Background: Paramphistomiasis (Rumen fluke disease) in ruminants is a major health problem, while documented reports on Paramphistomum species are limited in Asian countries. The present study aimed to identify paramphistomoid flukes that infects buffaloes with the goal of characterization of prevalence in Pakistan and its comparison with neighbor countries.Materials, Methods & Results: In 2018, a total of 178 slaughtered buffaloes aged four to six years were examined and flukes were collected from their infected rumen and reticulum using sterilized forceps. After amplification and sequencing of 18S rRNA partial fragment, the generated sequences were edited (810bp) and aligned with the other sequences of Paramphistomum species retrieved from NCBI. A phylogenetic tree was constructed using maximum likelihood method in MEGA X. The 18S rRNA sequence was found 100% similar with Paramphistomum cervi of China and 98% with Paramphistomum epiclitum and other Paramphistomum species of India. The parasitic Pharamphistomum species was identified molecularly as P. cervi.Discussion: Molecular studies provide insight into the biology and phylogenetic relationship among various parasites. These studies are reliable in the genetic-based identification and description of several disease causing agents. The 18S rRNA sequence of P. cervi generated in this study was found closely identical to the P. cervi of the neighbor countries (China and India) which may be due to the similar geographical, environmental conditions and transboundary movement of infected hosts. This is the first nature of study which provides the molecular-based evidence of P. cervi existence in Pakistan and revealed the 18S rRNA as novel molecular marker for the identification and further characterization of Paramphistomum species across Pakistan. The submitted sequence of this study will provide a baseline for further molecular characterization and to compare with other Paramphistoma species from different regions of Pakistan.

The present analysis explores the chemical constituents and determines the in vitro antimicrobial, antidiabetic, and antioxidant significance of the essential oils (EOs) of the stem, leaves, and flowers of Ochradenus arabicus for the first time. The EOs of the flowers presented seventy-four constituents contributing to 81.46% of the total EOs, with the major compounds being 24-norursa-3,12-diene (13.06%), 24-norursa-3,12-dien-11-one (6.61%), and 24-noroleana-3,12-diene (6.25%). The stem EOs with sixty-one compounds contributed 95.95% of the total oil, whose main bioactive compounds were (+)-camphene (21.50%), eremophilene (5.87%), and δ-selinene (5.03%), while a minimum of fifty-one compounds in the leaves’ EOs (98.75%) were found, with the main constituents being n-hexadecanoic acid (12.32%), octacosane (8.62%), tetradecanoic acid (8.54%), and prehydro fersenyl acetone (7.27%). The antimicrobial activity of the EOs of O. arabicus stem, leaves, and flowers was assessed against two bacterial strains (Escherichia coli and Streptococcus aureus) and two fungal strains (Penicillium simplicissimum and Rhizoctonia solani) via the disc diffusion assay. However, the EOs extracted from the stem were found effective against one bacterial strain, E. coli, and one fungal strain, R. Solani, among the examined microbes in comparison to the standard and negative control. The tested EOs samples of the O. arabicus stem displayed a maximum potential to cure diabetes with an IC50 = 0.40 ± 0.10 µg/mL, followed by leaves and flowers with an IC50 = 0.71 ± 0.11 µg/mL and IC50 = 10.57 ± 0.18 µg/mL, respectively, as compared to the standard acarbose (IC50 = 377.26 ± 1.20 µg/mL). In addition, the EOs of O. arabicus flowers had the highest antioxidant activity (IC50 = 106.40 ± 0.19 µg/mL) as compared to the standard ascorbic acid (IC50 = 73.20 ± 0.17 µg/mL) using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. In the ABTS assay, the EOs of the same sample (flower) depicted the utmost potential to scavenge the free radicals with an IC50 = 178.0 ± 0.14 µg/mL as compared with the ascorbic acid, having an IC50 of 87.34 ± 0.10 µg/mL the using 2,2-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic acid (ABTS) assay. The EOs of all parts of O. arabicus have useful bioactive components due to which they present antidiabetic and antioxidant significance. Furthermore, additional investigations are considered necessary to expose the responsible components of the examined biological capabilities, which would be effective in the production of innovative drugs.

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Microplastics (MPs) are an emerging environmental health concern due to their widespread occurrence in food sources such as fish, meat, chicken, honey, sugar, salt, tea and drinking water, thereby posing possible risks to human health. This study aimed to observe the existence of MPs in the crop and gizzard of the farm chicken, a significant food source in Pakistan. Twenty-four chicken samples were taken from eight poultry farms across Punjab, Pakistan. A total of 1227 MP particles were found from 24 samples (crop and gizzards) originating from the 8 poultry farms. In all, 429 MP particles were found in 24 chicken crops, with a mean of 17.8 ± 12.1 MPs/crop. In contrast, 798 MP particles were found in 24 chicken gizzards, with a mean of 33.25 ± 17.8 MPs/gizzard. Comparatively larger particles, ranging between 300–500 µm, were more abundant (63%) than other considered sizes (300–150 µm [21%] and 150–50 µm [16%]). Additionally, fragments were the dominant type of shape in both sample types (crop [64%] and gizzard [53%]). The predominant colours of particles extracted from gizzards and crops were yellow (32%) and red (32%), respectively. Chemical characterisation of these particles detected four types of polymers: polyvinyl chloride (PVC) at 51.2%, followed by low-density polyethylene (LDPE) at 30.7%, polystyrene (PS) at 13.6% and polypropylene homopolymer (PPH) at 4.5%. In conclusion, we provide evidence for MPs in the gizzards and crops of farmed chickens which may originate from contaminated poultry feed. Only a few studies have been reported globally to assess MPs ingestion in chickens. The current study is the first report from Pakistan. It could be a valuable addition to support MPs literature to establish a relationship between MPs contamination and intake through the food chain.

Paramphistomosis is a neglected ruminant parasitic disease caused by trematodes known as Paramphistomum, which has a diheteroxenic life cycle involving freshwater snail genera, i.e., Planorbis and Lymnaea as the intermediate host and mammals as the definitive host. Snail vector distribution, infection with Paramphistomum spp. cercariae, preferred habitat, and their relationship with certain meteorological factors were not investigated in the province Khyber Pakhtunkhwa of Pakistan. Therefore, this study is designed to evaluate the effects of meteorological factors on the occurrence and severity of Paramphistomum spp. cercariae in Indoplanorbis and Lymnaea intermediate snail hosts. For this purpose, a cross-sectional survey was conducted from October 2018 to September 2019, and snails were collected and then identified using snail shell morphology; their infection with Paramphistomum spp. cercariae was determined through microscopy; and descriptive statistics were used to estimate the prevalence of infection and evaluate their occurrence relationship with a certain meteorological factors including temperature, humidity, rainfall, and pan evaporation in different districts of the above-mentioned province of Pakistan, i.e., adjacent areas of Bara and Kabul rivers in district Nowshehra, Kabul River (Sardaryab) of district Charsadda, Kalpani River of district Mardan, and Indus River (Hund) of district Swabi. A total of 2,706 Indoplanorbis (1539) and Lymnaea (1167) snails were collected, in which overall 10.30% shed Paramphistomum spp. cercariae. The highest infection rate was found in the river of district Swabi (13.20%), while the lowest in adjacent rivers of district Nowshehra (8.19%). Meteorological factors play an essential role in the causation of Paramphistomum spp. infection, parasitic reproduction, vector growth, and survival. Due to these factors, high significant prevalence was found in the summer season (11.83), followed by autumn (11.25), which might be due to optimum temperature, relative humidity, and rainfall ( p < 0.05 ). It was concluded from the study that meteorological factors contribute to the prevalence of Paramphistomum species in the Indoplanorbis and Lymnaea, which act as vectors for the disease paramphistomosis, which may lead to the increased intensity of infection outbreaks of the parasite population in humans and domestic animals.