Aeman Jilani¹, Mirza Imran Shahzad^1*, Muhammad Mohsin Zaman¹, Areeba Yousaf¹, Gildardo Rivera²

¹Institute of Biochemistry, Biotechnology and Bioinformatics, The Islamia University of Bahawalpur, Pakistan

2Laboratorio de Biotecnología Farmacéutica, Centro de Biotecnología Genómica, Instituto Politécnico Nacional, México

Abstract

Tuberculosis (TB), the white plague of Europe is still uncontrolled and fatal in many parts of the world including Pakistan. It is a major cause of morbidity and mortality in human and domestic animals in Pakistan. No new vaccine in the last hundred years has been developed except for a few encouraging results from recombinant and DNA vaccines in the past two decades. Five Mycobacterium specific genes (Rv0379, Rv3914, Rv3006, Rv0432+SP, and Rv0432-SP) were selected to develop DNA vaccine(s). All the constructs were tested on mice using both naked DNA and prime-boost methodologies. Forty-five BALB/c mice were divided into three main groups; DNA vaccine group, BCG Prime boost group, and Control group. Post-vaccine (PV) and post-challenge (PC) immune responses were confirmed through cytokine ELISA and qRT PCR. IFN-γ was additionally checked in plasma as well. Based on cytokine ELISA PC immune responses showed significant differences in TNF-α levels for both naked DNA vaccine groups (Rv0379, Rv3006, and Rv0432-SP) and BCG primed Rv3914 group in comparison to the BCG control group (p<0.05). Based on qRT PCR, IL-6, TNF-α, IFN-γ, and IL-1β showed no significant difference among all the vaccines and BCG control groups (CT range 25- 30). IFN-γ levels in plasma were analyzed PC; two vaccines Rv3006/LppZ and BCG primed Rv0432/SodC-SP (highest mean value 1360.35 pg/ml) have shown significant results (cutoff value 21pg/ml) at 63 days. All the vaccine construct(s) alone or in combination have significant therapeutic effects in comparison to BCG and negative control groups.

Keywords: Mycobacterium tuberculosis, DNA vaccine, Tumor necrosis factor-alpha, Interleukin-6, Interferon-gamma, Interleukin-1beta, Bacille Calmette-Guérin (BCG)

Maria Rasool, Muhammad Hidayat Rasool^*, Mohsin Khurshid, Bilal Aslam

Institute of Microbiology, Government College University Faisalabad, Faisalabad-38000, Pakistan

Abstract

Multi-drug resistant (MDR) bacterial infections significantly increase mortality, morbidity, and treatment costs when they persist. Therefore, there is a pressing need to discover, modify, or search for antimicrobial agents with the ability to combat MDR bacteria. Silver nanoparticles used in this study were synthesized by Bacillus subtilis and characterized through different techniques. MDR strains underwent antibacterial activity, antioxidant activity, and time-kill kinetic assays to assess susceptibility to silver nanoparticles. Furthermore, the synergistic impact of silver nanoparticles and antibiotics was examined using the two-dimensional checkerboard method to calculate the Fractional Inhibitory Concentration Index (FICI). Scanning Electron Microscopy (SEM) results revealed a circular shape of synthesized AgNPs, with an average length and area of 76.78 and 47.10 nm, respectively. UV analysis showed an optimum peak at 420 nm. XRD analysis indicated the crystalline nature of nanoparticles with diversity in size. Remarkable antioxidant potential (55% of AgNPs) was observed at a concentration of 1000 µg/ml, while minimum activity (18%) was noted at 62.6 µg/ml. Silver nanoparticles demonstrated a synergistic interaction with the antibiotic Cefixime against Staphylococcus aureus, Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae, with FICI values of 0.37, 0.3, 0.25, and 0.49, respectively, and an additive effect against Pseudomonas aeruginosa, with a FICI value of 0.7. Moreover, this research explores the anti-inflammatory potential of silver nanoparticles (AgNPs) in two distinct models: formaldehyde-induced inflammation and carrageenan-induced inflammation, along with an assessment of their in vitro anti-inflammatory activity. The findings shed light on the multifaceted role of AgNPs in mitigating inflammatory responses, offering promising avenues for therapeutic interventions.

Keywords: Green synthesis, Silver, Bacillus subtilis, Synergistic effect, Cefixime, XRD

Urairat Mongmonsin¹, Prapenpuksiri Rungsa², Withan Teajaroen¹, Theerasak Somdee³, Jureerut Daduang⁴, Sakda Daduang^2,5*

¹Department of Biomedical Sciences, Faculty of Graduate School, Khon Kaen University, Khon Kaen, Thailand

²Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Khon Kaen University, Khon Kaen, Thailand

³Department of Microbiology, Faculty of Science, Khon Kaen University, Khon Kaen, Thailand

⁴Department of Clinical Chemistry, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand

⁵Division of Pharmacognosy and Toxicology, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, Thailand

Abstract

Cordyceps militaris has been used in traditional medicine due to its immune-boosting functions, especially in elderly and cancer patients. The study aims to assess the impact of C. militaris extract on the immune response in LPS-induced splenocytes and male Sprague Dawley rats. The study found that CME contains various bioactive compounds such as adenosine, cordycepin (3′ deoxyadenosine), and phenolic compounds. In vitro studies showed that CME has the potential to enhance CD3⁺ T cell and CD45RA⁺ B cell populations in LPS-induced splenocytes with a cell viability of over 80%. Moreover, we found that CME can enhance the immune response in rats by increasing CD3⁺ T cell proliferation and up-regulating IL-4, IL-6, and IL-17a expression, without affecting the rat’s body weight. The findings suggest that CME could be a promising immune enhancer for patients with weakened immune systems. However, further animal experiments with varying doses are essential to determine the optimal dosage for the successful development of CME as a therapeutic agent.

Keywords: Cordyceps militaris, Immunostimulatory effects, Pro-inflammatory cytokines, Cordycepin

Jian Huang¹, Xiaohua Chen¹, Qinglian Zhong^1*

[1] Department of Gastroenterology, The Eighth Affiliated Hospital, Sun Yat-sen University, Shenzhen, Guangdong 518033, China

Abstract

The dysbiosis of the gut microbiota caused by drug metabolism and diets may influence the gastrointestinal (GI) barrier and their ability for normal attachment and further immunity system, which all could be associated with the medical efficacy of colorectal cancer (CRC) during chemotherapy.

This study aims to investigate Trimethylamine N-oxide (TMAO), a gastrointestinal product readily entering the bloodstream, as a potential risk factor for various diseases, including CRC.

To investigate the relationship between gut microbiota dysbiosis and carcinogenesis in CRC patients, we analyzed taxonomic alterations in the gut microbiota of 77 subjects, including 36 CRC patients and 41 normal controls. We collected samples of the participants’ microbiome from their fecal material and utilized 16S rRNA sequencing to identify the microbial composition. Additionally, to predict the functions of the GI microbiota, Phylogenetic Investigation of Communities by Reconstructing Unobserved States (PICRUSt) was employed. This could serve as a promising biomarker for colorectal cancer. Moreover, the serum level of TMAO between the CRC and healthy controls was also compared, and it was observed that the intestinal microbiome was changed in CRC patients; however, the serum level of TMAO was not correlated with the progression of CRC.

In conclusion, in the present study, instead of relying solely on TMAO, which is a convenient clinical test, we focused on the treatment of CRC by emphasizing the modification of the intestinal microbiome.

Keywords: CRC, Intestinal microbiome, TMAO, 16s rRNA, PICRUSt

Muhammad Amjad Iqbal^{1, 2}, Noreen Sarwar^1*, Sohail Raza¹, Sehrish Firyal³, Rabia Riaz⁴, Afia Muhammad Akram⁵, Rashad Munir⁴, Aamir Riaz Khan⁴, Mobeen Sarwar⁶, Muhammad Imran Arshad⁷

¹Institute of Microbiology, University of Veterinary and Animal Sciences, Lahore, Pakistan

²Veterinary Research Institute, Lahore, Pakistan

³Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore, Pakistan

⁴Foot and Mouth Disease Research Centre, Lahore, Pakistan

⁵Department of Zoology, Division of Science and Technology, University of Education, Lahore, Pakistan

⁶Livestock and Dairy Development, Government of Punjab, Lahore, Pakistan

⁷Institute of Microbiology, University of Agriculture, Faisalabad, Pakistan

Abstract

Inactivation of foot-and-mouth disease virus (FMDV) with Formalin (FA) and binary ethyleneimine (BEI) is a slow process and takes long time. The purpose of this study was to evaluate β-Propiolactone (BPL), an alkylating agent that is frequently used in vaccine development and production, as a candidate for the inactivation of FMDV serotype O. Virus was grown on confluent monolayer of BHK-21 cell line. Harvesting was performed between 18 and 20 hours after infection, when CPE was between 90 and 95%. The virus was subjected to inactivation with formalin (0.02%), BEI (2mM, 2.5mM, 3mM) and BPL (0.1%, 0.2 and 0.4%) at 4°C and 37°C. Samples (05 mL/each) were collected after 0, 2, 4, 8, 16, and 24 hours post treatment. The biological titer (TCID50/ml) of each inactivated sample was measured. An innocuity test was used to further confirm the inactivation. RT-PCR was used to detect viral genome damage by amplification of VP1 gene. Data were analyzed by one-way analysis of variance and pair wise comparison was made by post hoc Tukey’s HSD test. Linear regression was used in Microsoft Excel Version 2010. The virus titers after 0.02% formalin treatment were 7.172±0.298 and 4.584±1.362 at 4°C and 37°C respectively. The virus titers were 6.036±0.513, 5.622±0.298, and 5.150±0.449 with 2mM, 2.5mM, 3mM BEI at 4°C and 1.646±1.0210, 1.050±0.644, 0.492±0.492 at 37°C respectively. Inactivation with 0.1%, 0.2% and 0.4% BPL at 4°C resulted in virus titers of 2.386±1.1104, 1.4400±0.9445 and 0.6960±0.6960 respectively. Rapid inactivation of virus with all three BPL concentrations at 37°C gave mean titer of 0.00±0.00. There was a significant difference (P<0.05) among all treatment groups with highest inactivation rates recorded for BPL.

Keywords: FMDV, BHK-21, Inactivation, BEI, BPL, Formalin

Imran Bodlah^1*, Ammara Gull E Fareen², Umer Ayyaz Aslam Sheikh³, Muhammad Adnan Bodlah⁴, Rehana Bibi⁵, Hesham F. Alharby^{6, 7}, Habeeb M. Al-Solami⁶, Naser A. Alkenani⁶, Abdullah G. Al-Ghamdi⁶

¹Insect Biodiversity and Conservation Group, Department of Entomology, PMAS Arid Agriculture University, Rawalpindi, Pakistan

²Department of Environmental Sciences, PMAS Arid Agriculture University, Rawalpindi, Pakistan

³Department of Entomology, University of Poonch, Rawalakot, Azad Jammu and Kashmir, Pakistan

⁴Fareed Biodiversity Conservation Centre, Department of Agricultural Engineering, Khawaja Fareed University of Engineering and Information Technology, Rahim Yar Khan, Punjab, Pakistan
⁵Department of Plant Protection, Faculty of Agricultural Sciences, Ghazi University D. G. Khan

⁶Department of Biological Sciences, Faculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia

⁷Plant Biology Research Group, Department of Biological Sciences, Faculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia

Abstract

Bumble bees are among the insects that play a significant role in the pollination of several agricultural crops, fruits, vegetables, and wild flowering plants. Climate change has been discussed as a prospective threat to the biodiversity of these pollinators in different parts of the planet during the next several decades. As a result of expected climate change circumstances, a number of studies have determined that the distribution range of bumble bees will alter, with some species becoming extinct and majority of them relocating to the higher mountainous areas. Numerous countries around the globe have developed conservation measures for these ecologically and economically significant organisms. In Pakistan, Bombus haemorrhoidalis (Hymenoptera: Apidae) is more likely to be used as a pollinator in greenhouse vegetable production, as compared to other documented species. Our research aimed to find moderately and highly favorable locations for B. haemorrhoidalis in 2050 and 2070 based on two representative concentration pathways (RCP4.5 and RCP8.5) using Maxent. Five key contributing variables including Bio1 (Annual Mean Temperature), Bio12 (Annual Precipitation), Bio14 (Precipitation of Driest Month) and Bio18 (Precipitation of Warmest Quarter) along with 52 occurrence record of the species were utilized in the modeling procedure for determining potential distribution of B. haemorrhoidalis. Performance of the model was assessed by calculating the area under the curves (AUC), the partial ROC, the omission rates (E=5%), and the AICc (Model complexity). Regularization multiplier of finally selected model was 2. Based on the results of the Jackknife test, it was determined that only four climatic factors, namely Bio4, Bio12, Bio14 and Bio18 contributed 89.0% to the prediction of the species’ prospective distribution. The results indicated that highly suitable distribution areas of this species would be concentrated in upper the mountainous areas of Pakistan under the influence of climate change. The suitability of its habitat, however, will decrease under the forecasted climatic conditions of future scenarios (RCP 4.5 and RCP 8.5) for 2050 and 2070. According to the findings, the area that is anticipated to be moderately suitable will shrink by 320.492083 km²between the years 2050 and 2070 under RCP 4.5 (70), as compared to RCP 4.5. (50). In the same pattern, according to RCP 8.5 (70), it would shrink by a total of 260.764698 km² as compared to RCP 8.5. (50). A similar pattern would be observed for forecasted highly suitable areas, which would shrink by a total of 2492.820215 km² under RCP 4.5. (70) compared to RCP 4.5 (50), and by a total of 1363.441658 km² under RCP 8.5 (70) in contrast to RCP 8.5. (50). Results indicated that suitable areas for this species would decrease during two scenarios of year 2050 and 2070. This species would leave many areas of its current distribution under the influence of climate change and move upward towards upper mountainous areas of Azad Kashmir, Gilgit Baltistan and Khyber Pakhtunkhwa. Based on the results of our studies, Government along with other stakeholders of bee pollination may develop climate mitigation strategies to conserve pollination services of B. haemorrhoidalis on a sustainable basis. More extensive surveys are needed along with other tools of remote sensing for more reliable predictions.

Keywords: Bombus haemorrhoidalis, Habitat suitability, Environmental predictors, Maxent, Climate change, Pakistan

Nguyen-Huan Pham-Khanh¹, Nhat-Quynh Huynh¹, Hong-Ngoc-Bao Le¹, Thi-Kim-Quy Ha^1*

¹College of Natural Sciences, Can Tho University, Campus II, 3/2 street, Ninh Kieu district, Can Tho City 94000, Vietnam

Abstract

The hazardous heavy metal ion Cr(VI) is harmful and easily mobile in the environment. Cr(VI) poisoning can cause delayed seed germination and damaged plant growth. This study suggested a green and simple synthesis method of ZnO microparticles (ZnO MPs) from Zn(CH₃COO)₂ solution and aqueous leaf extract of Dolichandrone spathacea (L. f.) K. Schum for protecting the legume plant (Vigna radiata) against the Cr(VI) stress. The optimized conditions for the synthesis of these MPs were determined using computational and experimental approaches. The characterization of ZnO MPs was analyzed by surface morphology, particle sizes, and elemental components using modern methods. The Zn MPs successfully exhibited the potential protective effects on the seed germination and seedling vigor of V. radiata under Cr(VI) stress. The results of PPR and ABTS assays also indicated that the antioxidant capacity of non-enzymatic antioxidants from leaves under Cr(VI) stress significantly reduced (46.83 ± 1.938% and 69.60 ± 2.17%, respectively) as compared to supplement of ZnO-MPs increased (55.44 ± 2.624% and 78.07 ± 0.820%, respectively). This study is an essential report for the agricultural field, which can apply further the new and green zinc-micronutrient fertilizer to mitigate the adverse effects of heavy metal contamination on crop cultivation.

Keywords: Green synthesis, Zinc oxide microparticles, Dolichandrone spathacea, Cr(VI) stress, Vigna radiata.

Wang Dongjing^1*, Su Zhonghua², Yuan Zhenjie¹, Mikhlid H. Almutairi³

¹Institute of Animal Husbandry and Veterinary, Tibet Autonomous Regional Academy of Agricultural Sciences, Lhasa 850009, China

²Tibet Autonomous Region Animal Disease prevention and Control Center, Lhasa 850009, China

³Zoology Department, College of Science, King Saud University, P.O. Box: 2455, 11451, Riyadh, Saudi Arabia

Abstract

This study was conducted to identify the biological characteristics of pathogen responsible for the death of Tibetan sheep in Nagarze County, Tibet, China. For this purpose, samples were collected from diseased animals and for bacterial culture and isolation. The isolated strains were subjected to several tests which included gram staining, biochemical identification, PCR amplification, genetic evolution analysis, in-vitro drug sensitivity and in-vivo pathogenicity tests. The results revealed that 7 strains of gram-positive cocci were isolated from Tibetan sheep, named TS-1, TS-2, TS-3, TS-4, TS-5, TS-6, and TS-7. These strains exhibited specific biochemical characteristics consistent with Enterococcus faecalis. Whereas, PCR amplification results were consistent with the expected outcomes on target band of approximately 1500 bp. Genetic evolutionary analysis revealed a significant homology (96.0%-99.9%) between the isolates and Enterococcus faecium. In-vitro drug sensitivity tests demonstrated that all the isolates exhibiting multiple drug resistance. Furthermore, the isolated strains displayed varying degrees of pathogenicity in mice. This study confirms that Enterococcus faecium is the causative agent for the deaths of Tibetan sheep. These findings enhance our understanding of the disease and suggest valuable insights for its prevention, control, and future research.

Keywords: Tibetan sheep, Enterococcus faecium, Isolation, Identification, Drug resistance, Pathogenicity

Muhammad Bilal¹, Shafia Tehseen Gul^1*, Muhammad Tariq Javed¹, Muhammad Saqib²

¹Department of Pathology, Faculty of Veterinary Sciences, University of Agriculture, Faisalabad, Pakistan

²Department of Clinical Medicine and Surgery, Faculty of Veterinary Sciences, University of Agriculture, Faisalabad, Pakistan

Abstract

Brucellosis is considered as a highly contagious and zoonotic disease globally and various diagnostic tests are available for its diagnosis. Keeping in view, the limitations of currently used serological techniques, a more precise, sensitive, and reliable loop mediated isothermal amplification (LAMP) assay was evaluated as an emerging diagnostic tool. In the current study, serum samples from cows (n=1989) and buffaloes (n=1467) were collected from the study area i.e., District Faisalabad and Toba Tek Singh in Punjab, Pakistan. As these two districts are present around the river Ravi in Punjab, Pakistan and known as the house of Nili Ravi breed. A number of dairy farms of local and imported cows are also present in this area. Initially the samples were screened by the RBPT and then subjected to c-ELISA for confirmation. Overall, 12.16 and 9.3% cows and buffaloes were seropositive through RBPT while 11.21 and 7.70% cows and buffaloes were seropositive via c-ELISA. The positive samples from c-ELISA were further subjected to molecular amplification at 1.5% agarose gel through LAMP assay. The current study concludes that LAMP assay is more sensitive as compared to other conventional PCR techniques while detecting true positives for brucellosis, so it can be used for confirmation of Brucella abortus as compared to PCR. In addition to the sensitivity and specificity and qualitative results can be observed through naked eye in LAMP, which is not possible in PCR.

Keywords: Brucellosis, LAMP, Quick diagnosis, Serological techniques, Rapid point of care

Hina Ali Ahmed¹, Nazeer Ahmed², Shahjahan Shabbir Ahmed², Shagufta Saddozai¹, Afroz Rais³,

Imran Ali Sani², Dawood Shahid², Shahbaz Khan^4*

¹Department of Zoology, Sardar Bahadur Khan Women’s University, Quetta, Pakistan

²Department of Biotechnology, Faculty of Life Sciences, BUITEMS, Quetta, Pakistan

³Department of Botany, Sardar Bahadur Khan Women’s University, Quetta, Pakistan

⁴Colorado Water Center, Colorado State University, Fort Collins, Colorado 80523, USA

Abstract

Deoxyribonucleic acid (DNA) sequencing is an emerging approach for revealing species diversity that has made species identification possible. This technique is an amazing and useful tool for studies in taxonomy, evolutionary biology, and biodiversity. Sequences of DNA can be used as genetic “barcodes” which could be used to  identify all animals, including insects. True flies, belonging to the order Diptera, are widely distributed and crucial components of ecosystems worldwide. Despite the rich biodiversity of Pakistan, our knowledge of various insect groups particularly flies remains limited. The current study was conducted in Quetta, Pakistan, from June 2017 to May 2018, to use DNA barcoding technique for the determination of the diversity of flies (658 bp sequence from the 5′-end of cytochrome oxidase I). Our analysis focused on a specific region of the cytochrome c oxidase 1 (COI) gene, known as the barcode region which provides valuable information for inferring evolutionary relationships and identifying species. The obtained sequences of 2,195 fly specimens were then compared and matched against the Barcode of Life Data System (BOLD), which assigned the specimens to 309 Barcode Index Numbers (BINs), which operates as a counterpart in the BOLD database. Among the families identified, Muscidae was the most dominant, with 283 specimens, followed by Cecidomyiidae with 184 specimens, and Ceratopogonidae with 164 specimens. A total 82 number of species were identified with Tricimba humeralis with the maximum catch.

Keywords: Arthropod, BOLD, Barcode index number, Biodiversity, Cytochrome c oxidase I, DNA barcoding