Assessment of Cytotoxic, Genotoxic, and Oxidative Stress of Dibutyl Phthalate on Cultured Bovine Peripheral Lymphocytes.

Recently, there have been numerous reports showing that phthalates have negative human health impacts and may cause several diseases such as asthma, breast cancer, obesity, type II diabetes, and male infertility. Animals are also exposed to phthalates through the environment and can cause adverse health effects on them. Several studies have been found on the cytogenetic effects of dibutyl phthalate (DBP) on different organisms but no documented evidence has been found on the cytotoxic and genotoxic effects of dibutyl phthalate (DBP) on bovine cultured lymphocytes. MTT assay was performed on different series of DBP concentrations (10 µM, 20 µM, 30 µM, 50 µM, 70 µM, 100 µM). A concentration-dependent decrease in cell viability was observed by the DBP. The LD50, LD50/2, and 2∗LD50 were found to be 50 µM, 30 µM, and 80 µM on bovine lymphocytes, respectively. Then, these concentrations of DBP were utilized to perform comet, micronucleus assays, and oxidative stress. A concentration-dependent increase in DNA damage, oxidative stress, and micronuclei formation was observed in lymphocytes by the DBP as compared to the control group. Highest genotoxic effects were observed at a concentration of 2∗LD50. Similarly, total oxidative stress was found higher, and antioxidative stress was lower in concentration-dependent manner by the DBP. The current study revealed a significant cytotoxic, genotoxic, and oxidative stress of DBP on cultured bovine lymphocytes.

FMD virus spillover from domestic livestock caused outbreak in captive wild ungulates: First report from Pakistan.

The potential spillover of foot and mouth disease (FMD) virus at the wildlife-livestock interface is mainly responsible for the outbreaks in captive wild ungulates. The current study was planned to investigate an FMD outbreak in the wild ungulate species in the Jallo Wildlife Park and breeding facility, Lahore, Pakistan from Mar 2021- Jun 2021. The disease was confirmed based on ELISA through the detection of antibodies against the non-structural protein of FMD virus and typical clinical signs of oral and feet lesions, and even partial or complete hoof shedding in severe cases. To investigate the possible cause of FMD spillover and its pattern, a series of interviews were conducted with wildlife practitioners, animal handlers, and the local veterinarians in villages adjacent to the park. The data revealed neither vaccination nor any FMD outbreak in the last 10 years. The epidemic curve and time-series analysis showed a mixed outbreak characterized by the introduction of disease and then propagative spread from infected to susceptible animals. After the first reports of clinical signs in Blackbuck, Urial and Mouflon sheep were infected while in the end, the cases were reported from the enclosures of Sambar deer and Spotted deer. The morbidity rate among all the wild ungulate species was 92% and the mean mortality rate was 27%. The study concluded that possible sources for primary disease incidence might be animal handlers having FMD infected animals in their houses and transport vehicles coming from adjacent disease-affected villages. For the disease spread between enclosures, another possibility of wind-borne FMD virus spread could also be considered. This is the first report regarding the FMD outbreak and its spillover pattern in captive wild ungulates in Pakistan. These findings will be helpful in understanding the importance of wildlife livestock interface in FMD transmission and to design effective strategies to control the disease.

Epi-Gene: An R-Package for Easy Pan-Genome Analysis.

The main aim of this study was to develop a set of functions that can analyze the genomic data with less time consumption and memory. Epi-gene is presented as a solution to large sequence file handling and computational time problems. It uses less time and less programming skills in order to work with a large number of genomes. In the current study, some features of the Epi-gene R-package were described and illustrated by using a dataset of the 14 Aeromonas hydrophila genomes. The joining, relabeling, and conversion functions were also included in this package to handle the FASTA formatted sequences. To calculate the subsets of core genes, accessory genes, and unique genes, various Epi-gene functions have been used. Heat maps and phylogenetic genome trees were also constructed. This whole procedure was completed in less than 30 minutes. This package can only work on Windows operating systems. Different functions from other packages such as dplyr and ggtree were also used that were available in R computing environment.

In Silico Analysis of Potential Outer Membrane Beta-Barrel Proteins in Aeromonas hydrophila Pangenome.

Outer membrane proteins (OMPs) of Aeromonas hydrophila have a variety of functional roles in virulence and pathogenesis and represent promising targets for vaccine development. The main objective of this study was to develop an in-silico model of beta-barrel OMP present among the valid A. hydrophila pangenomes (n = 22). With a program named the ß-barrel Outer Membrane Protein Predictor (BOMP), total beta-barrel OMPs (n = 3127) were predicted across 22 genomes with the estimated median number of 64 per genome. In pangenome analysis, only 32 OMPs were found to be conserved. These beta-barrel OMPs also showed variations among source of isolation, COG and KEGG classes. Among 32 conserved OMPs, a highly antigenic protein was identified by utilizing Vaxijen. With B cell epitope predictions, two fragments of amino acid sequences i.e. GLTLGAQFTGNNDPQNADRSN (21 mer) and FKPSLAYLRTDVKDNARGI DDTATEY (26 mer) bearing B-cell binding sites were selected. Further, an epitope (12 amino acids: GLTLGAQFTGNN) that complexes to maximum MHC alleles with a higher antigenicity was determined. The analysis of evolutionary forces on the identified OMP sequence and epitope indicated that none of basic amino acid sites has shown significantly different substitution ratios. This conserved protein and epitope will be helpful in developing a vaccine that may be effective against all the A. hydrophila strains. Also, this study provides a theoretical basis for vaccine design against other pathogenic bacteria. Supplementary Information: The online version contains supplementary material available at 10.1007/s10989-021-10259-z.

Status of Bioinformatics Education in South Asia: Past and Present.

Bioinformatics education has been a hot topic in South Asia, and the interest in this education peaks with the start of the 21st century. The governments of South Asian countries had a systematic effort for bioinformatics. They developed the infrastructures to provide maximum facility to the scientific community to gain maximum output in this field. This article renders bioinformatics, measures, and its importance of implementation in South Asia with proper ways of improving bioinformatics education flaws. It also addresses the problems faced in South Asia and proposes some recommendations regarding bioinformatics education. The information regarding bioinformatics education and institutes was collected from different existing research papers, databases, and surveys. The information was then confirmed by visiting each institution's website, while problems and solutions displayed in the article are mostly in line with South Asian bioinformatics conferences and institutions' objectives. Among South Asian countries, India and Pakistan have developed infrastructure and education regarding bioinformatics rapidly as compared to other countries, whereas Bangladesh, Sri Lanka, and Nepal are still in a progressing phase in this field. To advance in a different sector, the bioinformatics industry has to be revolutionized, and it will contribute to strengthening the pharmaceutical, agricultural, and molecular sectors in South Asia. To advance in bioinformatics, universities' infrastructure needs to be on a par with the current international standards, which will produce well-trained professionals with skills in multiple fields like biotechnology, mathematics, statistics, and computer science. The bioinformatics industry has revolutionized and strengthened the pharmaceutical, agricultural, and molecular sectors in South Asia, and it will serve as the standard of education increases in the South Asian countries. A framework for developing a centralized database is suggested after the literature review to collect and store the information on the current status of South Asian bioinformatics education. This will be named as the South Asian Bioinformatics Education Database (SABE). This will provide comprehensive information regarding the bioinformatics in South Asian countries by the country name, the experts of this field, and the university name to explore the top-ranked outputs relevant to queries.

Molecular Epidemiology and Clone Transmission of Carbapenem-Resistant Acinetobacter baumannii in ICU Rooms.

Carbapenem-resistant Acinetobacter baumannii (CRAB) is a major cause of nosocomial infections and hospital outbreaks worldwide, remaining a critical clinical concern. Here we characterized and investigated the phylogenetic relationships of 105 CRAB isolates from an intensive care unit from one hospital in China collected over six years. All strains carried blaOXA-23 , blaOXA-66 genes for carbapenem resistance, also had high resistance gene, virulence factor, and insertion sequence burdens. Whole-genome sequencing revealed all strains belonged to ST2, the global clone CC2. The phylogenetic analysis based on the core genome showed all isolates were dominated by a single lineage of three clusters and eight different clones. Two clones were popular during the collection time. Using chi-square test to identify the epidemiologically meaningful groupings, we found the significant difference in community structure only existed in strains from separation time. The haplotype and median-joining network analysis revealed genetic differences appeared among clusters and changes occurred overtime in the dominating cluster. Our results highlighted substantial multidrug-resistant CRAB burden in the hospital ICU environment demonstrating potential clone outbreak in the hospital.

Prevalence and Distribution Characteristics of blaKPC-2 and blaNDM-1 Genes in Klebsiella pneumoniae.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae infections have caused major concern and posed a global threat to public health. As blaKPC-2 and blaNDM-1 genes are the most widely reported carbapenem resistant genes in K. pneumonia, it is crucial to study the prevalence and geographical distribution of these two genes for further understanding of their transmission mode and mechanism. PURPOSE: Here, we investigated the prevalence and distribution of blaKPC-2 and blaNDM-1 genes in carbapenem-resistant K. pneumoniae strains from a tertiary hospital and from 1579 genomes available in the NCBI database, and further analyzed the possible core structure of blaKPC-2 or blaNDM-1 genes among global genome data. MATERIALS AND METHODS: K. pneumoniae strains from a tertiary hospital in China during 2013-2018 were collected and their antimicrobial susceptibility testing for 28 antibiotics was determined. Whole-genome sequencing of carbapenem-resistant K. pneumoniae strains was used to investigate the genetic characterization. The phylogenetic relationships of these strains were investigated through pan-genome analysis. The epidemiology and distribution of blaKPC-2 and blaNDM-1 genes in K. pneumoniae based on 1579 global genomes and carbapenem-resistant K. pneumoniae strains from hospital were analyzed using bioinformatics. The possible core structure carrying blaKPC-2 or blaNDM-1 genes was investigated among global data. RESULTS: A total of 19 carbapenem-resistant K. pneumoniae were isolated in a tertiary hospital. All isolates had a multi-resistant pattern and eight kinds of resistance genes. The phylogenetic analysis showed all isolates in the hospital were dominated by two lineages composed of ST11 and ST25, respectively. ST11 and ST25 were the major ST type carrying blaKPC-2 and blaNDM-1 genes, respectively. Among 1579 global genomes data, 147 known ST types (1195 genomes) have been identified, while ST258 (23.6%) and ST11 (22.1%) were the globally prevalent clones among the known ST types. Genetic environment analysis showed that the ISKpn7-dnaA/ISKpn27 -blaKPC-2-ISkpn6 and blaNDM-1-ble-trpf-nagA may be the core structure in the horizontal transfer of blaKPC-2 and blaNDM-1 , respectively. In addition, DNA transferase (hin) may be involved in the horizontal transfer or the expression of blaNDM-1 . CONCLUSION: There was clonal transmission of carbapenem-resistant K. pneumoniae in the tertiary hospital in China. The prevalence and distribution of blaKPC-2 and blaNDM-1 varied by countries and were driven by different transposons carrying the core structure. This study shed light on the genetic environment of blaKPC-2 and blaNDM-1 and offered basic information about the mechanism of carbapenem-resistant K. pneumoniae dissemination.

Preliminary view of the global distribution and spread of the tet(X) family of tigecycline resistance genes.

BACKGROUND: The emergence of plasmid-mediated tet(X3)/tet(X4) genes is threatening the role of tigecycline as a last-resort antibiotic to treat clinical infections caused by XDR bacteria. Considering the possible public health threat posed by tet(X) and its variants [which we collectively call 'tet(X) genes' in this study], global monitoring and surveillance are urgently required. OBJECTIVES: Here we conducted a worldwide survey of the global distribution and spread of tet(X) genes. METHODS: We analysed a comprehensive dataset of bacterial genomes in conjunction with surveillance data from our laboratory and the NCBI database, as well as sufficient metadata to characterize the results. RESULTS: The global distribution features of tet(X) genes were revealed. We clustered three types of genetic backbones of tet(X) genes embedded or transferred in bacterial genomes. Our pan-genome analyses revealed a large genetic pool composed of tet(X)-carrying sequences. Moreover, phylogenetic trees of tet(X) genes and tet(X)-like proteins were built. CONCLUSIONS: To the best of our knowledge, our results provide the first view of the global distribution of tet(X) genes, demonstrate the features of tet(X)-carrying fragments and highlight the possible evolution of tigecycline-inactivation enzymes in diverse bacterial species and habitats.

Roles of three TonB systems in the iron utilization and virulence of the Aeromonas hydrophila Chinese epidemic strain NJ-35.

The TonB system functions in iron transport and has been identified in certain Gram-negative bacteria. Recently, we reported three TonB systems in the Aeromonas hydrophila Chinese epidemic strain NJ-35, but the functions of these systems have not been thoroughly elucidated to date. In this study, we investigated the role of these TonB systems in A. hydrophila iron utilization and virulence. We found that tonB1 and tonB2 were preferentially transcribed in iron-chelated conditions, where gene expression levels were approximately 8- and 68-fold higher compared with iron-rich conditions, respectively; tonB3 was consistently transcribed at a low level under iron-repleted and iron-depleted conditions. Only the TonB2 system was required to utilize iron-binding proteins. The tonB123 mutant showed increased susceptibility to erythromycin and roxithromycin. In addition, all three tonB genes were involved in A. hydrophila virulence in zebrafish, and various phenotypes associated with environmental survival were changed with varying degrees in each tonB mutant. TonB2 plays a relatively major role in adhesion, motility, and biofilm formation, while TonB3 is more involved in the anti-phagocytosis of A. hydrophila. In each observed phenotype, no significant difference was found between the single- and double-deletion mutants, whereas the triple-deletion mutant exhibited the most serious defects, indicating that all three TonB systems of A. hydrophila coordinately complement one another. In conclusion, this study elucidates the importance of TonB in iron acquisition and virulence of A. hydrophila, which lays the foundation for future studies regarding the survival mechanisms of this bacterium in iron-restricted environments.

Comparative genome analysis provides deep insights into Aeromonas hydrophila taxonomy and virulence-related factors.

BACKGROUND: Aeromonas hydrophila is a potential zoonotic pathogen and primary fish pathogen. With overlapping characteristics, multiple isolates are often mislabelled and misclassified. Moreover, the potential pathogenic factors among the publicly available genomes in A. hydrophila strains of different origins have not yet been investigated. RESULTS: To identify the valid strains of A. hydrophila and their pathogenic factors, we performed a pan-genomic study. It revealed that there were 13 mislabelled strains and 49 valid strains that were further verified by Average nucleotide identity (ANI), digital DNA-DNA hybridization (dDDH) and in silico multiple locus strain typing (MLST). Multiple numbers of phages were detected among the strains and among them Aeromonas phi 018 was frequently present. The diversity in type III secretion system (T3SS) and conservation of type II and type VI secretion systems (T2SS and T6SS, respectively) among all the strains are important to study for designing future strategies. The most prevalent antibiotic resistances were found to be beta-lactamase, polymyxin and colistin resistances. The comparative analyses of sequence type (ST) 251 and other ST groups revealed that there were higher numbers of virulence factors in ST-251 than in other STs group. CONCLUSION: Publicly available genomes have 13 mislabelled organisms, and there are only 49 valid A. hydrophila strains. This valid pan-genome identifies multiple prophages that can be further utilized. Different A. hydrophila strains harbour multiple virulence factors and antibiotic resistance genes. Identification of such factors is important for designing future treatment regimes.

Discovery of lahS as a Global Regulator of Environmental Adaptation and Virulence in Aeromonas hydrophila.

Aeromonas hydrophila is an important aquatic microorganism that can cause fish hemorrhagic septicemia. In this study, we identified a novel LysR family transcriptional regulator (LahS) in the A. hydrophila Chinese epidemic strain NJ-35 from a library of 947 mutant strains. The deletion of lahS caused bacteria to exhibit significantly decreased hemolytic activity, motility, biofilm formation, protease production, and anti-bacterial competition ability when compared to the wild-type strain. In addition, the determination of the fifty percent lethal dose (LD50) in zebrafish demonstrated that the lahS deletion mutant (ΔlahS) was highly attenuated in virulence, with an approximately 200-fold increase in LD50 observed as compared with that of the wild-type strain. However, the ΔlahS strain exhibited significantly increased antioxidant activity (six-fold). Label-free quantitative proteome analysis resulted in the identification of 34 differentially expressed proteins in the ΔlahS strain. The differentially expressed proteins were involved in flagellum assembly, metabolism, redox reactions, and cell density induction. The data indicated that LahS might act as a global regulator to directly or indirectly regulate various biological processes in A. hydrophila NJ-35, contributing to a greater understanding the pathogenic mechanisms of A. hydrophila.

Inhibition of Aeromonas hydrophila-induced intestinal inflammation and mucosal barrier function damage in crucian carp by oral administration of Lactococcus lactis.

This study explored the immunomodulatory effect and inhibition effects of the candidate probiotic Lactococcus lactis 16-7, which was isolated from crucian carp, on Aeromonas hydrophila infection in crucian carp. The experimental fish were divided into two groups; one was fed a diet supplemented with L. lactis, while the other was fed the control probiotic-free diet. After feeding for 42 d with the experimental diets, the fish that received the diet supplemented with probiotics exhibited a significantly enhanced serum superoxide dismutase activity, phagocytic activities of innate immune cells, and the expression levels of immune-related genes [interferon-γ (INF-γ), interleukin-1ß (IL-1ß), interleukin-11 (IL-11), tumour necrosis factor α (TNF-α) and myeloid differentiation factor 88 (MyD88)], indicating that L. lactis 16-7 could activate the non-specific immune system of crucian carp. At the end of the feeding trial, the crucian carps in each group were orally infected with A. hydrophila NJ-35. The results show that L. lactis 16-7 could prevent the increase in d-lactic acid concentration and inflammatory response caused by A. hydrophila in crucian carp. Compared with A. hydrophila group, L. lactis 16-7 preserved the integrity of intestinal villi and mitigated A. hydrophila-induced reduce in the transcriptional levels of tight junction (TJ) proteins zonula occludens-1 (ZO-1) and occludin, indicating that L. lactis 16-7 could reduce intestinal mucosal barrier damage and inflammation induced by A. hydrophila in crucian carp. In addition, L. lactis 16-7 could effectively antagonize the colonization of A. hydrophila in the intestine. Overall, these data clearly indicate that L. lactis 16-7 has the potential to be developed as a probiotic agent against A. hydrophila infection in aquaculture.

Diverse roles of Hcp family proteins in the environmental fitness and pathogenicity of Aeromonas hydrophila Chinese epidemic strain NJ-35.

The type VI secretion system (T6SS) has been considered as a crucial factor in bacterial competition and virulence. The hemolysin co-regulated protein (Hcp) is the hallmark of T6SS. The secretion of Hcp in Aeromonas hydrophila Chinese epidemic strain NJ-35 indicated a functional T6SS. In this study, three copies of the hcp gene were identified in the genome of strain NJ-35. We targeted these Hcp family proteins for generating deletion mutants. These mutants showed varying levels in Hcp production, the interaction with other bacteria or eukaryotic cells, and bacterial virulence. Hcp1 was necessary for T6SS assembly and played a predominant role in the bacterial competition; Hcp2 negatively functioned in the biofilm formation and bacterial adhesion and was more involved in the A. hydrophila virulence in zebrafish and survival against the predation of Tetrahymena, and Hcp3 positively influenced the biofilm formation and bacterial adhesion. These findings illustrate that the T6SS of A. hydrophila NJ-35 is active, and the three Hcp family proteins take part in different processes in environmental adaptation and virulence of this bacterium. This study will provide valuable insights into our understanding of microbial interactions and thus contribute to a broader effort to manipulate these interactions for therapeutic or environmental benefit.

Tetrahymena thermophila Predation Enhances Environmental Adaptation of the Carp Pathogenic Strain Aeromonas hydrophila NJ-35.

Persistence of Aeromonas hydrophila in aquatic environments is the principle cause of fish hemorrhagic septicemia. Protistan predation has been considered to be a strong driving force for the evolution of bacterial defense strategies. In this study, we investigated the adaptive traits of A. hydrophila NJ-35, a carp pathogenic strain, in response to Tetrahymena thermophila predation. After subculturing with Tetrahymena, over 70% of A. hydrophila colonies were small colony variants (SCVs). The SCVs displayed enhanced biofilm formation, adhesion, fitness, and resistance to bacteriophage infection and oxidative stress as compared to the non-Tetrahymena-exposed strains. In contrast, the SCVs exhibited decreased intracellular bacterial number in RAW264.7 macrophages and were highly attenuated for virulence in zebrafish. Considering the outer membrane proteins (OMPs) are directly involved in bacterial interaction with the external surroundings, we investigated the roles of OMPs in the antipredator fitness behaviors of A. hydrophila. A total of 38 differentially expressed proteins were identified in the SCVs by quantitative proteomics. Among them, three lipoproteins including SurA, Slp, and LpoB, and a serine/threonine protein kinase (Stpk) were evidenced to be associated with environmental adaptation of the SCVs. Also, the three lipoproteins were involved in attenuated virulence of SCVs through the proinflammatory immune response mediated by TLR2. This study provides an important contribution to the understanding of the defensive traits of A. hydrophila against protistan predators.

The fight for invincibility: Environmental stress response mechanisms and Aeromonas hydrophila.

Aeromonas hydrophila is a freshwater-dwelling zoonotic bacterium that has economic importance in aquaculture. In the past decade, Aeromonas hydrophila has become increasingly important because of its emergence as a food-borne zoonotic pathogen that is resistant to different treatment regimes. Being an aquatic bacterium, Aeromonas hydrophila is frequently subjected to several stressful environmental conditions, including changes in temperature, acidic pH and starvation that challenge its survival. To cope with these stressful conditions, like every cell, A. hydrophila possesses stress response mechanisms, such as alternative sigma factors, two-component systems, heat shock proteins, cold shock proteins, and acid tolerance response systems that eventually lead the fittest to survive. Moreover, the establishment of genetic variations among the strains related to environmental stress is also of great concern. This review presents the understandings based on inter-strain variations and stress response behavior of A. hydrophila that are important to control the increasing outbreaks of this bacterium in both human populations and aquaculture.

Catecholamine-Stimulated Growth of Aeromonas hydrophila Requires the TonB2 Energy Transduction System but Is Independent of the Amonabactin Siderophore.

The growth-stimulating effects of catecholamine stress hormones have been demonstrated in many pathogens. However, catecholamine-induced growth and its underlying mechanisms remain poorly understood in Aeromonas hydrophila. The present study sought to demonstrate that norepinephrine (NE), epinephrine (Epi), dopamine (Dopa), and L-dopa stimulate the growth of A. hydrophila in iron-restricted media containing serum. NE exhibited the strongest growth stimulation, which could be blocked by adrenergic antagonists. Furthermore, it was demonstrated that NE could sequester iron from transferrin, thereby providing a more accessible iron source for utilization by A. hydrophila. The deletion of the amoA gene associated with amonabactin synthesis revealed that the amonabactin siderophore is not required for NE-stimulated growth. However, the deletion of the TonB2 energy transduction system resulted in the loss of growth promotion by NE, indicating that a specific TonB-dependent outer membrane receptor might be involved in the transport of iron from transferrin. Collectively, our data show that catecholamine sensing promotes the growth of A. hydrophila in a manner that is dependent on the TonB2 energy transduction system.

Identification and Characterization of an Aeromonas hydrophila Oligopeptidase Gene pepF Negatively Related to Biofilm Formation.

Bacterial biofilms are involved in adaptation to complex environments and are responsible for persistent bacterial infections. Biofilm formation is a highly complex process during which multifarious genes work together regularly. In this study, we screened the EZ-Tn5 transposon mutant library to identify genes involved in biofilm formation of Aeromonas hydrophila. A total of 24 biofilm-associated genes were identified, the majority of which encoded proteins related to cell structure, transcription and translation, gene regulation, growth and metabolism. The mutant strain TM90, in which a gene encoding oligopeptidase F (pepF) was disturbed, showed significant upregulation of biofilm formation compared to the parental strain. The TM90 colony phenotype was smaller, more transparent, and splendent. The adhesive ability of TM90 to HEp-2 cells was significantly increased compared with the parental strain. Fifty percent lethal dose (LD50) determinations in zebrafish demonstrated that the enhanced-biofilm mutant TM90 was highly attenuated relative to the wild-type strain. In conclusion, the pepF gene is demonstrated for the first time to be a negative factor for biofilm formation and is involved in A. hydrophila pathogenicity.

Molecular epidemiology and viral load of HCV in different regions of Punjab, Pakistan.

BACKGROUND: Hepatitis C virus (HCV) is highly infectious pathogen which is responsible for causing Hepatitis around 200 million individuals worldwide. In Pakistan, 4.7% of HCV prevalence has been reported and HCV genotype 3a has been found to be the major source of infection in Pakistan but still there is lack of information on distribution of HCV genotypes and viral load in various geographical regions of Pakistan. Therefore, current study was designed to determine distribution of HCV genotypes as well viral load in different areas of Punjab province of Pakistan. FINDINGS: A total of 995 serum samples were taken from those individuals in which antibodies against HCV were detected through ELISA, from different regions of Punjab i.e. Lahore 317(31.85%), Faisalabad 70(7.03%), Gujranwala 129(12.96%), Gujrat 106(10.65%), Sialkot 94(9.44%), Sargodha 60(6.03%), Mandibaha-ud-din 135(13.56%), Jhang 86(8.64%). Qualitative PCR was performed to determine viral load and genotyping was performed using Nested PCR. Chi-square test was used to determine the age and sex-wise prevalence of HCV. Out of 995 samples, 888 samples were found positive for HCV RNA. In all regions, genotype 3a showed highest prevalence (82.81%) followed by genotype 1 (3.41%), mixed genotypes (2.41%), genotype 2 (0.50%), genotype 5 (0.1%) and unclassified genotypes (10.75%). Viral load in 29.5% patients infected with genotype 3a was less than 600,000 IU/mL, while it was between 600,000-800,000 IU/mL in 27.9% patients and 25.22% patients had more than 800,000 IU/mL viral load. CONCLUSION: HCV genotype 3a is the most prevalent genotype in various regions of Punjab. Viral load of HCV patients in these different regions of Punjab are reported for the first time. Moreover, based upon these results the Patients having viral load below 800,000 IU/mL would be expected to show better response of anti-HCV therapy.

Some challenges in progressive control of livestock originated zoonotic diseases in Pakistan-a pilot survey

Objective: To document the recent livestock related practices and possible unhygienic ways of pathogen entry. Identification of the potential risk factors for the spread of infection is important to design an evidence-based disease control programme. Methods:Rapid assessment method was adopted and a purposive sample of 60 dairy farmers were interviewed. The following factors were noted for contributing in primary and secondary transmission of zoonotic infections: (i) persons who come in close contact with animals and their secretions, (ii) management strategies of farm animals (sheds and environment), (iii) management practices adopted at farms, (iv) small scale farmers and rural livestock production systems, (v) milk collection systems. Results:This research unveiled the certain routes of zoonotic disease transmission. Certain management practices, precautionary measures and strategies were the pivotal risk factors. Conclusions:The study emphasizes the need to educate the poor livestock keepers.