for MCF-7, Hep-2, HUH-7 and HCEC cell lines. Relative expression variation of the apoptotic pathway-associated genes was done by qRT-PCR. SEM revealed spherical shape of 56.62 ± 8.04, 143 ± 11.54 and 83.36 ± 38.73 nm size and zeta potential - 18.6, - 15.5 and - 18.3 mV for liposomes, silver and albumin nanoparticles. Silver nanoparticles showed highest anticancer activity in vitro than albumin and liposomes nanoparticles with IC50 0.101 ± 0.004, 0.177 ± 0.03 and 0.434 ± 0.022 mg/mL in MCF-7, Hep-2 and HUH-7 respectively. F. cretica albumin and silver nanoparticles upregulated the in vitro TRAIL, DR4, DR5 and FADD gene expression at statistically significant levels in Hep-2 cell lines. Nano-formulations of F. cretica proved therapeutically important biomolecules in vitro. The hypothesized modulation of extrinsic apoptosis pathway genes through the plant nanoparticles proved novel medicinal options for effective treatment of cancer and enhancing the bioavailability of the active plant metabolites.

Epidemiological survey of bovine gammaherpesvirus 4 (BoHV-4) infection in cattle and buffalo from Pakistan.

This study was conducted to evaluate the prevalence of bovine gammaherpesvirus 4 (BoHV-4) among healthy cattle and buffaloes as well as those associated with different diseases (respiratory tract infection, mastitis and reproductive tract infection) in District Chakwal, Pakistan. Blood, swab and milk samples of cattle and buffaloes were randomly collected from different areas of Chakwal. DNA was isolated from the samples and subjected to nested PCR using thymidine kinase gene primers. Out of 300 samples (200 blood, 50 swab and 50 milk samples) from both species (cattle and buffalo), an overall prevalence of BoHV-4 of 3.33% was obtained. Samples from cattle showed a higher species-specific prevalence (4.16%) than samples from buffalo (2.78%). One sample out of 50 swab samples and 1 out of 50 milk samples were also positive for BoHV-4. DNA sequencing of a positive PCR product from cattle confirmed that the sequence was from the thymidine kinase gene of BoHV-4. Phylogenetic analysis also revealed close similarities with other BOHV-4 thymidine kinase sequences. To detect BoHV-4 antibodies, an indirect ELISA was also performed. Two hundred blood samples were also collected from the same animals in nonanticoagulant-containing tubes for the isolation of serum and were subjected to indirect ELISA. Sixteen samples (8%) were positive for BoHV-4 antibodies. This study will be useful in further diagnoses of BoHV-4 in Pakistan and in devising measures to control the spread of BoHV-4.

Disadvantaged Economic Conditions and Stricter Border Rules Shape Afghan Refugees' Ethnobotany: Insights from Kohat District, NW Pakistan.

The study of migrants' ethnobotany can help to address the diverse socio-ecological factors affecting temporal and spatial changes in local ecological knowledge (LEK). Through semi-structured and in-depth conversations with ninety interviewees among local Pathans and Afghan refugees in Kohat District, NW Pakistan, one hundred and forty-five wild plant and mushroom folk taxa were recorded. The plants quoted by Afghan refugees living inside and outside the camps tend to converge, while the Afghan data showed significant differences with those collected by local Pakistani Pathans. Interviewees mentioned two main driving factors potentially eroding folk plant knowledge: (a) recent stricter border policies have made it more difficult for refugees to visit their home regions in Afghanistan and therefore to also procure plants in their native country; (b) their disadvantaged economic conditions have forced them to engage more and more in urban activities in the host country, leaving little time for farming and foraging practices. Stakeholders should foster the exposure that refugee communities have to their plant resources, try to increase their socio-economic status, and facilitate both their settling outside the camps and their transnational movement for enhancing their use of wild plants, ultimately leading to improvements in their food security and health status.