Potential of ZnO nanoparticles for multi-drug resistant Escherichia coli having CRISPR-Cas from poultry market in Lahore.

BACKGROUND AND OBJECTIVES: Apart from known factors such as irrational use of antibiotics and horizontal gene transfer, it is now reported that clustered regularly interspaced short palindromic repeats (CRISPR) are also associated with increased antimicrobial resistance. Hence, it is critical to explore alternatives to antibiotics to control economic losses. Therefore, the present study aimed to determine not only the association of CRISPR-Cas system with antibiotic resistance but also the potential of Zinc Oxide nanoparticles (ZnO-NPs) for avian pathogenic Escherichia coli (APEC) isolated from poultry market Lahore. MATERIALS AND METHODS: Samples (n = 100) were collected from live bird markets of Lahore, and isolates were confirmed as Escherichia coli (E. coli) using the Remel One fast kit, and APEC was identified using PCR. The antibiotic resistance pattern in APEC was determined using the minimum inhibitory concentration (MIC), followed by genotypic confirmation of antibiotic-resistant genes using the PCR. The CRISPR-Cas system was also identified in multidrug-resistant (MDR) isolates, and its association with antibiotics was determined using qRT-PCR. The potential of ZnO-NPs was evaluated for multidrug-resistant (MDR) isolates by MIC. RESULTS: All isolates of APEC were resistant to nalidixic acid, whereas 95% were resistant to chloramphenicol and 89% were resistant to streptomycin. Nineteen MDR APEC were found in the present study and the CRISPR-Cas system was detected in all of these MDR isolates. In addition, an increased expression of CRISPR-related genes was observed in the standard strain and MDR isolates of APEC. ZnO-NPs inhibited the growth of resistant isolates. CONCLUSIONS: The findings showed the presence of the CRISPR-Cas system in MDR strains of APEC, along with the potential of ZnO-NPs for a possible solution to proceed. This highlights the importance of regulating antimicrobial resistance in poultry to reduce potential health consequences.

Targeting inflammation for the treatment of endometritis in bovines.

The uterine endometrial surface of bovines is in constant exposureconstantly exposed with to a multitude ofmany microbial populations that changes throughout the post-partum phase in terms of complexity and dynamics. These microbes contribute to the host pathology, leading to severe economic losses along withnd reproductive capabilities. The basic primary interface that occurs between the internal tissues of the body of the hostbetween the host body's internal tissues and the microbes is the endometrial surface of the uterus. As a result of the infinite pathogenic population, there is always a danger for the opportunistic organisms to attack. Therefore, it is paramount that any interactions, especially microbial microbes with the endometrial surface, are regulated by the host cells. However, the inflammatory response as the defense mechanism contributes a pivotal roleis pivotal in host immunity and pathology. The inflammatory cascade and pathways are important essential to eliminate this clinical problem. In this review, we will discuss and explain how the inflammation and the various components of the immune system play their role in host pathology and therapeutic strategies, taking into account the interface between the host and the microbes on the surface of the endometrium. This review is also instrumental in further explanation of inflammatory uterine disease by discussing the response of inflammation to external insult.

Exploring the Interplay of the CRISPR-CAS System with Antibiotic Resistance in Staphylococcus aureus: A Poultry Meat Study from Lahore, Pakistan.

Staphylococcus aureus is one of the major pathogens responsible for causing food poisoning worldwide. The emergence of antibiotic resistance in this bacterium is influenced by various factors. Among them, bacterial acquired defense systems described as clustered regularly interspaced short palindromic repeats (CRISPR)-cas system might be involved in antibiotic resistance development in bacteria. The current study was designed to assess the prevalence of S. aureus and its antibiotic resistance profile and identify the relationship of the CRISPR-cas system with antimicrobial resistance, followed by phylogenetic analysis. Total samples (n = 188) of poultry meat were collected from the poultry bird market of Lahore, Punjab, Pakistan. We used both phenotypic (antibiotic disc diffusion) and genotypic methods (PCR) to identify multi-drug resistant (MDR) strains of S. aureus. Additionally, the role of the CRISPR-Cas system in the isolated MDR S. aureus was also assessed. In addition, real-time quantitative PCR (qRT-PCR) was used to evaluate the association of the CRISPR-cas system with antimicrobial resistance. All of the S. aureus isolates showed 100% resistance against erythromycin, 97.5% were resistant to tetracycline, and 75% were resistant to methicillin. Eleven isolates were MDR in the current study. The CRISPR system was found in all MDR isolates, and fifteen spacers were identified within the CRISPR locus. Furthermore, MDR S. aureus isolates and the standard strain showed higher expression levels of CRISPR-associated genes. The correlation of said system with MDR isolates points to foreign gene acquisition by horizontal transfer. Current knowledge could be utilized to tackle antibiotic-resistant bacteria, mainly S. aureus.

Patterns of Common Dermatological Conditions among Children and Adolescents in Pakistan.

Background and Objectives: Dermatological disorders are highly prevalent among children in Pakistan. The present cross-sectional study aims to identify the spectrum of dermatological conditions among children and adolescents in Pakistan. Materials and Methods: A total of 582 patients (50.9% males; 49.1% females) were included in the study based on their age (5.7 ± 4.1 years), dermatological condition, and epidemiology. The youngest patient was aged ten days, whereas the eldest was seventeen. Age criteria were further stratified into three categories: infants and toddlers (≤5 years), children (≥5 to <12 years), and adolescents (≥12 to <18 years). Amongst them, the majority was from Punjab (81.6%), while the other regions included were Azad Jammu and Kashmir (14.4%), Islamabad (3.3%), and Khyber Pakhtunkhwa (0.7%). Results: Scabies was the highest reported skin condition with 281 (45.55%) patients, followed by 114 (19.6%) with eczema, 60 (10.3%) with dermatitis, 33 (5.7%) with tinea capitis, 17 (2.9%) with tinea corporis, 16 (2.7%) with impetigo, and 15 (2.6%) with folliculitis. Other conditions include urticaria, burns, infections, pediculosis, tinea inguinalis, tinea faciei, nappy rashes, alopecia, warts, tinea incognito, tinea cruris, and acne vulgaris. The chi-squared test showed a high prevalence of tinea corporis and acne among adolescents (12-17 years), whereas eczema, dermatitis, and impetigo were more prevalent among infants and toddlers. Conclusions: Pets or livestock and poor hygiene were found to be highly reported risk factors for many dermatological conditions like scabies and fungal infections. Dermatological conditions are common in younger individuals, but unfortunately, many children do not receive the desired medical assistance.

RNA-seq-based transcriptome analysis of a cefquinome-treated, highly resistant, and virulent MRSA strain.

The emergence and dissemination of methicillin-resistant Staphylococcus aureus (MRSA) strains of animal origin that are resistant to several antibiotics is of great concern. Cefquinome is a fourth-generation cephalosporin developed specifically for veterinary use. The mechanism of MRSA resistance to cefquinome is still not established. Therefore, we designed this study to evaluate the effect of cefquinome on the transcriptome of MRSA1679a, a strain that was isolated from a chicken. The transcriptome analysis indicated that multiple efflux pumps (QacA, NorB, Bcr, and ABCb) were upregulated in MRSA1679a as a resistance mechanism to expel cefquinome. Additionally, penicillin-binding protein 1A was overexpressed, which conferred resistance to cefquinome, a ß-lactam antibiotic. Adhesion and the biofilm-forming capacity of the MRSA strain was also enhanced in addition to overexpression of many stress-related genes. Genes related to carbohydrate metabolism, secretion systems, and transport activity were also significantly upregulated in MRSA1679a. In conclusion, global transcription was triggered to overcome the stress induced by cefquinome, and the MRSA1679a showed a great genetic potential to survive in this challenging environment. This study provides a profound understanding of MRSA1679a as a potentially important pathogen and identifies key resistance characteristics of MRSA against cefquinome. Studies should be aimed to demonstrate multidrug resistance mechanisms of virulent strains by exposing to different antibiotic combinations.

An in vitro antiviral activity of iodine complexes against SARS-CoV-2.

Since the emergence of COVID-19 pandemic in China in late 2019, scientists are striving hard to explore non-toxic, viable anti-SARS-CoV-2 compounds or medicines. We determined In vitro anti-SARS-CoV-2 activity of oral formulations (syrup and capsule)of an Iodine-complex (Renessans). First, cell cytotoxicity of Renessans on the Vero cells was determined using MTT assay. Afterwards, the antiviral activity of Renessans was determined using viral inhibition assays and TCID50. For this, nontoxic concentrations of the Renessans were used. The results showed that Renessans is nontoxic to the cells up to 50 µg/mL. At 1.5 µg/mL concentration, SARS-CoV-2 production was significantly reduced to 101.43 TCID50 and 101.58 TCID50 for the syrup and capsule, respectively, as compare to virus infected control cells 106.08 TCID50 and we found the dose dependent inhibition of virus replication in the presence of Renessans. Renessans inhibited SARS-CoV-2 with an EC50 value of 0.425 µg/mL and 0.505 µg/mL for syrup and capsule, respectively. Furthermore, there was no virus detected at concentration of 50 µg/mL of Renessans. This study indicates that Renessans, containing iodine, have potential activity against SARS-CoV-2 which needs to be further investigated in human clinical trials.

A comparative phylogenomic analysis of SARS-CoV-2 strains reported from non-human mammalian species and environmental samples.

Coronaviruses (CoVs) infect a wide range of domestic and wild mammals. These viruses have a potential and tendency to cross-species barriers and infect humans. Novel human coronavirus 2019-nCoV (hCoV-19) emerged from Wuhan, China, and has caused a global pandemic. Genomic features of SARS-CoV-2 may attribute inter-species transmission and adaptation to a novel host, and therefore is imperative to explicate the evolutionary dynamics of the viral genome and its propensity for differential host selection. We conducted an in silico analysis of all the coding gene sequences of SARS-CoV-2 strains (n = 39) originating from a range of non-human mammalian species, including pangolin, bat, dog, cat, tiger, mink, mouse, and the environmental samples such as wastewater, air and surface samples from the door handle and seafood market. Compared to the reference SARS-CoV-2 strain (MN908947; Wuhan-Hu-1), phylogenetic and comparative residue analysis revealed the circulation of three variants, including hCoV-19 virus from humans and two hCoV-19-related precursors from bats and pangolins. A lack of obvious differences as well as a maximum genetic homology among dog-, cat-, tiger-, mink-, mouse-, bat- and pangolin-derived SARS-CoV-2 sequences suggested a likely evolution of these strains from a common ancestor. Several residue substitutions were observed in the receptor-binding domain (RBD) of the spike protein, concluding a promiscuous nature of the virus for host species where genomic alternations may be required for the adaptation to novel host/s. However, such speculation needs in vitro investigations to unleash the influence of substitutions towards species-jump and disease pathogenesis.

Disposition of cyadox in domesticated cats following oral, intramuscular, and intravenous administration.

Cyadox (CYX) is a synthetic antibacterial agent of quinoxaline with much lower toxic effects. A safety criterion of CYX for clinical use was established by studying the pharmacokinetics and metabolism of CYX after oral (PO), intramuscular (IM), and intravenous (IV) administration. CYX was administered in six domesticated cats (three males and three females) by PO (40 mg/kg.b.w.), IM (10 mg/kg.b.w.), and IV (10 mg/kg.b.w.) routes in a crossover pattern. Highly sensitive liquid chromatography with ultraviolet detection (HPLC-UV) method was developed for detection of CYX and its metabolites present in plasma, urine, and feces. The bioavailability of CYX after PO and IM routes was 4.37% and 84.4%. The area under curves (AUC), mean resident time (MRT), and clearance (CL) of CYX and its metabolites revealed that CYX quickly metabolized into its metabolites. The total recovery of CYX and its main metabolites was >60% after each route. PO delivery suggesting first pass effect in cats that might make this route suitable for intestinal infection and IM injection could be better choice for systemic infections. Less ability of glucuronidation did not show any impact on CYX metabolism. The findings of present study provide detailed information for evaluation of CYX.

A comparative evaluation of serum biochemistry profile and antigenic relatedness among velogenic and mesogenic Avian avulavirus 1 infection in chickens and pigeons.

Newcastle disease (ND), caused by virulent Avian avulavirus 1 (AAvV 1), affects variety of avian species around the globe. Several AAvV 1 viruses of different genotypes have recently emerged with varying clinical impacts on their susceptible hosts. Although experimental infection with velogenic and mesogenic strains in chickens and pigeons is well-studied, nevertheless, there exists a paucity of data for comparative variations in serum biochemistry profile of susceptible hosts upon challenge with isolates of varying pathogenicities. With this background, a comparative assessment of a range of serum biochemical parameters was made following challenge with duck-originated velogenic strain (sub-genotype VIIi; MF437287) and pigeon-originated mesogenic strain (sub-genotype VIm; KU885949) in chickens and pigeons. For each of the isolate, commercial broiler chickens and wild pigeons were challenged (10-6.51 EID50/0.1 mL for sub-genotype VIIi and 10-6.87 EID50/0.1 mL sub-genotype Vim) separately via intranasal and intraocular route. Sera were collected on 0, 3rd, 5th, 7th, and 9th day post-infection (dpi), and processed for quantitative analysis of different biochemical parameters. By day 3 post-infection (pi), a substantial decrease (p < 0.0001) in serum alkaline phosphatase (ALP) was observed in chickens and pigeons challenged with velogenic isolate. On the other hand, from day 5 pi and onward, a significant increase (p < 0.001) in serum ALP and total protein concentration was observed exclusively in pigeons challenged with mesogenic isolate. For serum aspartate aminotransferase (AST), a significant increase (p < 0.05) in concentration was observed on day 3 pi which decreased from day 5 pi and onward in pigeons and chickens challenged with mesogenic isolate. Also, to reveal antigenic differences among homologous and heterologous vaccine and field-prevalent strains, cross-hemagglutination inhibition assay demonstrated antigenically diverse nature (R-value < 0.5) of both strains from vaccine strain (LaSota, genotype II). The study concludes antigenic differences among prevalent genotypes than vaccine strain and, although requires further studies to ascertain study outcomes, the serum biochemical profile may facilitate presumptive diagnosis of disease in their susceptible hosts.

Resistance and Virulence Mechanisms of Escherichia coli Selected by Enrofloxacin in Chicken.

This study aimed to investigate the genetic characteristics, antibiotic resistance patterns, and novel mechanisms involved in fluoroquinolone (FQ) resistance in commensal Escherichia coli isolates. The E. coli isolates were recovered from a previous clinical study and subjected to antimicrobial susceptibility testing and molecular typing. Known mechanisms of FQ resistance (target site mutations, plasmid-mediated quinolone resistance [PMQR] genes, relative expression levels of efflux pumps and porins) were detected using DNA sequencing of PCR products and real-time quantitative PCR. Whole-genome shotgun sequencing was performed on 11 representative strains to screen for single nucleotide polymorphisms (SNPs). The function of a key SNP (A1541G) was investigated by site-directed mutagenesis and allelic exchange. The results showed that long-term enrofloxacin treatment selected multidrug-resistant (MDR) E. coli isolates in the chicken gut and that these E. coli isolates had diverse genetic backgrounds. Multiple genetic alterations, including double mutations on GyrA (S83L and D87N), a single mutation on ParC (S80I) and ParE (S458E), activation of efflux pumps, and the presence of the QnrS1 protein, contributed to the high-level FQ resistance (enrofloxacin MIC [MICENR] ≥ 128 µg/ml), while the relatively low-level FQ resistance (MICENR = 8 or 16 µg/ml) was commonly mediated by decreased expression of the porin OmpF, besides enhancement of the efflux pumps. No significant relationship was observed between resistance mechanisms and virulence genes. Introduction of the A1541G mutation on aegA was able to increase FQ susceptibility by 2-fold. This study contributes to a better understanding of the development of MDR and the differences underlying the mechanisms of high-level and low-level FQ resistance in E. coli.

CRISPR-cas system: biological function in microbes and its use to treat antimicrobial resistant pathogens.

The development of antibiotic resistance in bacteria is a major public health threat. Infection rates of resistant pathogens continue to rise against nearly all antimicrobials, which has led to development of different strategies to combat the antimicrobial resistance. In this review, we discuss how the newly popular CRISPR-cas system has been applied to combat antibiotic resistance in both extracellular and intracellular pathogens. We also review a recently developed method in which nano-size CRISPR complex was used without any phage to target the mecA gene. However, there is still challenge to practice these methods in field against emerging antimicrobial resistant pathogens.

A microbiological inhibition method for the rapid, broad-spectrum, and high-throughput screening of 34 antibiotic residues in milk.

In this study, we developed a microbiological inhibition method for the rapid screening of antibiotics in milk with Geobacillus stearothermophilus ATCC12980 as an indicator bacterium and an easy sample pretreatment. We observed that the limits of detection of the kit for 34 common antibiotic residues in milk, including ß-lactams (13), aminoglycosides (6), tetracyclines (4), sulfonamides (6), macrolides (4), lincosamides (1), were lower than or close to the maximum residue limits formulated by the European Union and China. Moreover, the false-positive rate was 1% and the false-negative rates were less than 5%. The ruggedness of the method (the reproducibility of detection capability of different batches of medium) met requirements at determined levels and residual limits. The shelf life of the kit was more than 6 mo at 4°C. Additionally, we observed good correlations between the kit results and ultra-high-performance liquid chromatography-tandem mass spectrometry results for incurred milk (samples taken from animals treated with antibiotics according to the pre-slaughter medication data), which indicated that the kit was reliable for screening antibiotics in incurred samples. In conclusion, the kit has a broad application potential with high sensitivity, specificity, and reproducibility, stability, and reliability, combined with simple operation, low cost, and high-throughput capacity.

Aqueous two-phase system (ATPS): an overview and advances in its applications.

Aqueous two-phase system (ATPS) is a liquid-liquid fractionation technique and has gained an interest because of great potential for the extraction, separation, purification and enrichment of proteins, membranes, viruses, enzymes, nucleic acids and other biomolecules both in industry and academia. Although, the partition behavior involved in the method is complex and difficult to predict. Current research shows that it has also been successfully used in the detection of veterinary drug residues in food, separation of precious metals, sewage treatment and a variety of other purposes. The ATPS is able to give high recovery yield and is easily to scale up. It is also very economic and environment friendly method. The aim of this review is to overview the basics of ATPS, optimization and its applications.

Microbial communities present in the lower respiratory tract of clinically healthy birds in Pakistan.

Commercial poultry is an important agricultural industry worldwide. Although dense living conditions and large flocks increase meat and egg production, they also increase the risk of disease outbreaks and zoonoses. Current pathogen identification methods mostly rely on culture-dependent techniques and, therefore, are limited to a very small number of bacteria present in the environment. Next Generation Sequencing allows for culture-independent characterization of lower respiratory microbiome of birds including the identification of novel commensals and potentially emerging pathogens. In this study, we collected tracheo-bronchoalveolar lavage of 14 birds raised at 3 different farms in the Punjab province of Pakistan. To characterize the lower respiratory microbiome of these birds, we sequenced hyper-variable regions of the 16S ribosomal subunit gene. Although dominated by bacteria belonging to a small number of taxonomic classifications, the lower respiratory microbiome from each farm was far more diverse and novel than previously known. The differences in microbiome among farms suggest that inter-farm differences affect the microbiome of birds more than breed, geographic location, or management system. The presence of potential and known pathogens in genetically similar specialty breeds of chickens kept at unnaturally high densities and under variable conditions presents an extraordinary opportunity for the selection of highly pathogenic bacteria. In some instances, opportunistic respiratory pathogens were observed in apparently healthy birds. Understanding and monitoring the respiratory microbiome of such populations may allow the early detection of future disease threats.

Complete genome sequencing of a velogenic viscerotropic avian paramyxovirus 1 isolated from pheasants (Pucrasia macrolopha) in Lahore, Pakistan.

We report the complete genome sequence of avian paramyxovirus 1 (APMV-1) isolated from an acute and highly contagious outbreak in pheasants (Pucrasia macrolopha) in Lahore, Pakistan. Biological and serological characterization showed a velogenic strain of APMV-1, which was further confirmed by the sequence analysis of the cleavage site in the fusion protein. Complete genome sequencing and phylogenetic analysis indicated that this isolate belonged to genotype VII, specifically to subgenotype VIIa, and clustered closely with isolates characterized from Indonesia. Notably, the isolate showed significant differences from previously characterized APMV-1 from Pakistani commercial and rural chicken.

Genetic diversity of Newcastle disease virus in Pakistan: a countrywide perspective.

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 (112RK/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.

Insights into the Prognostic Role of Serum Interleukin-6 and Hematobiochemical Alterations in Cattle during Recent Outbreaks of Lumpy Skin Disease in Lodhran District, Pakistan.

Lumpy skin disease (LSD) is a highly infectious disease of cattle caused by a virus of the Poxviridae family, genus Capripoxvirus. The present study was designed to determine the prognostic ability of serum IL-6 in LSD using a binary logistic regression model at baseline sampling. A 17-day cohort study was conducted on a recent outbreak of LSD among cattle in the Lodhran District of Punjab, Pakistan. Infected cattle were divided into two categories based on their clinical status on day 17 as recovered (n = 33) or unrecovered (n = 17). Nodular lesions and scab specimens (n = 50) were used for the isolation of the lumpy skin disease virus and were confirmed by PCR. In recovered animals, hematological results showed marked leukocytosis, eosinophilia, lymphocytosis, neutrophilia, and monocytopenia. However, marked erythrocytosis, leukopenia, and thrombocytopenia were observed in the unrecovered animals at the final sampling point of the study. Serum levels of total protein, albumin, and glucose were significantly higher in the recovered animals. Meanwhile, aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenase, creatinine phosphokinase, total bilirubin, and direct bilirubin were found considerably higher in the unrecovered group. Receiver-operating characteristic curve analysis for serum IL-6 at baseline predicts the extended clinical conditions at the cut-off value of 85.16 pg/mL (55% specificity, 94% sensitivity, area under the curve 0.8039, respectively). In conclusion, the disease-induced hematological and biochemical alterations were significantly ameliorated in the recovered animals. In addition, the study revealed that serum IL-6 can be used as a valid marker for predicting the clinical worsening of LSD in cattle.

Molecular Characterization and Selection of Indigenous SARS-CoV-2 Delta Variant for the Development of the First Inactivated SARS-CoV-2 Vaccine of Pakistan.

Vaccines are one of the efficient means available so far for preventing and controlling the infection rate of COVID-19. Several researchers have focused on the whole virus's (SARS-CoV-2) inactivated vaccines which are economically efficient to produce. In Pakistan, multiple variants of SARS-CoV-2 have been reported since the start of the pandemic in February 2020. Due to the continuous evolution of the virus and economic recessions, the present study was designed to develop an indigenous inactivated SARS-CoV-2 vaccine that might help not only to prevent the COVID-19 in Pakistan, it will also save the country's economic resources. The SARS-CoV-2 were isolated and characterized using the Vero-E6 cell culture system. The seed selection was carried out using cross-neutralization assay and phylogenetic analysis. The selected isolate of SARS-CoV-2 (hCoV-19/Pakistan/UHSPK3-UVAS268/2021) was inactivated using beta-propiolactone followed by vaccine formulation using Alum adjuvant, keeping the S protein concentration as 5 µg/dose. The vaccine efficacy was evaluated by in vivo immunogenicity testing in laboratory animals and in in vitro microneutralization test. The phylogenetic analysis revealed that all the SARS-CoV-2 isolates reported from Pakistan nested into different clades, representing multiple introductions of the virus into Pakistan. The antisera raised against various isolates from different waves in Pakistan showed a varied level of neutralization titers. However, the antisera produced against a variant (hCoV-19/Pakistan/UHSPK3-UVAS268/2021; fourth wave) efficiently neutralized (1:64-1:512) all the tested SARS-CoV-2 isolates. The inactivated whole virus vaccine of SARS-CoV-2 was safe and it also elicited a protective immune response in rabbits and rhesus macaques on the 35th-day post-vaccination. The activity of neutralizing antibodies of vaccinated animals was found at 1:256-1:1024 at 35 days post-vaccination, indicating the effectiveness of the double-dose regime of the indigenous SARS-CoV-2 vaccine.

Seroprevalence of Newcastle disease virus and avian influenza virus in poultry and captive wild birds in poultry-dense regions of Pakistan.

Newcastle disease virus (NDV) and avian influenza virus (AIV) are causing contagious diseases in chickens and wild birds worldwide; however, there is a paucity of information on the current status of seropositivity of Newcastle and avian influenza diseases in chickens and wild birds of Pakistan. Therefore, the present study aimed to investigate the serological evidence of both diseases in commercial poultry (broiler, layer chickens), backyard poultry, and captive wild birds in poultry­dense regions of Punjab, Pakistan. Enzyme­linked immunosorbent (ELISA) and haemagglutination inhibition (HI) assays were performed for the determination of antibodies against NDV and AIV and their genotyping and subtyping, respectively. Overall, 47.5% and 67.4% seroprevalence of NDV and AIV, respectively, was observed in both poultry and wild birds. Based on bird's category, layer chickens had the highest seroprevalence of NDV (60.8%, 95% CI: 52.95­68.22, OR: 0.71) followed by backyard poultry (56.8%, 95% CI: 47.92­65.32, OR: 0.82), broilers (52.7%, 95% CI: 46.84­58.64), pigeons (41.3%, 95% CI: 30.53­52.81, OR: 1.59), peafowls (26.1%, 95% CI: 11.09­48.69, OR: 3.16), ducks (23.8%, 95% CI: 12.59­39.8, OR: 3.57), turkeys (16.7%, 95% CI: 4.41­42.27, OR: 5.58), parrots (14.3%, 95% CI: 2.52­43.85, OR: 6.70) and quails (2.3%, 95% CI: 0.2­13.51, OR: 4.8). Comparatively, backyard chickens had the highest seroprevalence of AIV (78.8%, 95% CI: 70.64­85.22, OR: 0.63) followed by ducks (73.8%, 95% CI: 57.68­85.6, OR: 0.83), layers (73.5%, 95% CI: 65.98­79.89, OR: 0.84), pigeons (72.5%, 95% CI: 61.2­81.61, OR: 0.89), broilers (70.1%, 95% CI: 64.44­75.29), turkeys (55.5%, 95% CI: 31.35­77.6, OR: 1.87), peafowls (47.8%, 95% CI: 27.42­68.9, OR: 2.56) and parrots (42.8%, 95% CI: 18.8­70.3, OR: 3.1). Overall, 40.1%, 34.2%, 31.3%, and 25.1% sera were positive for H9 AIV, G­VII NDV, H7 AIV, and G­VI NDV, respectively. The current study revealed a widespread exposure to NDV and AIV in poultry and captive wild birds. Therefore, it is crucial to include captive wild birds in NDV and AIV surveillance programs to further strengthen disease control measures, particularly in endemic regions.

Temporal study of wastewater surveillance from September 2020 to March 2021: an estimation of COVID-19 patients in Lahore, Pakistan.

The first aim of study was to quantify the viral load in the wastewater samples by RT-qPCR testing in Lahore population to estimate the number of patients affected and predict the next resurgence of COVID-19 wave in the city. The second aim of the study was to determine the hotspot areas of Lahore which remained positive more often for virus with high viral load. In this study, n = 420 sewage samples were collected on an average of two weeks intervals from 30 different sewage water disposal stations (14 sampling events) from Sept 2020 to March 2021. RNA was extracted and quantified by RT-qPCR without concentrating the virus in samples. Number of positive disposal sites (7-93%), viral load from sewage samples (100.296 to 103.034), and estimated patients (660-17,030) ranged from low to high according to the surge and restrain of 2nd and 3rd COVID-19 waves in the country. The viral load and estimated patients were reported high in January 2021 and March 2021 which were similar to the peak of 2nd and 3rd waves in Pakistan. Site 18 (Niaz Baig village DS) showed the highest viral load among all sites. Findings of the present study helped to estimate the number of patients and track the resurgence in COVID-19 waves in Lahore particularly, and in Punjab generally. Furthermore, it emphasizes the role of wastewater-based epidemiology to help policymakers strengthen the quarantine measures along with immunization to overcome enteric viral diseases. Local and national stake holders should work in collaboration to improve the environmental hygiene to control the disease.