The excessive application of various synthetic pesticides led to control difficulties, including insect resistance and environmental contamination. This study aimed to evaluate the insecticidal and repellent activities of twelve botanical powders and aqueous extracts against Tribolium castaneum, with a focus on acetylcholinesterase and glutathione-S-transferase detoxification enzyme activities. Toxicity tests revealed that Azadirachta indica dry powder was the most harmful, having the lowest LD50 value of 2.09% w/w, while in aqueous extract A. indica was the most toxic, with an LC50 of 2.20% after 24h. Repellency tests demonstrated that A. indica exhibited the highest repellent effect in both powder and aqueous forms (86.66%). As a result of the most effective botanical application, biochemical analyses showed that acetylcholinesterase activity remained highest (6.17 ± 0.17 U/min/g) in A. indica-treated insects, whereas glutathione-S-transferase enzyme activity peaked in response to Eucalyptus tereticornis (85.00 U/min/g), indicating a strong physiological defense response. These results indicate that plants like A. indica, D. stramonium, E. tereticornis, and Ar. nilagirica can be used as promising bio-insecticides options for controlling T. castaneum in stored products.

Lernaea spp., commonly referred to as anchor worms, are widespread ectoparasitic copepods known for their broad host range and high morphological variability, which complicates accurate taxonomic identification. The hypothesis of this study was to accurately identified lernaea and differentiate at the species level using 18S rRNA gene sequencing, thereby overcoming the limitations and ambiguities of conventional morphological identification. In this study, approximately 200 Lernaea specimens were collected from infected carps at fish farms in the Kasur district of Pakistan. Parasites were detached from hosts using potassium permanganate (KMnO₄) treatment and preserved in 70% ethanol for both morphological and molecular analyses. Initial morphological assessment was conducted under a stereomicroscope, focusing on key diagnostic features such as the structure of the anchor-shaped holdfast. For molecular characterization, genomic DNA was extracted using the Thermo Scientific GeneJET Genomic DNA Purification Kit. DNA quality and concentration were evaluated with a NanoDrop spectrophotometer. The partial 18S rRNA gene was amplified via polymerase chain reaction (PCR), and the products were visualized through agarose gel electrophoresis before sequencing. Sequence data were edited using BioEdit v7.0 and subjected to BLAST analysis against the NCBI GenBank database. Phylogenetic relationships were inferred using the neighbor-joining method, revealing the presence of three Lernaea species: L. cyprinacea, L. polymorpha, and L. ctenopharyngodonis. The findings underscore the effectiveness of molecular tools particularly 18S rRNA gene analysis in resolving morphological ambiguities and provide novel sequence data that helps to explain Lernaea diversity in Pakistan.

Eosinophilia is a hallmark of many helminthic infections. However, the underlying parasitic etiology often goes undetected-especially in immunosuppressed patients. We aimed to evaluate the helminthic seropositivity in immunosuppressed patients with peripheral eosinophilia at a tertiary care hospital in north India. In this retrospective observational cross-sectional study, a total of 115 immunosuppressed patients with eosinophilia were subjected to serological testing for six common helminths, i.e., IgG antibodies against Strongyloides stercoralis, Toxocara canis, Trichinella spiralis, Echinococcus granulosus, and Taenia solium by ELISA; IgG4 antibodies against Wuchereria bancrofti by ELISA; and W. bancrofti antigen by immunochromatographic assay. Absolute eosinophil counts (AEC) and clinical-demographic data were analyzed using non-parametric tests and Spearman correlation statistics. Overall, 34 patients (29.6%) were positive for at least one parasitic serology, with S. stercoralis being the most common (18.3%), followed by T. canis (11.3%), T. spiralis (6.1%), W. bancrofti (5.2%), E. granulosus (5.2%), and T. solium (1.7%). Multiple seropositivities were detected in 14 (12.2%) patients. Seropositive patients had significantly higher median AEC than seronegatives (3021 vs. 1495 cells/µL, Wilcoxon p = 0.008). A statistically significant correlation was found between AEC and number of positive serologies per patient (Spearman's ρ = 0.269, p = 0.003). A graded relationship between increasing seropositivity and severity of AEC was noted (Cochran-Armitage trend p = 0.006). Nearly 30% of immunosuppressed patients with peripheral eosinophilia had serological evidence of helminthic infections. Helminth serology panel testing for persistent or unexplained eosinophilia along with clinical evaluation may facilitate early diagnosis and management in immunocompromised hosts.

The liver fluke Opisthorchis viverrini is a major public health concern in Southeast Asia and a leading cause of cholangiocarcinoma. Its transmission relies on freshwater snails of the genus Bithynia as the first intermediate hosts. However, accurately identifying these morphologically similar snails remains challenging. This study aimed to develop species-specific primers for the accurate molecular identification of Bithynia siamensis siamensis, which serves as an important first intermediate host of O. viverrini. Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) was employed to isolate a unique DNA fragment specific to B. siamensis siamensis. A distinct 820bp RAPD-derived fragment was obtained, sequenced, and analyzed. From this fragment, a conserved internal region was selected and used to design a species-specific primer pair (BSS2F/BSS2R), which produces a 280bp diagnostic amplicon. The specificity and sensitivity of the newly developed assay were subsequently evaluated against other freshwater snail species. The designed primers consistently amplified a 280bp fragment exclusive to B. siamensis siamensis, with no cross-reactivity to B. siamensis goniomphalos, B. funiculata, or other cohabiting snail species. The assay detected as little as 5 ng of genomic DNA and achieved 90% amplification success across samples from four provinces. The RAPD-derived primer pair provides a rapid, reliable, and cost-effective molecular tool for the specific detection of B. siamensis siamensis. This approach enhances epidemiological surveillance of O. viverrini transmission and supports integrated One Health strategies for controlling opisthorchiasis in endemic regions.

Toxoplasma gondii is considered the second most common pathogen causing abortions in sheep, resulting in great economic losses. Thus, timely, sensitive and specific diagnosis of ovine toxoplasmosis is very important. In this study, diagnostic performance of an ELISA using a T. gondii GRA6 peptide (GRA6 ELISA) was investigated using serum samples (n = 61) collected from sheep diagnosed with toxoplasmosis using a commercial ELISA kit. Additionally, an in house ELISA using T. gondii tachyzoite lysate antigen (TLA ELISA) was also used to compare its performance with that of the GRA6 ELISA. Using the commercial ELISA kit as reference, the TLA ELISA and the GRA6 ELISA showed sensitivities of 83.7% and 88.4%, and specificities of 85% and 80.0%, respectively. Cohen's kappa coefficient between TLA ELISA/ GRA6 ELISA and commercial ELISA kit was 0.652 and 0.675, respectively, indicating that both tests have good agreements. When we analyzed serum samples (n = 19) collected from a sheep group that had experienced abortions and were diagnosed with toxoplasmosis using a commercial ELISA kit, sensitivity values for the TLA ELISA and the GRA6 ELISA reached 100% and 89.5%, respectively. According to the seroprevalence results in serum samples analyzed, the commercial ELISA kit detected seroprevalence as 68.2% whereas the GRA6 ELISA and TLA ELISA detected seroprevalence as 66.7% and 61.2%, respectively. Furthermore, among the serum samples that yielded compatible results across the three different ELISA assays (n = 45), 31 were identified as seropositive, corresponding to a seroprevalence rate of 68.9% (31/45). In conclusion, the GRA6 ELISA detected ovine toxoplasmosis with high sensitivity and specificity.