Narumon Tangthirasunun^1*, Darbhe Jayarama Bhat^2,3 , Supattra Poeaim¹

¹Department of Biology, School of Science, King Mongkut’s Institute of Technology Ladkrabang, Bangkok, 10520, Thailand

²Department of Botany and Microbiology, College of Science, King Saud University, P.O. Box 2455, Riyadh, 11451, Saudi Arabia

³Vishnugupta Vishwavidyapeetam, Ashoke, Gokarna, 581326, India

Abstract

Twenty-seven coprophilous fungi, isolated from field-fed cow dung in an organic farm in Thailand, were identified using morphology and ITS barcode. A total of five genera viz. Aspergillus, Hamigera, Paecilomyces, Penicillium, and Talaromyces were identified with varying numbers and growth rates. These fungi were evaluated for their antibacterial properties against Gram-positive (Bacillus subtilis, Kocuria rhizophila, Staphylococcus aureus and St. epidermidis) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria. Pe. javanicum NTD-SP2-01 and Talaromyces sp. NTD-SP5-48 exhibited activity against all bacteria when tested with agar plug diffusion method. Talaromyces sp. NTD-SP5-48 was particularly effective against Gram-negative bacteria. As. terreus NTD-NG1-05 displayed the highest activity against five bacterial strains, except Ps. aeruginosa. Notably, As. terreus NTD-NG1-05 and Talaromyces sp. NTD-SP5-48 demonstrated extended antibacterial activity in the agar disk diffusion method, with fermented broth (FB) showing superior inhibitory effects compared to mycelial extract (MY). Both isolates demonstrated significant antibacterial activity against B. Subtilis. Furthermore, all isolates exhibited significant antibacterial activity against B. subtilis, with a diffusion of 0.125 mg/disk. Only Talaromyces sp. NTD-SP5-48 (FB) displayed the highest inhibition activity against Ps. aeruginosa, with a diffusion of 1 mg/disk (100 mg/mL). In terms of enzyme activity, all isolates exhibited cellulase activity, with Talaromyces sp. showing the highest cellulase activity, followed by As. terreus. Laccase activity was only observed in the unidentified isolate NTD-SP5-34, while none of the isolates showed pectinase activity.

Keywords: Antimicrobial activity, Coprophilous fungi, Heat-resistant fungi, Lignocellulolytic enzymes

Masood Ur Rehman¹, Syed Muhammad Suhail¹, Iqbal Munir², Rajwali Khan^1*

¹Department of Livestock Management, Breeding and Genetics, The University of Agriculture, Peshawar, 25130, Khyber Pakhtunkhwa, Pakistan

²Institute of Biotechnology and Genetic Engineering, The University of Agriculture, Peshawar, 25130, Khyber Pakhtunkhwa, Pakistan

Abstract

The current study aimed to evaluate the impact of three different agroecological zones on the productive and reproductive performance of Holstein-Friesian (HF) cows, along with their comparative analysis of physiological and molecular markers under subtropical conditions of Pakistan. The productive (milk yield and composition) and reproductive (service per conception and calving interval) performance of genetically identical (n=210) HF cows placed across three agroecological zones: irrigated lowlands (Okara), wet (Abbottabad), and dry (Queta) highlands were analyzed. Additionally, heat stress markers (superoxide dismutase, glutathione peroxidase, serum cortisol, bovine heat shock protein 70 (HSP-70), and blood glucose) from five cows in each location (n=15 in total) were also investigated in early and late summers using commercial ELISA and calorimetric kits. Furthermore, the mRNA levels of heat shock protein genes HSPA8 and HSP90AB1 were also quantified in three agroecological zones through qRT-PCR.  The results revealed that cows raised in the wet highlands of Abbottabad exhibited significantly higher (P<0.05) milk production (daily and total lactational yield), and the lowest service period, calving interval and number of services per conception as compared to the cows from the other two agroecological zones. Furthermore, there was a significant effect (P<0.05) of agroecological zones on the heat stress indicators (SOD, GPX, cortisol, blood glucose, and HSP70) profiles and expression of heat shock protein genes in HF cows. The dairy cows from Abbottabad (Wet highlands) showed a significantly lower profile of heat stress indicators as compared to the cows from the other two agroecological zones. It can be inferred that HF cows function better in moderate agroecological zones of subtropical countries.

Keywords: Heat stress indicators, Subtropics, Holstein-Friesian cows, Agroecological zone

Heer Baloch¹, Irfan Ali Sabir², Saadullah Khan Leghari¹, Muhammad Sohail Saddiq^3*, Pravej Alam⁴, Shahbaz Khan^5*, Esha Mehik Fatima⁶, Mateen Sajid⁷, Muhammad Hammad Raza⁸, Muhammad Arif Hussain¹, Muhammad Ayoub¹, Rashid Iqbal^9,10

¹Department of Botany, Ghazi University, Dera Ghazi Khan, Pakistan

²College of Horticulture, South China Agricultural University, Guangzhou, 510642, China

³Department of Agronomy, Ghazi University, Dera Ghazi Khan, Pakistan

⁴Department of Biology, College of Sciences and Humanities, Prince Sattam Bin Abdulaziz University, Al- Kharj 11942, Saudi Arabia

⁵Colorado Water Center, Colorado State University, Fort Collins, CO, USA

⁶Department of Entomology, Muhammad Nawaz Shareef University of Agriculture, Multan, Pakistan

⁷Department of Horticulture, Ghazi University, Dera Ghazi Khan, Pakistan

⁸Department of Agri. Extension and Education, Ghazi University, Dera Ghazi Khan, Pakistan

⁹Department of Agronomy, Faculty of Agriculture and Environment, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan

¹⁰Department of Life Sciences, Western Caspian University, Baku, Azerbaijan

Abstract

Moringa leaf extract (MLE) as a biostimulant has demonstrated success in boosting the productivity of several agronomic crops, but its impact on Buckwheat crops remains unexplored. Buckwheat, recognized as an essential nutritional and functional food crop, often exhibits lower yields compared to major cereal crops grown in similar environments. Therefore, this research aimed to investigate the impact of different concentrations (1%, 2%, and 3%) of MLE on the agricultural performance of common buckwheat (CB) and tartary buckwheat (TB). A pot experiment was carried out according to completely randomized design with factorial arrangements having three replications. Results demonstrated significant improvements in growth parameters (branches, leaves, nodes, and internodes) for MLE-treated plants compared to the control group. Foliar treatment MLE 2% also increased chlorophyll content, improved membrane stability index (MSI) and relative water content (RWC), and enhanced biochemical composition (phenolic compounds, free amino acids, leaf proline, and soluble sugars) in both buckwheat genotypes followed by MLE 3% and MLE 1%. TB produced significantly higher grain yield (0.74 g) as compared to CB (0.43 g). The findings showed that a foliar treatment of MLE 2% led to increased grain yield in both TB (0.97 g) and CB (0.55 g) as compared to control group plants (0.37g TB and 0.22 g CB) respectively. This increase was associated with elevated activities of photosynthetic pigments, phenolic content, RWC, free amino acids, soluble sugars, and catalase in both buckwheat genotypes. In conclusion, MLE application at 2% significantly boosted the agriculture performance of buckwheat, and this study unlocked new insights into optimizing the productivity of the vital food crop.

Keywords: Biostimulant, Free amino acid, Phenolic, Buckwheat, Foliar application, Grain yield

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)

Tayyaba Asghar¹, Mudassar Mohiuddin^1*, Ayesha Mohiud Din^2,3, Tahira Kamal⁴, Muhammad Khalid Mansoor¹,

Abubakar Siddique^5,6,7, Mudasser Habib⁸, Riaz Hussain⁹, Muhammad Taslim Ghori¹⁰, Hira Hameed¹, Humaira Rizwana¹¹,

Islem Abid¹², Muhammad Ehsan¹³, Aroosh Shabbir¹⁴

¹Department of Microbiology, Faculty of Veterinary and Animal Sciences, The Islamia University of Bahawalpur, Pakistan

²Department of Biotechnology, Virtual University of Pakistan, Lahore, Pakistan

³School of Biological Sciences, University of Southampton, Southampton, UK

⁴National Institute for Genomics and Advanced Biotechnology, National Agricultural Research Centre (NARC), Islamabad, Pakistan

⁵Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Science and Technology (NUST), Islamabad, Pakistan

⁶Department of Veterinary Medicine, College of Animal Sciences, Zhejiang University, China

⁷Hainan Institute of Zhejiang University, Sanya, China

⁸Animal Sciences Division, Nuclear Institute for Agriculture and Biology, Faisalabad, Pakistan

⁹Department of Pathology, Faculty of Veterinary and Animal Sciences, The Islamia University of Bahawalpur, Pakistan

¹⁰Department of Clinical Medicine and Surgery, Faculty of Veterinary and Animal Sciences, The Islamia University of Bahawalpur, Pakistan

¹¹Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia

¹²Centre of Excellence in Biotechnology Research, King Saud University, P.O Box 2455, Riyad 11495, Saudi Arabia

¹³Department of Parasitology, Faculty of Veterinary and Animal Sciences, The Islamia University of Bahawalpur, Pakistan

¹⁴Institute of Molecular Biology and Biotechnology (IMBB), The University of Lahore, Pakistan

Abstract

The lumpy skin disease virus (LSDV) is a member of the Capripox genus of the Poxviridae family. It is the causative agent of lumpy skin, a highly contagious disease of cattle, water buffalo, sheep, and goats. In 2022, several outbreaks of LSD were reported in the Cholistan region of Pakistan, which has a large population of livestock living in arid conditions. A total of 230 blood/serum and scab samples were collected from three LSD outbreak locations. Clinically, affected cattle showed acute clinical signs characterized by skin nodules, fever, enlarged lymph nodes, emaciation, and lower leg edema. Hematological findings revealed non-significant changes in red blood cell and white blood cell counts (some animals had leukocytosis while others were leukopenic) whereas, hemoglobin level were significantly low. Platelet count, MPV, PCT, P-LRC, and P-LCC were elevated. Granulocytes were significantly low in LSD affected cattle while lymphocyte counts were significantly high. Serological findings revealed elevated protein levels, along with high creatinine and ALT concentrations. Amplification of DNA-dependent RNA polymerase 30 kDa subunit gene (RPO30) confirmed the presence of LSD virus in all suspected samples. Phylogenetic analysis showed that all Pakistani isolates clustered closely with isolates from neighboring countries. The SNPs differences were less than 20 among these isolates, indicating their close resemblance with each other. It can, therefore, be inferred that our LSD strains might be originated from neighboring Asian countries, that were affected by LSD in previous years.

Keywords: Lumpy skin disease virus (LSDV), Cholistan, Skin lesions, Hematology, Phylogenetic analysis

Pozdnyakova Yelena^1*, Solyanov Dmitry², Tatina Yelena¹, Britko Valeriy¹, Omarbekova Nazgul³, Korshukova Marina³

¹Department of Biomedicine, Karaganda Medical University, Karaganda, The Republic of Kazakhstan

²Department of Pharmacy, Karaganda Medical University, Karaganda, The Republic of Kazakhstan

³Department of Informatics and Biostatistics, Karaganda Medical University, Karaganda, The Republic of Kazakhstan

Abstract

The aim of this study is to investigate the effects on the nervous system of the use of Stachys sieboldii root powder, in growing male rats on a high fat and sucrose (HFHS) diet. The animals were divided into three groups: Intact – normal rodent chow, HFHS – high fat and sucrose diet, HFHS + Stachys – high fat and sucrose diet with Stachys supplementation. After 30 days of the experiment, the animals were subjected to the “Open Field” test to study the changes in behavioural responses. Biochemical studies of changes in the concentration of lipid peroxidation products in brain homogenates of growing rats were also carried out. Growing males on the HFHS diet exhibit altered nervous system functioning. Animals show apathy, their motor activity and indicators of exploratory behaviour are reduced and the level of emotional reactivity increases. On the part of biochemical changes – the level of all indicators of lipid peroxidation increases significantly. The introduction of Stachys root powder reduces the negative effect of the diet on the organism of animals – they significantly increase the indicators of exploratory behaviour, increase locomotor activity and reduce emotional reactivity, as well as there is a decrease in the concentration of all products of free-radical oxidation in the brain homogenate. Our study showed that diets high in fat and sucrose had negative effects on growing male rats. The introduction of Stachys sieboldii root powder into the diet improved both behavioural response scores and antioxidant processes in the brain.

Keywords: Stachys sieboldii, Open field, Lipid peroxidation, Conjugated dienes, Ketodienes

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

Khaled Mohamed Lela, Abdellatif Saleh El-Sebaay, Shaimaa Hassan Abd-Elrahman^*, Mahmoud Mohamed Elbordiny

Soil and Water Department, Faculty of Agriculture, Ain Shams University, P.O. Box 68–Hadayek Shoubra, Cairo 11241, Egypt

Abstract

Global attention is shifting to using fertilizers organically produced from available wastes in the surrounding ecosystem to provide sustainability in agriculture and conserve the environment. The aim of this study is to investigate the possible changes in the yield and quality of cucumber resulting from different combinations of organic solid and liquid fertilizers prepared from different organic wastes, as compared to applying mineral fertilizers. Furthermore, detecting available N concentration in sandy loam soil and total N concentration in cucumber leaves considering the effect of the studied treatments every 15 days after transplanting (DAT) until 120 DAT. Four fertilizer treatments (3 organic fertilizers, i.e., vermicompost (VC), pigeon manure (PM), and compost (COMP) in addition to mineral fertilizers) were tested in a randomized complete blocks design with three replicates. The organic fertilizer treatments were divided into three categories, the first was solid added through two equal doses. The second category was like the first one plus adding vermicompost tea (VCT) through drip irrigation (DI). The third category was like the second one, but the VCT was substituted with pigeon manure tea (PMT). Obtained results showed that the treatments of PM_50/50, and VC_50/50 as ground applications plus PMT or VCT through DI were given significant increases in available N in the studied soil, and enhanced the cucumber plant growth parameters, yield traits, and the total concentration of N in leaves throughout the experimental period. Additionally, the highest benefit/cost ratio (5.68) was achieved with the application of organic treatments compared to traditional ones (3.28).

Keywords: Vermicompost, Pigeon manure, Organic aqueous extract, Organic farming, Detecting N in soil and plant, Cucumber plants

Duy Ha Nguyen¹, Ngoc Anh Thi Ta², Huong Giang Van², Dinh Toi Chu³, Thai Son Nguyen¹ , Van Mao Can¹, Quynh Uyen Nguyen^2*, Hoang Van Vinh^2*

¹Vietnam Military Medical University, Hanoi, Vietnam

²VNU, Institute of Microbiology and Biotechnology, Hanoi, Vietnam

³International School, Vietnam National University, Hanoi, Vietnam

Abstract

Diarrhea is a side effect of antibiotic misuse and is frequently associated with intestinal inflammation and imbalanced gut microbiota. Many studies have demonstrated that probiotics can exhibit potential to mitigate the effects of antibiotic-associated diarrhea (AAD). In this study, we employed Lincomycin to induce AAD in the rats and subsequently assessed the impact of the multi-strain probiotic preparation LabMix on this model. The rat groups, including healthy control rats, AAD-induced rats, AAD rats with no treatment (natural recovery rats), and AAD rats treated by LabMix preparation, were evaluated regarding the general assessments, some immune indices, and intestinal microbiota analysis. The results revealed that the LabMix preparation considerably lowered the effects of the antibiotic regarding the diarrhea score and the thickness of the ceca in the rats treated by LabMix preparation. Additionally, the LabMix preparation reduced inflammatory cytokines, including TNF-a, and IL-6, while increasing the IgA in sera and in intestinal mucosae. Furthermore, it altered the compositions and abundance of intestinal bacteria of the rats. In particular, the AAD rats treated by LabMix preparation decreased the levels of potentially harmful genera such as Bacteroides, Escherichia-Shigella, and Pseudomonas. They also increased the levels of beneficial genera including Lactobacillus, Bacillus, Romboutsia, and Clostridium innocuum. In general, the multi-strain probiotic preparation LabMix showed the effective mitigation and the improvement of the intestinal microbiota of the AAD rat model.

Keywords: Antibiotic Associated Diarrhea (AAD), Cytokine, LabMix, Microbiota, Probiotic

Amina Foughalia¹, Sakina Zerizer^1*, Boutheyna Aribi¹, Zahia Kabouche², Chawki Bensouici³

¹Département de Biologie Animale, Laboratoire d’Immunologie et Activités Biologiques des Substances Naturelles, Université des frères Mentouri-Constantine 1, 25000 Constantine, Algeria

²Département de Chimie, Laboratoire d’Obtention de Substances Thérapeutiques, Université des frères Mentouri-Constantine1, 25000 Constantine, Algeria

³Centre de Recherche en Biotechnologie (C.R.Bt), Ali Mendjli Nouvelle Ville BPE.73 Constantine, Alegria

Abstract

Calendula stellata (Asteraceae family), growing in North-East Algeria was investigated for its biological activities in laboratory animal model studies. The n-butanol extract was prepared from aerial parts using ethanol maceration followed by liquid-liquid extraction then, the total phenolic and flavonoid contents were measured, and the antioxidant activity was evaluated using DPPH, ABTS, CUPRAC, and reducing power assay. Then acute toxicity was tested in mice using the Up and Down test and, the anti-inflammatory anti-arthritic activity was evaluated using formalin induced arthritis (FIA) in Wistar rats. Results indicated that, the extract was rich in phenolic and flavonoid contents (224.097 ±7.31 mg GAE/g and 207.36±10.081mg QE/g, respectively). It possessed considerable antioxidant activity, the extract showed no visible toxicity or mortality signs, and the LD₅₀ was > 2000mg/kg body weight. Furthermore, in the FIA, the extract showed significant dose-dependent inhibition in paw edema. It also it decreased the C-reactive protein (CRP) plasmatic, preventing cartilage destruction and liver injury. In conclusion, C. stellata n-butanol extract possesses antioxidant and anti-arthritic activities, in addition to protective properties in hepatic tissue.

Keywords: Calendula stellata, Antioxidant, Anti-inflammatory, Anti-arthritic, Algeria